Lori Maguire

CHAPTER SIX "FREEDOM, DEMOCRACY AND THE RULE OF LAW": THE DEBATE IN GREAT BRITAIN AROUND

Hong Kong, Britain"s last major colony, returned to China, marking the effective end of the long history of the British Empire. 1 But the word "return" tells us a great deal about how Hong Kong"s fate differed from that of other imperial possessions. All the others had become "independent" and U.K. policy had been to ensure that they received preparation and training in establishing a political system that reflected British values-even if that system did not always last long. Chris Patten, the last governor of Hong Kong, described this process in both admiring and ironic tones:

 But this one-size-fits-all module could not work in the case of Hong Kong for it was being "returned"; there was a "handover" to China-a nation not renowned for its record on human rights. Paradoxically, the civil service, police and independent courts already existed, as did a very real demand for democracy on the part of the population but there were very real fears that these elements would not survive the handover.

Unlike a number of other colonies, Hong Kong had never preoccupied British domestic opinion very much.

destiny or the achievement of economic and social modernisation at home." [START_REF] Darwin | Hong Kong in British Decolonisation[END_REF] Very little academic work on Hong Kong appeared in the U.K. before the 1980s, and it was largely ignored in the United States and elsewhere. [START_REF]Hong Kong in Transition: The Handover Years[END_REF] So it only figured significantly in British political discourse at the end of its time as a colony. [START_REF]And even then in only a limited way[END_REF] Debate in Britain about the handover centred on two areas. First, the question of democracy and human rights which preoccupied much of the press and most lawmakers. The other major subject of debate was that of immigration. Because the people of Hong Kong worried about their future under Chinese rule, many of them sought to leave. As the familiar colonial power, Britain was the first choice for many of these and this fact would shape British discourse and policy on the question. Interestingly enough, there was very little difference between the parties on either of these two subjects. With very few dissenting voices, they all agreed on the need to support the reforms of Chris Patten. And, once again with few dissenting voices (but one of these was Michael Howard, Home Secretary from 1993 to 1997) basic agreement existed on immigration policies. Patten summarised the situation in his memoirs:

If Britain was to deny any moral obligation to Hong Kong that raised issues of race (and this was the real purport of its policy on nationality and passports), it clearly recognized its duty to defend Hong Kong"s bonds to the economic and political values that had shaped it and that defined its difference from the rest of China. From the outset in 1982 of its negotiations with China on Hong Kong"s future, Britain made plain its commitment to the maintenance of capitalism and freedom in the territory. 6 Although there was a difference in emphasis between the parties, almost everyone agreed that Britain"s last major act of decolonization should be conducted with dignity and honour-which meant bequeathing to Hong Kong a legacy of "freedom, democracy and the rule of law", as Chris Patten put it-whatever China"s objections might be. 7 To begin with, let us briefly examine the history of the return of Hong Kong. Although the United Kingdom had an internationally recognised title to Hong Kong Island and the Kowloon Peninsula, most of the rest of the territory was held under a 99 year lease from China, which expired in 1997. During the premiership of Thatcher, the government realised that they had to tackle the issue of the future of Hong Kong. Given the relative strength of Britain and China at the time, the former had no choice but to negotiate and try to get the best deal possible for its colony. By this time, Deng Xiaoping had embarked on his economic reforms and many people in the West were optimistic about the future of China. They hoped that, as the economy developed so would political liberty. Indeed, the British were, on the whole, satisfied with the Joint Declaration, signed in 1984, that enshrined the concept of "one country, two systems"-Hong Kong would be part of China but as a special administrative region for fifty years and would enjoy considerable independence. This principle was given shape in the Basic Law, promulgated in 1990, which became the constitutional document of Hong Kong after its transfer.

In June 1989 the Chinese government violently repressed prodemocracy demonstrators at Tiananmen Square. The event traumatised much of the population of Hong Kong who, quite understandably, worried about their own future. Numerous protests took place there and strong calls for democracy developed in the colony. Indeed, London had been singularly lax in introducing self-government into Hong Kong: few people possessed the right to vote and all of the colony"s governors had been civil servants appointed by London without local consultation. Of course, the British could justify this democratic deficit: chiefly because of their fear of how the Chinese government would react. Still, the British government had declared in 1984 that they would "build up a firmly-based, democratic administration in Hong Kong in the years between now and 1997" but in reality did little about this. [START_REF] Luce | House of Commons Debates[END_REF] Added to this, as we shall see, a major reform of British citizenship in 1981 had deprived most people in the colony of full British nationality and thus the right of abode in the United Kingdom. By the late 1980s, this, plus the governmental reluctance to put into effect democratic reforms, had caused many people in Hong Kong to lose confidence in London. In such circumstances, most British leaders felt they had to make an attempt to remedy these deficiencies and try to provide some form of future protection to Hong Kong. John Major, recently appointed Foreign Secretary and soon-to-be Prime Minister, described the situation as follows: Before Tiananmen Square it was possible to be optimistic. After it, trust was shattered. A mood of near despair gripped the territory. Its stock exchange fell 30%, and business investment was held back. [START_REF] Major | The Autobiography[END_REF] For this reason, Major decided to keep a previously scheduled meeting with Qian Qichen, the Chinese Foreign Minister, a few weeks after the massacre, as he felt that cancelling it would only hurt Hong Kong. He received criticism in the British press for this which, like most of the western media, became more hostile to China after Tiananmen Square.

In September 1991, Major, now Prime Minister, became the first important Western leader to visit Beijing since the massacre. He did so to discuss the construction of a new airport in Hong Kong which the Chinese had been blocking. The visit also received much criticism in the British media and its only tangible result was a "Memorandum of Understanding" on the new airport which, it was hoped, would clear Chinese obstacles to its construction. 10 Furthermore, at around the same time, on 17 September, Legislative Council elections were held in Hong Kong and the democrats got 16 of 18 seats while no pro-China candidate won. However, Governor Wilson failed to appoint any of the victorious democrats to the Executive Council, which functioned as his cabinet. Although only 39.2% of voters actually cast their ballots, the result showed much support for democracy and the governor looked out of touch. [START_REF] Chang | The Politics of Hong Kong's Reversion to China[END_REF] These factors all seem to have weighed on Major who decided, with the accord of Douglas Hurd, the Foreign Secretary, to replace the current governor with a heavyweight politician. He made the announcement that this change would occur after the next election. Furthermore, Sir Percy Cradock, the government"s major adviser on Hong Kong, was pensioned off. London had clearly signalled a significant change in the direction of its policy.

Many in the press saw this as a direct result of Major"s visit: he had gained little from the Chinese and only embarrassed himself, and had come to the conclusion that there was little to be gained from attempting to placate China. Hurd, however, has downplayed the importance of the visit in the policy change. As we have already seen, many other factors weighed in the decision. Indeed, it reflects ideas that Hurd already held. In his book, The Arrow War: An Anglo-Chinese Confusion, 1856 to 1860, written in 1967, he described the Chinese as possessing an "assumption of superiority". In the nineteenth century, Western nations had dealt with this by obliging the Chinese, sometimes through the use of force, to treat them as equals. [START_REF] Stuart | The Public Servant[END_REF] He does not seem to have found any advantage in following Cradock"s belief in placating China. Hurd felt that Great Britain"s honour was at stake. In December 1989 the Foreign Secretary announced that: This is just about the last chapter in the story of this country"s empire. I am rather keen… that the last chapter should not end in a shabby way. [START_REF][END_REF] Previously, the strong concentration on seeking accommodation with the Chinese government had meant that the aspirations to self-government of Hong Kong"s population had been neglected. This had not caused instability in earlier years when the colony had been poor and most residents had focused on improving their economic position. But with wealth and education they desired some say in their own government. Most British people felt that, as a democratic country, they had to make an effort to show respect for what were their own fundamental values. They had failed earlier to introduce democracy and self-government into Hong Kong but in their last hours of governance-and especially after the shock of Tiananmen Square-they felt it necessary to push through reforms and to be seen as responsive to the local inhabitants. As Major said:

It was right that as Hong Kong changed, its constitution should change too. Throughout the 1960s and 1970s the colony had a low-cost manufacturing base, with no demand for political reform. By the 1990s it was a prosperous, educated financial centre of global importance, and expectations were far greater. The citizens of Hong Kong were now eager for political reform, and it would have been wrong to deny it, although the changes we implemented had to be within the terms of the Joint Declaration. [START_REF] Major | Steve Tsang has shown how the colonial government of Hong Kong, by the 1980s, came closer than any purely Chinese government ever had to fulfilling Confucian ideals. Paradoxically, by this time it was not enough and[END_REF] In this policy the British government had the full support of the other parties. So the discourse on Hong Kong during this period is remarkably one-sided.

After the Conservative victory in the 1992 elections, Major and Hurd chose to appoint Chris Patten as the last governor of Hong Kong. Patten, a former cabinet minister, rising star in the Conservative party and close personal friend of the prime minister, had lost his seat in the 1992 election. He was, thus, a governor with considerable political weight and one with strong ideas about his job. Patten was given the mission of reassuring the populace of Hong Kong about the handover as well as increasing democracy there. Hurd informed Parliament in May 1992:

The new governor will find the political development of Hong Kong high on his list of priorities. With his advice, we shall need soon to start putting in place arrangements for the 1995 elections to the Legislative Council. As the House knows, we will raise with the Chinese the need for a faster pace of democratisation… We want-this is familiar ground to the House-to promote the political development of Hong Kong in a way that is capable of enduring beyond 1997-a through train. Reconciling these two requirements will be one of the main tasks in Hong Kong over the next year or so. 15 Hurd had no illusions about the difficulty involved. When asked earlier about the impediments to introducing universal suffrage in Hong Kong, he had replied simply: "That it will come to an abrupt end in 1997." 16 Patten became a governor unlike any previous one. He refused the governor"s formal dress (which he described as making one look like "a recently deceased hen") and went around in a plain suit. 17 Being a politician, he also worked the crowds and made a point of leaving Government House to meet ordinary people and see conditions in Hong Kong for himself. By autumn 1992 he had prepared his proposals to extend democracy and gave them the maximum of publicity in the world press. He clearly wished to draw the eyes of the world on Hong Kong. China was furious. In the past, agreements had been worked out quietly, behind the scenes, between the two governments. Although China had been given advance notice, they had not been consulted and so immediately rejected the changes.

Patten"s proposals were modest enough and probably did not violate either the Joint Declaration or the Basic Law but he pushed both as far as he could. Furthermore, Patten clothed his proposals in the rhetoric of democracy and did his best to attract the attention of the world"s pressespecially that of the United States-to Hong Kong. Jonathan Dimbleby stated that: "Patten"s goal, commonplace in Western democracies, but hitherto untested in Hong Kong, was to charm the media into unwitting complicity with his efforts to woo public opinion, and thereby protect his flank from potential critics within the foreign-policy establishment in Britain and the business community in Hong Kong." [START_REF] Dimbleby | The Last Governor: Chris Patten and the Handover of Hong Kong[END_REF] Patten clearly realized the key economic role played by the colony which enjoyed significant business and investment interests, not only from Great Britain and China, but from the United States, Canada, Australia, Japan and the European Union. Furthermore, because of recent large-scale emigration, there were large numbers of its citizens in the first three of these countries where they could exercise pressure in domestic politics. [START_REF]For more on this see Brian Hook[END_REF] Patten certainly succeeded in getting international attention and received near universal praise in the western media for his proposals. The American press in particular flocked to the colony. Larry King actually broadcast a special edition of his CNN show from there with Patten present. Newsweek published an article on Patten entitled "The "God of Democracy"". [START_REF] Strasser | [END_REF] Time, Business Week, U.S. News and World Report, The New Yorker and major newspapers all featured articles on the subject. Patten also made numerous trips abroad to explain his position. At around the same time as he was presenting his proposals, the United States Congress also decided to act and voted the United States-Hong Kong Policy Act. Among other things, it outlined how the U.S. would relate to Hong Kong after the handover: Hong Kong would be treated as a distinct entity on its own and all previous agreements would continue. The law also expressed backing for democracy and human rights and required the Secretary of State to report regularly on conditions in Hong Kong relating to U.S. interests. These later reports generally espoused the process of democratisation.

After the Chinese had expressed their hostility, they turned to trying to hurt Hong Kong economically in order to show their own power. For the first month or so, Chinese threats had little impact on investor confidence but in mid November the Hong Kong stock market began to decline and this trend continued. As The Economist explained: "The market has fallen by 23% in less than three weeks…. falling markets-property is likely to go down next-step up the pressure on Mr. Patten and undercut his support in the colony." [START_REF]Patten"s Chinese Torture[END_REF] A number of Hong Kong businessmen certainly urged the governor to give in but, on the whole, public opinion in both the colony and Britain remained firm. The Financial Times succinctly explained why:

Worrying as its threats may be, however, they do not constitute an argument for changing course. For one thing, they are still largely rhetorical. In saying that it will, after the handover, reverse reforms and refuse to honour contracts, Beijing is underlining that, notwithstanding the Sino-British deal on Hong Kong"s future, it will be in charge and will do as it pleases. The hope must be that in practice its policies will be shaped by an economic self-interest based on the status quo in Hong Kong. For another thing, Mr Patten"s ideas for improving democratic accountability still enjoy -so far as can be gauged-overwhelming support from the Hong Kong people. His proclaimed object is to ensure that they have a say in their destiny. So long as they, and their representatives in the colony"s Legislative Council, continue to express the wish for greater political freedom, that is what he should be striving to provide, even if the price is friction with China and instability.

Nothing in recent events has undermined Mr Patten"s judgment that trying "in a modest way" to accommodate the Hong Kong people"s aspirations is the best way to promote political stability. [START_REF]Dragon"s Wrath[END_REF] Many felt that, by attracting the eyes of the world on Hong Kong, Patten had made it more difficult for the Chinese to destroy its freedoms. As Malcolm Rifkind, who later replaced Hurd as Foreign Secretary, put it: "There will be great global interest in what happens and the Chinese government will need to reassure the entire international community that the welfare of Hong Kong will be properly safeguarded." 23 Of course, the British insisted on placing the onus for the failure to reach an agreement on the Chinese. Patten publicly called on China to make its own proposals and, when they failed to do so, complained about the difficulty of playing tennis "unless the ball comes back over the net". [START_REF] Beijing Vs | Lords Debates[END_REF] Hurd also stressed this:

The Governor"s proposals represent our judgment of the right way forward for Hong Kong, but we have said from the start that we are open to alternative ideas, from the people of Hong Kong or from the Chinese side. We have had a wide range of suggestions from people in Hong Kong. The Chinese side have opposed the proposals without offering anything in their place. Since last October, we and the Governor have been urging the Chinese side to discuss those electoral issues with us in order to reach an understanding. We are ready to enter such discussion without preconditions. We want to see as much continuity as possible in Hong Kong"s electoral arrangements before and after 1997… We cannot and do not accept what some Chinese officials have said in the past few days-that the role of people from Hong Kong in discussions about Hong Kong"s future should be downgraded. 25 Hurd was clear: the blame lay squarely with the Chinese. The British were simply standing up for the rights of the people of Hong Kong and trying to ensure that they had a role in determining their own future.

A number of MPs, especially in the Conservative party, saw policy on Hong Kong in almost mystical terms and they frequently used words like "mission". The Conservative MP, Timothy Renton, chairman of the allparty Hong Kong parliamentary group, described his view of Patten"s role: "The mission was to leave a legacy of a very well-founded democracy in Hong Kong from 1 July 1997 onwards." Note also his use of the word "legacy" another important term which focuses on Britain and on Britain"s involvement. The stress is clearly on Britain"s honour-in her own eyes and in that of the world"s-and on the survival of its values and this became an important theme, primarily of the Conservatives. David Howell, chairman of the Select Committee on Foreign Affairs insisted that: "The issue at the forefront of our relations with China, and which has understandably been the subject of most comments today because it blocks our longer-term vision, is Hong Kong and how we can do the right thing and fulfil our duty from Hong Kong"s point of view." 26 For Nigel Waterson: "The only realistic bulwark in favour of Hong Kong, to protect it after 1997, is the rule of law. That is the lasting legacy of this country in Hong Kong." 27 The former journalist, Lady Olga Maitland concluded that when 1997 comes: "We should feel proud that we have more than honoured our obligations. We shall have strived to the end… We shall leave Hong Kong with pride and honour." 28 This line of reasoning, though, was not limited to the Conservatives. The Labour peer Lord Dubs talked of Britain"s "enormous responsibility to do the right thing by the people of Hong Kong". 29 And no less a figure than Robin Cook insisted that: "It is important to show that we have discharged our obligation to the people of Hong Kong that we will surrender sovereignty of the territory of Hong Kong without surrendering the liberty of the people of Hong Kong." 30 Certainly one must see much of this rhetoric in the context of a long history of British discourse on Empire. By the late eighteenth century, many had come to see imperialism in a paternalist light; not only should the Mother Country benefit from her colonies but the colonies should themselves gain from the system. William Pitt the Younger told Parliament in 1784 that there was a need "to render that connection a blessing to the native Indians". This theme grew in the nineteenth century and many argued that what Britain offered its colonies was a superior economic and political system. This idea was perhaps most famously expressed by the writer Rudyard Kipling who contended that western nations had a duty to bring their superior civilization to less-developed countries. He called this "the White Man"s Burden". Imperial masters had to improve the life of colonial inhabitants; they had "To seek another"s profit/And work another"s gain". They had to "Fill full the mouths of Famine/And bid the sickness cease". The debate on Hong Kong follows this tradition but generally avoids taking a patronising tone towards the people of the colony, who are widely recognised as sophisticated and educated. It also helped that a genuine demand for democracy existed there. Therefore, Britain was honour-bound to do its best to give them her traditions. Since the UK was not granting independence to Hong Kong but returning it to a nation known for its political repression, they also had a duty to ensure that the colony would have some protection in the future-a protection that they could not guarantee themselves-and, therefore, the need for international recognition of the importance of the question. Certainly there is a self-congratulatory air to much of the discourse. Pride in the imperial past had not totally died, at least among the Conservatives, as can be seen from Edward Leigh, scion of a gentry family and descendant of Henry VII:

This may be an historic occasion-the last occasion on which the House debates the future of a major British colony. This is the last of the debatesconducted over perhaps half a millennium-that have affected the fate of millions of people around the world. Before he [Malcolm Rifkind] finishes his speech and hauls down the flag of empire, will he pay tribute to the many men and women who created something that was special, not only in its extent, but as a great example in world history of good government and justice? 31 Rifkind, now Foreign Secretary, was happy to do so, talking about the "historic achievement" of the Empire, "the provision of the rule of law and democratic government, and a massive increase in prosperity for all territories". 32 John Redwood, the Eurosceptic who challenged Major for the party leadership in 1995, went even further, suggesting that the government should levy a "success fee" on the colony so that China would not benefit so much from Britain"s good work. 33 Many Conservatives seem to have felt that the "White Man"s Burden" had been fulfilled and that democracy was virtually a British invention. Of course, the Labour Party showed markedly less enthusiasm for imperial praise. Robin Cook even suggested that the main reason for Patten"s popularity was his social policy: "The programme that he has pursued in Hong Kong could be written as an illustration in a textbook of social democracy." 34 Many people argued that Hong Kong must retain freedom and the rule of law in order to keep its economic prosperity and that, in fact, the Chinese would only hurt themselves by curtailing its liberty. Baroness Dunn, who was of Chinese origin herself and had been one of the most senior politicians in the colony, serving on both the Executive and the Legislative Councils, stated that: "One of Hong Kong"s main attractions to investors is its reputation as a free, fair and clean place to do business." The Liberal Democrat peer, Lord Thomson of Monifieth, former Commonwealth secretary, went even further:

The inter-dependence in Hong Kong of its various facets-the integrity of the Civil Service, the inviolability of the commercial law system, the academic freedom of the universities, its citizens" right to travel-all hang together. They form a seamless web essential to the continuation of Hong Kong"s economic success. As Hong Kong"s seven million people generate a GDP nearly a quarter of that generated by China"s one billion plus population, any damage to Hong Kong"s economy as a world trading city will do massive damage to the whole Chinese economy. That is by far the best argument to Chinese self-interest in living up to its commitment to "one country, two systems", under the Joint Declaration. 36 The warning-echoed by many others-was clear: China should tread softly in Hong Kong or risk destroying both the goose and its golden egg. The press also echoed this argument. The Times, for example, in an article entitled "Bone-Headed China" wrote that: "Businessmen will not wait until June 30, 1997 to decide whether they are confident that the rule of law will continue to be impartially upheld. Some are already moving the legal domicile of their companies elsewhere." 37 As The Economist put it: "China"s economic ambitions depend on a prospering Hong Kong." Chris Patten followed this line of reasoning. He later explained in an interview with the Institute of International Studies at Berkeley that: I thought British interests were pretty clear. Britain had to be seen to withdraw honourably from its last colonial responsibility, even though what it was obliged to do, by history, would appear to a lot of people to be dishonourable… the handing over of a free society to a society which was not free. I always reckoned that honor and short-term interest and longerterm interest, in every sense, went hand-in-hand. If we weren"t to behave honourably, for example, it would help to produce political instability, which would be extremely bad for the economy in Hong Kong. Britain had a large stake in that economy; a lot of firms on the Hong Kong Stock market, a lot of British firms, and big investments of three billion also. Huge commercial stake in Hong Kong"s continuing success. So I never saw what some [of] my business critics argued-a distinction between trying to do what was right and what was, in every sense, in Britain"s interest-its commercial interest and its political interest as well. 39 So Britain"s economic interests and her sense of honour led her in the same direction.

The term "one country, two systems" had obvious implications for Taiwan and many believed that China needed to show it could respect freedom if it wanted to have any chance of getting back this even greater prize. The independence of Taiwan, although not internationally recognized, was, and is, protected by American military strength. If China wished to avoid World War III, it would have to convince the Taiwanese to join them voluntarily. As Patten noted in his memoirs: "Taiwan will watch closely what happens further down the coast: can "one country, two systems" work in the former colony, and if it cannot do so there, the Taiwanese will ask, how could it possibly work for them?" 40 So, once again, it was in China"s own long-term, and particularly economic, interest to keep freedom and the rule of law in Hong Kong.

Very few dissented from Patten"s policy and he enjoyed the support of all the major parties. Many Labour M.P.s took a keen interest in the fate of Chinese dissidents and frequently asked questions about them. People like Jeremy Bray of the Labour Party, who had been born in Hong Kong, confessed that:

A question faced the Opposition when the Chris Patten whom we knew so well appeared as a somewhat belated knight in shining armour in Hong Kong, seeking ingeniously to squeeze the last ounce of democracy out of the arrangements that he found had already been made there, even at the risk of upsetting Chinese friends in Beijing. I was in Shanghai at the time, and I must confess my reaction was to cheer. 41 He expressed quite clearly here his support for government policy and admiration for Patten. In April 1995, Robin Cook, Shadow Foreign Secretary, stressed the cross party nature of the policy:

We do not approach the debate in a party political spirit. I hope that I do not disappoint the House when I say that I do not intend to make this a partisan occasion. It is a subject that we should try to pursue with the least party political disagreement between us, and with the fullest unity on a national basis. 42 He went on to underline the human dimension of the question and the anxiety felt in Hong Kong over the handover. Tiananmen, he argued perceptively, had pushed China and Hong Kong in different directions:

The events of Tiananmen square had a profound effect on the views of people resident in Hong Kong, and appear to have had a profound effect on the rulers of China as well. One of the issues with which we must now grapple is that those events appear to have left the rulers of China perhaps more hostile to democratic reform and more nervous about the impact on the rest of China of the political processes in Hong Kong…. There is a paradox in the fact that the events of five years ago have made progress towards democratic rights in Hong Kong more pressing, but have also made it more difficult to obtain the agreement of the Government of China.

In November 1996, Cook told Parliament that, if a Labour government came to power in May 1997, there would be no change on policy towards Hong Kong. 43 As we shall see, Cook would follow a more moderate line towards China after becoming Foreign Secretary but, while the colony remained British, he and the Labour Party resolutely supported the Conservative government"s policies, as did the Liberal Democrats.

This, of course, did not mean that there was no opposition in Britain to Patten"s policies, only that this disagreement did not follow party lines. Many businessmen who had investments in the colony voiced their hostility. Obviously, most of those who had constructed the earlier policy, and in particular the Joint Declaration resented the change and predicted dire consequences. The strongest critic of the government"s policy was undoubtedly Sir Percy Cradock who had been one of the major architects of the handover. He wrote a number of articles in the press that scathingly attacked Patten"s policy. In his memoirs he went as far as to suggest it had an irrational and even racist base:

The episode [the Patten governorship] might be seen as an irruption of irrational forces, a reminder of the still unexhausted reserves of prejudice and emotion on both sides arising from a long, painful and too often uncomprehending relationship. It is also possible to see British policy as an example of nostalgia in action, an attempted reversion to times when Britain was in a position to impose solutions. Others may see the failure to read Chinese intentions as only another example of that chronic inability to put ourselves in the shoes of the other side which has manifested itself in our European as much as our Far Eastern dilemma. 44 There is an element of truth here for, as we have seen, many of the comments on Hong Kong resembled earlier imperial discourse. But there is also something spiteful about many of Cradock"s comments. Others insisted that Patten"s policies were aimed more at pleasing the U.S. and the British press than at ensuring the future of Hong Kong. 45 Within Parliament, the main dissident was the former Conservative Prime Minister, Edward Heath who took an extremely pragmatic view of the question, notably stressing the importance of cultivating and increasing British economic interests with China: I take the example of China, which has a rapid rate of expansion of between 8.5 per cent and 12.5 per cent. We are not establishing ourselves in those markets as we should be. When the Governor of Hong Kong said that when Hong Kong returns to China on 1 July next year, we shall still have responsibility for looking after Hong Kong, he could not have chosen a better way of upsetting Beijing and affecting the Chinese Government"s future attitude towards Hong Kong and towards us. That undoubtedly affects out trading arrangements with Hong Kong. As I know from my discussions with officials in Beijing and elsewhere in China, such comments affect where they place their orders. 46 His main argument was simple: China was potentially a massive market so do not upset the Chinese government or there will be little or no economic profit for Britain. He also insisted on the limits of British power, which were very real. As he put it: "They [the Chinese] know the power they have, and what they can bring about." 47 Heath also stressed that Britain had been in Hong Kong for a long time and asked the difficult question, why now?

We have had Hong Kong for nearly 150 years, and what did we do about all those issues? We did nothing. Only when the time came to hand it back did we say that they all should be addressed immediately and in exactly the way we wanted. The House and the Government cannot get away with that. We must be realistic. 48 And so realism and the economic importance of China were the cornerstones of those who opposed the government"s Hong Kong policy. But, as we have already seen, those who supported the policy felt that this was an exaggeration or, indeed, wrong and that Britain"s, as well as China"s, economic interest lay in supporting Patten"s proposals.

Let us now turn to the other major subject of debate-immigration and nationality. Much of this discourse had been settled before this period. The British Nationality Act of 1981 created a British Dependent Territories citizenship for residents of those regions which took away the right of abode in the United Kingdom. Its principal target was obviously Hong Kong. Thatcher justified this by insisting that there was a real danger "that financial confidence would evaporate and that money and in due course key personnel would flee the Colony, impoverishing and destablizing it well before the lease of the New Territories came to an end. 49 There was some truth in her assertion. About one per cent of the population emigrated in 1990 and this figure actually increased in 1992. U.S. News and World Report even ran a cover story asking, "Will the last one to leave please turn out the lights?" 50 This mass emigration was certainly something that the Chinese government did not want either.

Further complicating the situation was the plight of the Vietnamese boat people. After the Vietnam War ended in 1975 with the reunification of the country under a communist government, hundreds of thousands of people decided to leave. Over the next two decades nearly 200,000 took refuge in Hong Kong and most of them spent years in refugee camps there. 51 Dealing with these refugees placed a considerable strain on the colony"s resources. In 1988, the Hong Kong government announced a plan to screen the arrivals to see if they were genuine refugees or simply looking for economic improvement. Those deemed to fall in the latter category would be repatriated. There was much criticism of this plan in the British press as well as in the American press and government. Douglas Hurd justified his decision in his memoirs:

The Americans, whose hostile policy towards Vietnam was one reason for the country"s poverty and the outflow of boat people, began to object on humanitarian grounds to what we intended. Despite this, I decided in December 1989 that we must begin to fly even unwilling Vietnamese home from Hong Kong. There were by then 57,000 boat people in Hong Kong. This seemed the only way of deterring larger numbers from risking the voyage. We also needed to put paid to the stories current in Vietnam that once in Hong Kong the boat people would be generously treated and perhaps offered a golden life across the Pacific in California. 52 Britain took about 15,000 of these immigrants but there was hostility among many people to taking more.

Policy towards immigration from Hong Kong changed slightly after the Tiananmen massacre and Thatcher herself insisted on legislation in 1990 to give 50,000 key civil servants and business people and their families British passports. The expressed hope was that this would reassure them and that they would therefore stay in Hong Kong. Although Patten, in October 1995, publicly called for the government to grant full British nationality to all Hong Kong citizens, a general consensus prevailed among the parties against such a policy. Immigration discourse, then, tended to focus on certain small groups who were perceived as being especially deserving or as facing particular difficulties. One of these was the wives and widows of British servicemen who had fought to defend Hong Kong during the Second World War. According to Major there were only about 29 of these and it struck many as particularly ungenerous of the Home Office to block such a small number from receiving full citizenship. The Labour peer, Lord Dubs called it "a disgrace to us as a country". 53 The Prime Minister finally made the announcement granting this during his visit to Hong Kong in 1996. Many, though, criticized the fact that-instead of making this a government bill-it was only a Private Member"s bill. On the 8 th of May 1996, V-E Day, the Conservative Tim Renton, with bipartisan support, introduced the bill to grant these women full British citizenship which quickly passed both houses of Parliament and received the Royal Assent. The question was resolved but it left a bitter taste for some. Sir Peter Lloyd, a former Conservative minister of state at the Home Office, summed up the feelings of many: "Although the substance was conceded, with an undertaking that they could come to the UK whenever they wanted, the desire for citizenship was insensitively and pointlessly denied for years." 54 Another group on which there was a near consensus was that of the non-Chinese ethnic minorities who numbered around 7000 people. Primarily of Asian descent, they had often been settled in Hong Kong for several generations and most of them worked for the British military forces. As such, they had earlier signed a document renouncing any earlier nationality they might have possessed. They had British National (Overseas) citizenship which meant that, once Hong Kong returned to China, they would have no right of abode anywhere in the world. Nor could they receive Chinese nationality like the other inhabitants of Hong Kong since Chinese citizenship was based on ethnicity.

Both Patten and the Legislative Council of Hong Kong called on the government to grant them British nationality. To illustrate the widespread support for such a measure, the ultra-conservative Sir Teddy Taylor, member of the anti-immigration Monday Club, raised the question in the House of Commons in July 1993 and called on the government to grant citizenship to these people. He stated that: My real interest stems from my belief that Britain is in danger of flouting its inescapable duty to a group of citizens whose rights are being ignored and whose future will be unstable and constitutionally deprived unless we do something. It would be terrible if we told these 5000 people that they belong nowhere-that they are nationals of nowhere-especially as they have served Britain so well in the past. 55 In 1993 Lord Bonham-Carter, publisher and first chairman of the Race Relations Board, introduced a bill into the House of Lords to grant this nationality but this was defeated in the House of Commons because of government opposition. But numerous people continued to lobby the government. This resulted in John Major stating on 4 March 1996 that: "Her Majesty"s Government will guarantee admission and settlement should they come under pressure to leave Hong Kong". 56 This did not, however, solve the problem and lobbying continued. In December 1996, with the return of Hong Kong a little over six months away, the Eurosceptic 21 st Baron Willoughby de Broke, Conservative peer, introduced another bill. In his speech he criticized the government guarantee:

That statement implicitly recognises that that group is a special case, but I fear that the guarantee is seen in Hong Kong as having little value. For what is that pressure? Who is to decide it? Will that be decided in Hong Kong or in the remote comfort of a Whitehall office? This looks like an administrative and moral swamp, employing objective criteria that the Government refuse to define, leaving 5000 individuals uncertain of their eligibility. But, what is most important, that policy will result in the need for each person to put his or her case individually, exposing himself or herself to further discrimination if their specific circumstances do not fall into the, as yet, undetermined definition of "under pressure". In addition, that policy completely misses the point. That community does not want to rush into the UK. All it is asking for is a full British passport so that its status in Hong Kong will not be an anomaly that exposes it to discrimination and a subjective interpretation of their status. Ministerial assurances, however well-intentioned, are in this case just not good enough. 57 For Willoughby de Broke, it came down, once again, to a question of honour: "Therefore, if only for political reasons, let us resolve this issue which could blight our record and reputation in Hong Kong and tarnish our colonial legacy." 58 Another Conservative, David Howell, told the House of Commons that the government"s failure to grant citizenship to this group left "an overall feeling-an aroma-of meanness". 59 Sir Patrick Cormack, also a Conservative, used the term "moral obligation" to describe the situation and told the House of Commons that: "Moral obligations are real. What is morally wrong is never politically right. I think that we made a mistake in not granting passports." 60 The distinguished barrister, Sir Ivan Lawrence, yet another Conservative, could not understand "Why the United Kingdom Government are deliberately making it appear that they do not want to be seen to be magnanimous." 61 The question of honour was developed in more detail by the Conservative businesswoman, Baroness O"Cathain who spoke of Britain"s "responsibility":

We are talking about non-ethnic Chinese living in Hong Kong whose ancestors, let us recall, were taken to Hong Kong by the British founding fathers of the colony in the 1840s. About 2,000 Indian troops were there when the British flag was first raised in Hong Kong. They did not choose to go there; they were taken there by us. Since that time many of them have suffered as a result of their "British" nationality. They are regarded by the ethnic Chinese as British. During the Second World War some were taken to Japanese prisoner of war camps because they were perceived to be British. This House has great knowledge of the deprivation and the sheer horrors that occurred in those prisoner of war camps. We should remember that. They suffered alongside British prisoners. Their loyalty to the Crown was never in question. More of them served in the British Armed Forces. Again, their loyalty was never in question. And more of them gave sterling service to this country in the voluntary support services… Surely we in turn should show loyalty to them. 62 Clearly, many right-wing Conservatives were still using an imperial discourse and the issue for them was one of noblesse oblige. Their vocabulary reflects this belief, for they talk of "duty" and "loyalty". The British government has a duty to recognise and reward the past loyalty of the ethnic minorities. Major later said that there were many people in his own party who supported the government position but there is no sign of this in the records of parliamentary debate. 63 The other parties supported Willoughby de Broke"s bill and, indeed, there is not an enormous difference in their arguments. Paddy Ashdown of the Liberal Democrats, for example, called the government"s treatment of the non-ethnic minorities "disgraceful". 64 The Labour peer and celebrity lawyer, Lord Mishcon asserted that: "We shall lower the Union Jack on 30 th June; please may it be done with honour." 65 In the previous month, Robin Cook, Shadow Foreign Secretary, announced his own and his party"s support for granting them full citizenship. The arguments he used differed little from those put forward by the Conservative peers, although his language was less emotional:

The Labour party has written to the Prime Minister urging him to extend British citizenship now to that small group of people. Such a step would honour our obligation to them, because of their past contribution to our colony, and remove any uncertainty about their rights in future. I assured the Government that Labour would co-operate in facilitating the passage of such legislation. I repeat that offer.

He went on to say that "if a general election produces a Labour Government, we will be prepared to legislate to extend citizenship to the small Asian community of Hong Kong." 66 Cook recognized that this would be difficult in the short time between 1 May and the handover unless the Conservatives cooperated with him.

Amazingly enough, the only participant in the House of Lords debate to speak against granting nationality was the government spokesperson and her only argument was to say that the government did not think such a bill necessary. The only real opposition, in fact, appears to have come from Michael Howard, the Home Secretary. Baroness Dunn attacked him directly:

On 15 th July 1993, by a vote of 60 to 48, this House called upon the Government to give them full British citizenship. Those who supported the Motion spoke from all sides of the House and included two previous Governors of Hong Kong and a former Minister responsible for Hong Kong.

None of this has moved the Home Secretary. He has argued against granting full British citizenship on three main grounds: first, that this is not a special case; secondly, that giving them full citizenship would require legislation which is would be difficult to get through Parliament; and, thirdly, that existing arrangements give them sufficient protection.

None of these objections stands up to scrutiny. 67 Lord Dubs, went even further:

My understanding is that the Bill is widely supported in this House and in the other place; it is supported by the present governor of Hong Kong and two previous governors; it is supported by the Chinese Government, LegCo and the bulk of public opinion itself. Who is against it? I know that the Government of this country speak with one voice. But if one looks behind that voice, there is reason to believe that there are many voices in government who would urge that this Bill be supported. I know the Minister cannot comment on that, but my understanding for some time has been that the only thing that stands in the way of the passage of this Bill is the Home Office… I suppose there is one other argument-let us be frank-an election is coming. Is the Home Secretary nervous lest he be accused of being soft on immigration? 68 It became clear that the bill was likely to pass in the House of Commons in spite of government opposition. Howard finally decided to give in and on 4 February 1997 announced that non-ethnic minorities would be given full British citizenship. The Home Office then almost completely rewrote Willoughby de Broke"s bill, introducing requirements like the need for residence in Hong Kong before the date of his announcement and making it obligatory to register to become British subjects instead of having an automatic process. Willoughby de Broke accepted the changes graciously and with good humour:

One of the favourite expressions of the late Deng Xiaoping was, "It matters not what colour your cat is as long as it catches mice." So although the amendment before your lordships will substantially alter the original Bill, I am satisfied that even though the cat is a different sort of cat-a blow-dried, shampooed-and-set cat-it will still do its job and catch mice. 69 At midnight on 30 June 1997, amidst the pouring rain, Hong Kong became a special administrative region of China. The event received massive coverage in the world press. After the handover, British policy became much more conciliatory towards China. In May 1995 Robin Cook made his first visit to Beijing. According to John Kampfner, he decided then that Labour"s approach to China would differ from Chris Patten"s. 70 Indeed, as Foreign Secretary, Cook would establish a policy of constructive engagement with China. As such, Britain did not back the annual resolution at the United Nations Commission on Human Rights which criticized China. During a 1999 visit to the U.K. by the Chinese president, the Labour government was criticised for using strong arm tactics to hide protesters. Cook also announced in January 1998 that he was "too busy" to meet a leading Chinese dissident, now resident in the United States, Wei Jingsheng. When Wei returned on a second visit, Cook felt obligated to meet him but, due to an apparent mix-up, the press arrived too late and no photos were taken. 71 Essentially Cook followed the E.U. line on China-to such an extent that it was often difficult to distinguish a separate British policy. 72 Let us try now to evaluate the success or not of British policy. Major later wrote that Patten"s actions eventually had the result of making the Chinese more amenable-notably on finally reaching an agreement on the new airport. 73 Major also insisted that the dispute had little or no effect on trade with China for exports to there doubled during Patten"s term of office. Still, China refused to accept the Patten reforms of 1992. As they had promised, they dissolved the elected Legislative Council and replaced it with a hand-picked Provisional Legislative Council. The Shanghai-born millionaire businessman, Tung Chee-hwa became Hong Kong"s Chief executive. He also had been chosen by Beijing and possessed almost no political experience. In 2001 the popular Deputy Chief Executive Anson Chan, who had earlier assisted Patten, resigned under pressure from Beijing. The following year the Hong Kong administration tried to introduce an anti-subversion law, called Article 23. After widespread protests, the bill was withdrawn and, eventually, in 2005, Tung himself resigned. At the time of writing, the press remains free and the judiciary largely independent. Although democracy has not been achieved, civil liberties continue and the people of Hong Kong seem determined to defend them, frequently protesting in the streets. So perhaps Patten"s gamble paid off. Britain did leave its last colony with a sense of honour and the colony has retained most of its old freedom (although it might have done so anyway). Let us, therefore, give the last word to Patten:

Was the consequence fatal for Hong Kong?... The condition of Hong Kong in 1997 answered that question better than I could ever have done. We had stood up for Hong Kong, belatedly honouring the promises made to it about freedom, democracy and the rule of law. Where were the fatalities? Had the roof fallen in on us?... Here was Hong Kong in the summer of 1997, richer than we could ever have believed possible, with a good government guaranteed its passage through the transition, with an independent judiciary enjoying the same guarantee, with a rich fabric of civil society, and with no disorder on the streets. The demonstrations, when they occurred, were politely directed against China, not Britain. 74 For more on this see Kampfner. For more on this see Angela Bourne and Michelle Cini "Exporting the Third Way in Foreign Policy: New Lab, the EU and Human Rights Policy" in New Labour's Foreign Policy: A New Moral Crusade, ed. Richard Little and Mark Wickham-Jones (Manchester: Manchester University Press, 2000)

Major, 506 Patten, 82

I would like to thank Barbara Cartwright and my husband, Henri Zuber, for his help with this article.


Niamh H Foley

Isabella Bray

Karen M Watters

Sudipto Das

Kenneth Bryan

Tytus Bernas

Jochen H M Prehn

Raymond L Stallings
email: rstallings@rcsi.ie

MicroRNAs 10a and 10b Are Potent Inducers of Neuroblastoma Cell Differentiation Through Targeting of Nuclear Receptor Co-repressor 2

Keywords: ATRA, MYCN, neuroblastoma, miR-10a, miR-10b, NCOR2, differentiation

MicroRNAs function as negative regulators of post-transcriptional gene expression, playing major roles in cellular differentiation. Several neuroblastoma cell lines can be induced to undergo differentiation by all-trans-retinoic acid (ATRA) and are used for modelling signalling pathways involved in this process. To identify miRNAs contributing to differentiation, we profiled 364 loci following ATRA treatment of neuroblastoma cell lines and found miR-10a and miR-10b to be highly over-expressed in SK-N-BE, LAN5, and SHSY-5Y. Ectopic over-expression of these miRNAs led to a major reprogramming of the transcriptome and a differentiated phenotype that was similar to that induced by ATRA in each of these cell lines. One of the predicted downregulated miR-10a/b targets was nuclear receptor co-repressor 2 (NCOR2), a co-repressor of gene transcription which is known to suppress neurite outgrowth. NCOR2 was experimentally validated as a direct target of miR-10a/b, and siRNA mediated inhibition of this mRNA alone resulted in neural cell differentiation. Moreover, induction of differentiation could be blocked by ectopic up-regulation of NCOR2 using an expression construct lacking the miR-10a/b 3' UTR target site. We conclude that miR-10a/b play major roles in the process of neural cell differentiation through direct targeting of NCOR2, which in turn induces a cascade of primary and secondary transcriptional alterations, including the down-regulation of MYCN.

INTRODUCTION

MicroRNAs are small non-coding RNAs (18-25 nucleotides in length) that act as complex negative regulators of post-transcriptional gene expression by targeting regions of sequence similarity in mRNA 3'UTRs, leading to either degradation or translational inhibition of the mRNA [START_REF] Baek | The impact of microRNAs on protein output[END_REF][START_REF] Bartel | MicroRNAs: genomics, biogenesis, mechanism, and function[END_REF][START_REF] Lim | Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs[END_REF]. MiRNAs are expressed in a temporal and spatial specific manner in the development of the brain [START_REF] Miska | Microarray analysis of microRNA expression in the developing mammalian brain[END_REF][START_REF] Sempere | Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation[END_REF], thus contributing to the process of determining neuronal cell identity, as reviewed by Li et al 2010 [START_REF] Li | Roles of small regulatory RNAs in determining neuronal identity[END_REF].

Neuroblastomas are paediatric tumors originating from precursor cells of the sympathetic nervous system which have highly diverse clinical outcomes, ranging from spontaneous regression, to rapid progression. Cell lines derived from these tumors, such as SK-N-BE, SHSY-5Y and LAN-5, can be induced to undergo neural cell differentiation by treatment with all-transretinoic acid (ATRA) and are often used as a model system to study biochemical pathways involved in the process of differentiation [START_REF] Edsjo | Neuroblastoma as an experimental model for neuronal differentiation and hypoxia-induced tumor cell dedifferentiation[END_REF]. ATRA treatment of these cell lines leads to the outgrowth of neurites, the up-regulation of markers of neural differentiation such as TUBB3 and GAP43, and a decrease in the rate of cell proliferation. These morphological and functional changes are reflective of the large numbers of protein coding genes [START_REF] Nagai | Retinoic acid induces neuroblastoma cell death by inhibiting proteasomal degradation of retinoic acid receptor alpha[END_REF] and miRNAs (9) that have been altered in cells induced to undergo differentiation by ATRA. Most notably, ATRA leads to immediate transcriptional down-regulation of the MYCN transcription factor before the onset of cellular morphological differentiation [START_REF] Thiele | Decreased expression of N-myc precedes retinoic acid-induced morphological differentiation of human neuroblastoma[END_REF], which further contributes to the remodeling of the transcriptome. MYCN is amplified or expressed at high levels in the cell lines studied, and it is notable that MYCN amplification is one of the most potent genetic predictors of poor clinical outcome for neuroblastoma patients [START_REF] Seeger | Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas[END_REF][START_REF] Brodeur | Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage[END_REF].

Recently, it has been demonstrated that some of the miRNAs which undergo expressional changes following ATRA treatment appear to functionally contribute to the differentiation phenotype of neuroblastoma. Laneve et al [START_REF] Laneve | The interplay between microRNAs and the neurotrophin receptor tropomyosin-related kinase C controls proliferation of human neuroblastoma cells[END_REF] demonstrated that ATRA induced up-regulated miRNAs 9, 125a and 125b decrease cell proliferation rates through the common targeting of a truncated isoform of the neurotrophin receptor tropomyosin-related kinase C mRNA. It was further shown that miR-125b ectopic over-expression induces neurite outgrowth and neuronal gene expression through the inhibition of multiple mRNA targets [START_REF] Le | MicroRNA-125b promotes neuronal differentiation in human cells by repressing multiple targets[END_REF]. Another miRNA which is up-regulated in ATRA treated neuroblastoma cell lines, miR-128, was shown to reduce neuroblastoma cell motility and invasiveness through inhibiting the Reelin and DCX genes [START_REF] Evangelisti | MiR-128 up-regulation inhibits Reelin and DCX expression and reduces neuroblastoma cell motility and invasiveness[END_REF].

The miR-17-5p poly-cistronic cluster has been shown to be down-regulated in response to ATRA [START_REF] Beveridge | Down-regulation of miR-17 family expression in response to retinoic acid induced neuronal differentiation[END_REF], and knock-down of two family members, miR-18a and -19a, results in neuroblastoma cell differentiation through the targeting of estrogen receptor-α [START_REF] Loven | MYCN-regulated microRNAs repress estrogen receptor-alpha (ESR1) expression and neuronal differentiation in human neuroblastoma[END_REF]. Chen et al [START_REF] Chen | miR-7 and miR-214 are specifically expressed during neuroblastoma differentiation, cortical development and embryonic stem cells differentiation, and control neurite outgrowth in vitro[END_REF] showed that ATRA induced miR-214 over-expression in neuroblastoma SHSY-5Y cells can modulate neuronal differentiation, and also showed that ATRA repression of miR-7 also induces neurite outgrowth. In addition, it was recently demonstrated that some miRNAs up-regulated in response to ATRA target DNA methytransferases, resulting in demethylation and up-regulation of specific genes which are known to be involved with ATRA induced differentiation [START_REF] Das | MicroRNA Mediates DNA Demethylation Events Triggered by Retinoic Acid during Neuroblastoma Cell Differentiation[END_REF]. From all of these studies it is apparent that the contribution of miRNAs towards an ATRA induced differentiated phenotype is an essential, highly complex, but poorly understood process.

Up-regulation of miR-10a in response to ATRA was reported by Beveridge et al [START_REF] Beveridge | Down-regulation of miR-17 family expression in response to retinoic acid induced neuronal differentiation[END_REF] for SHSY-5Y cells, although this group did not carry out functional studies to assess the role of miR-10 family members in ATRA induced differentiation. Here, we have carried out a detailed functional analysis of the biological effects of miR-10a and 10b, which we determine to be also up-regulated in SK-N-BE and LAN-5 cells in response to ATRA. Interestingly, both miR-10a and 10b are imbedded in HOX gene clusters on chromosomes 17q and 2q, respectively. HOX genes are transcription factors that are expressed in embryogenesis in distinct regions during head-tail body axis of animal embryos. This cluster of genes is highly important in development, as precise regulation is necessary for efficient differentiation to occur [START_REF] Shah | The Hox genes and their roles in oncogenesis[END_REF]. In addition to being co-localized within HOX gene clusters, these miRNAs are also known to directly inhibit some of the HOX genes, with miR-10a targeting HOXA1 [START_REF] Garzon | MicroRNA fingerprints during human megakaryocytopoiesis[END_REF], and miR-10b targeting HOXD10 [START_REF] Ma | Therapeutic silencing of miR-10b inhibits metastasis in a mouse mammary tumor model[END_REF].

Tsang et al., [START_REF] Tsang | MicroRNA-mediated feedback and feedforward loops are recurrent network motifs in mammals[END_REF] have previously shown neuronal enriched miRNAs tend to be co-expressed with their target genes, suggesting a role for these miRNAs in neuronal homeostasis. Here, we investigate the importance of these miRNAs in ATRA induced neuroblastoma cell differentiation, including the identification of a direct target gene, nuclear receptor co-repressor 2 (NCOR2; also known as SMRT), whose silencing recapitulates an ATRA induced differentiated phenotype.

RESULTS

Differential expression of miRNAs after retinoic acid induced differentiation of SK-N-

BE cells. SK-N-BE, SHSY-5Y and LAN-5 neuroblastoma cells treated with all-trans retinoic acid (ATRA) exhibited extensive outgrowth of neurites (Figure 1). Further analysis of SK-N-BE cells revealed that ATRA induced differentiation resulted in the over-expression of markers of neuronal differentiation (GAP43 and TUBB3), a decrease in MYCN mRNA and protein, and a significant decrease in cell growth, all well known features of ATRA induced differentiation (Supplementary Figure 1). In order to gain further insight into miRNAs that might be involved in the process of neural cell differentiation, we profiled 364 mature miRNAs in ATRA treated and untreated SK-N-BE cells, using TaqMan low density arrays (Applied Biosystems). ATRA induced differentiation had a major impact on the expression levels of numerous miRNAs, with 30 loci having statistically significant expressional increases (>1.5 fold; P < 0.05), and 26 loci exhibiting expressional decreases (>1.5 fold; P < 0.05) in two biological replicates (Supplementary Figure 2a).

MiRNAs with the highest expressional changes (>10 fold at day 7) included miR-132, miR-10a, and miR-10b. Although miR-132 was also highly-up-regulated in response to ATRA, Chen et al [START_REF] Chen | miR-7 and miR-214 are specifically expressed during neuroblastoma differentiation, cortical development and embryonic stem cells differentiation, and control neurite outgrowth in vitro[END_REF] reported that ectopic over-expression of miR-132 in neuroblastoma cells had no consistent effect on neurite outgrowth, so we focused our studies on miR-10a and 10b for which functional studies in neuroblastoma cells have never been reported. Both of these miRNAs were also significantly up-regulated in SHSY-5Y and LAN5 cells in response to ATRA (Supplementary Figure 2b andc), indicating that this is a general feature of neuroblastoma cell lines undergoing ATRA induced differentiation. The high differential expression of miR-10a/b in these cell lines could not be attributable to absence of expression in untreated cells given that the Ct values for biological replicates were in the range of 30 to 31 (Ct values in the range of 35 to 40 would be indicative of no expression).

Ectopic over-expression of miR-10a and -10b in neuroblastoma SK-N-BE cells

recapitulates ATRA induced differentiation. Mature miR-10a or 10b mimics were transfected into SK-N-BE, LAN-5 and SHSY-5Y cells, both individually and in combination, resulting in ectopic over-expression. Over expression of both miRNAs resulted in pronounced increased neurite outgrowth by day 5 post-transfection (P values < 0.001 or better)(Figure 1). Functional studies using SK-N-BE cells transfected with miR10a, 10b or 10a/b displayed significant reduction in the rate of cell growth (ascertained by cell counts and acid phosphatase (Figure 2a; Supplementary Figure 3) and colony forming ability in soft agar (Figure 2b). Notably, expression of two markers of differentiation, GAP43 and TUBB3, increased following miR-10a or miR-10b transfections, consistent with what transpires following ATRA treatment (Figure 2c,d). In addition, there was a highly significant reduction in MYCN mRNA and protein levels (Figure 2e,f) following ectopic over-expression of the miRNAs, similar to what occurred following ATRA treatment. This is most likely an indirect effect given that the MYCN 3' UTR lacks target sites for either of these miRNAs. Anti-sense knock-down of miR-10a/b 24 hours prior to ATRA treatment led to a highly significant, but incomplete, reduction in the outgrowth of neurites (Supplementary Figure 4). It is not surprising that miR-10a/b knockdown did not completely reverse the effects of ATRA given that ectopic over-expression of other ATRA induced miRNAs, such as 125b, have been shown to result in neurite outgrowth [START_REF] Le | MicroRNA-125b promotes neuronal differentiation in human cells by repressing multiple targets[END_REF] and Supplementary Figure 2a.

We conclude, nevertheless, that ectopic over-expression of miR-10a and 10b, both individually and in combination, recapitulates many features of the ATRA induced differentiation phenotype of SK-N-BE cells.

mRNA expression profiling and gene ontology analysis following miR-10 transfection of SK-N-BE cells.

In order to experimentally identify direct targets down-regulated by miR-10a, mRNA expression profiling was performed using microarrays representing 24,000 protein coding genes. SK-N-BE cells at either 1 or 7 days post-transfection with miR-10a were analyzed. There was a statistically significant enrichment for miR-10a predicted target sites in the set of mRNA sequences which decreased by 2 fold or greater following miR-10a transfection at 24 hours (P = 0.03) and 7 days (P = 0.00001) (Table 1). Interestingly, there were more mRNAs down-regulated with predicted targets at day 7 than at day 1 post-transfection, indicating that specific mRNAs might be targeted at differential rates. However, the majority (91.7%) of mRNAs possessing miR-10a binding sites that were down-regulated after 24 hours remained down-regulated at day 7. There was also large numbers of mRNAs down-regulated at both 24 hours (n = 845) and 7 days post-transfection (n = 1,629) which lacked miR-10a target sites, indicating that ectopic upregulation of this miRNA results in substantial secondary effects. For up-regulated mRNAs, there were significantly greater numbers at day 7 than at day 1 post-transfection, which also would be due to secondary events. At the 7 day time point, the up-regulated mRNAs were significantly inversely-correlated with miR-10 binding sites, a correlation which was not statistically significant at day 1.

Gene ontology (GO) analysis on the most significantly up-regulated mRNAs (top 20%) following transfection of SK-N-BE cells with miR-10a indicated that the top enriched term was cell surface signal transduction following correction for multiple comparisons (Supplementary Table 1). For the most significantly down-regulated genes (top 20%; both direct and indirect effects), the most significant enrichment term was regulation of transcription (Supplementary Table 1), which explains why ectopic over-expression of miR-10a launches a cascade of secondary alterations to the transcriptome. The decrease in MYCN following miR-10a/b transfection or ATRA treatment would also impact mRNA and miRNAs levels. In this regard, some MYCN responsive miRNAs, including miR-184, 152 and 190 are up-regulated in response to ATRA and would result in further tertiary effects.

One of the down-regulated miR-10a/b targets, NCOR2, is a co-repressor of transcription, whose down-regulation could account for the up-regulation of many genes detected by the microarray analyses. From the standpoint of neurite outgrowth, NCOR2 (SMRT), is of immediate interest given that neural stem cells derived from NCOR2 homozygous knockout mice exhibit extensive neurite outgrowth and reduction in cell proliferation following 5 days in culture [START_REF] Jepsen | SMRT-mediated repression of an H3K27 demethylase in progression from neural stem cell to neuron[END_REF]. For these reasons, NCOR2 was selected for further functional studies.

The miR-10a/10b predicted target site in the 3' UTR of NCOR2 is broadly conserved and contains an 8 nucleotide match in the seed region of miR-10a/b. The down-regulation of NCOR2 mRNA following miR-10 over-expression in SK-N-BE was validated by qPCR and at protein level by immunoblotting (Figure 3a,b). Moreover, a luciferase reporter construct containing a 484 bp region of the NCOR2 3' UTR (designated Psi/miR-10) showed a significant decrease in luciferase activity when co-transfected with mature miR-10a, 10b or 10a/10b mimics in SK-N-BE cells (Figure 3c). The decrease in luciferase activity was completely abrogated by a 3 bp mutation in the seed region (designated Psi/miR-10mut), leading us to conclude that NCOR2 is a direct target of both miR-10a and 10b.

Effects of NCOR2 on neurite outgrowth and cell growth. As already discussed, miR-10a and -10b have many direct and indirect effects on the mRNA transcriptome. In order to assess how down-regulation of NCOR2 alone might contribute to neuroblastoma cell differentiation, we performed siRNA mediated knock-down of NCOR2 in SK-N-BE cells. Transfection of two independent NCOR2 siRNAs into SK-N-BE cells resulted in approximately 60% reduction in NCOR2 mRNA levels and a significant reduction in protein (Figure 4a). This level of NCOR2 knockdown resulted in a highly significant increase in neurite outgrowth (P < 0.0001)(Figure 1) and a reduction in cell growth (Figure 4b). Similar to ATRA treated cells, siRNA mediated NCOR2 knockdown resulted in reduced MYCN mRNA and protein (Figure 4c) and increased amounts of the neuronal markers GAP43 and TUBB3 (Figure 4d). Most interestingly, inhibition of NCOR2 resulted in a 14 fold increase in miR-10a (P < 0.0001) (Figure 4d), providing further evidence for the involvement of NCOR2 in the suppression of this miRNA (see proposed mechanism of action model Figure 5).

In order to further validate that NCOR2 is the only target of miR-10a that is required for yielding a differentiated phenotype, we co-transfected an NCOR2 expression construct lacking the 3' UTR region with miR-10a mature mimics. Transfection of the NCOR2 construct into SK-N-BE cells resulted in increased NCOR2 mRNA and protein, reflecting both endogenous and ectopic molecules (Figure 6a). Co-transfection of NCOR2 plasmid and miR-10a led to an approximate 2 fold increase in NCOR2 protein over the negative control at 72 hours, reflecting only ectopically derived protein (Figure 6a). Co-transfection completely blocked the effects of miR-10a, including over-expression of GAP43, TUBB3B and down-regulation of MYCN (Figure 6b). Furthermore, at day 5 following co-transfection there was no detectable increase in neurite outgrowth (Figure 6c) or decrease in the rate of accumulation of cell numbers (Figure 6d,e).

MiR-10a and 10b expression in primary neuroblastoma tumors and association with clinical features.

Given that patient survival is related to the degree of differentiation of neuroblastoma tumors, we examined the expression patterns of miR10a/b in a cohort of 145 primary neuroblastoma tumors. As illustrated in Supplementary Figure 5a, miR-10a was significantly under-expressed (1.4 to 1.9 fold; P <0.0001) in two genetic subtypes that are associated with poor clinical outcome, those with 11q deletion (n = 30; stage 4) or MYCN amplification (n =45; stage 4), relative to tumors lacking these abnormalities. MiR-10b displayed an inverse pattern of expression in the tumor subgroups from miR-10a (Supplementary Figure 5b). Interestingly, miR-10a maps within a chromosomal region on chromosome 17q that is gained in ~80 to 90% of tumors from patients with stage 4 disease, but is nevertheless expressed at significantly (P < 0.0001) lower levels in tumors with 17q gain in spite of increased dosage (Supplementary Figure 5c). This result indicates that although there might be a gene or set of genes on chromosome 17q that are selectively up-regulated by 17q gain, miR-10a expression is down-regulated by some unknown mechanism.

In the overall cohort of 145 patients, lower miR-10a expression in tumors was not significantly associated with worse overall (OS) or event free survival (EFS). However, within the subset of 11q-tumors, lower than median miR-10a expression was associated with poor OS (p = 0.045) and displayed a trend towards poor EFS (p=0.057) (Supplementary Figure 5d,e). The 11q-tumors represent a distinct genetic subtype of neuroblastoma which can be subdivided into clinically favourable and unfavourable groups on the basis of mRNA/miRNA expression patterns [START_REF] Buckley | Chromosomal and miRNA Expression Patterns Reveal Biologically Distinct Subgroups of 11q-Neuroblastoma[END_REF][START_REF] Fischer | Integrated genomic profiling identifies two distinct molecular subtypes with divergent outcome in neuroblastoma with loss of chromosome 11q[END_REF]. No association between miR-10b expression levels and patient survival was discernable.

We conclude that although the in vitro effects of miR-10a and 10b are indistinguishable, they have remarkably different patterns of expression in tumor subtypes, with under-expression of miR-10a having a greater influence on patient survival for the 11q-tumor subtype.

DISCUSSION

Prior studies of miRNA expression profiling of ATRA treated neuroblastoma cell lines differ in the use of cell lines (SK-N-BE versus SHSY-5Y), analysis platforms (qPCR, microarray or Northern blot), and the numbers of miRNAs that were profiled (ranging from 32 to 364), thus some variation in the miRNAs determined to be differentially regulated in response to ATRA would not be surprising [START_REF] Chen | Differential patterns of microRNA expression in neuroblastoma are correlated with prognosis, differentiation, and apoptosis[END_REF][START_REF] Laneve | The interplay between microRNAs and the neurotrophin receptor tropomyosin-related kinase C controls proliferation of human neuroblastoma cells[END_REF][START_REF] Le | MicroRNA-125b promotes neuronal differentiation in human cells by repressing multiple targets[END_REF][START_REF] Beveridge | Down-regulation of miR-17 family expression in response to retinoic acid induced neuronal differentiation[END_REF][START_REF] Chen | miR-7 and miR-214 are specifically expressed during neuroblastoma differentiation, cortical development and embryonic stem cells differentiation, and control neurite outgrowth in vitro[END_REF]. Nevertheless, a comparison between these studies revealed that 20 out of 56 miRNAs determined to be up or down-regulated in the current study were also similarly differentially regulated in at least one other study, including miR-10a (Supplementary Table 2). This is not to imply that the miRNAs identified as ATRA responsive in a single study are not valid, only that they have yet to be confirmed. This study has determined that endogenous over-expression of miR10a/b in response to ATRA plays a major role in the process of neuroblastoma cell differentiation through the specific targeting and inhibition of NCOR2. MiR-10a and 10b differ by a single nucleotide which occurs outside of the "seed" region, thus, it is not surprising that the in vitro biological effects of these miRNAs are indistinguishable following ectopic up-regulation in SK-N-BE cells. Both miRNAs lead to phenotypic changes similar to ATRA induced differentiation, including a significant increase in neurite out-growth, up-regulation of the neuronal differentiation markers GAP43 and TUBB3, the down-regulation of MYCN and a reduction of cell growth. In addition, miR-10a and 10b over-expression launches a cascade of mRNA transcript changes through a complex series of primary and secondary mechanisms. In spite of the vast re-programming of the SK-N-BE cell transcriptome following miR-10a/10b transfections, it is remarkable that siRNA mediated inhibition of one single direct target, NCOR2, can recapitulate the differentiating phenotypic effects of these miRNAs.

NCOR2 has the ability to bind to hormone receptors, including retinoic acid receptors, recruit co-repressor proteins and silence primary response genes [START_REF] Chen | A transcriptional co-repressor that interacts with nuclear hormone receptors[END_REF][START_REF] Chen | SMRT isoforms mediate repression and anti-repression of nuclear receptor heterodimers[END_REF]. NCOR2 functions as a transcriptional co-repressor through recruitment of a complex of proteins to DNA promoter regions, including SIN3A/B, and histone deacetylases HDAC1, HDAC2 and HDAC3 [START_REF] Nagy | Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase[END_REF].

Therapeutic levels of ATRA causes a disruption and displacement of the co-repressor complex with a co-activator complex at retinoic acid response elements and subsequent transcriptional activation of adjacent genes. It has been established that miR-10a, which is located on chromosome 17q within the HOXB3 gene, is a retinoic acid receptor target, as knock-down of a retinoic acid receptor in a pancreatic cancer cell line resulted in complete inhibition of miR-10a expression [START_REF] Weiss | Retinoic acid receptor antagonists inhibit miR-10a expression and block metastatic behavior of pancreatic cancer[END_REF]. Thus, miR-10a up-regulation in response to ATRA is a direct consequence of ATRA causing displacement of the co-repressor complex with a co-activator complex, leading to chromatin acetylation and concurrent miR-10a transcription (Figure 5). miR-10a can then posttranscriptionally down-regulate NCOR2, maintaining the disruption of the co-repressor complex in the absence of ATRA. siRNA or miR-10a/b mediated inhibition of NCOR2 therefore results in the same differentiated phenotype that is caused by ATRA treatment, along with a vast reprogramming of the cellular transcriptome. The involvement of NCOR2 in a co-repressor complex that down-regulates miR-10a expression indicates that a regulatory feed-back loop is in place.

MiR-125b has also recently been implicated in promoting neuronal differentiation of neuroblastoma SH-SY5Y cells through directly repressing multiple key target genes [START_REF] Le | MicroRNA-125b promotes neuronal differentiation in human cells by repressing multiple targets[END_REF]. There appears to be some level of redundancy in function of miR-125b with miR-10a/b as three of these key target genes (ITCH, GAB2, and MKNK2) have miR-10 seed sites in their 3' UTRs and are down-regulated following ectopic up-regulation of miR-10a/b in our studies. Although not noted in the study by Le et al (14), it is highly significant that miR-125b also has a 7-mer conserved seed match in the 3' UTR of NCOR2 (TargetScan), further explaining why both miRNAs cause neural cell differentiation.

Analysis of the expression of miR-10a and 10b in primary neuroblastoma tumors, along with patient survival, indicates that these miRNAs differ in both their patterns of expression in genetic subtypes and in their associations with clinical outcome. MiR-10b expression is not correlated with patient survival, whereas low-expression of miR-10a shows a trend towards lower OS and EFS for patients with 11q-tumors. Although miR-10a maps to a region on chromosome 17q that commonly undergoes increased copy-number in most high-risk neuroblastomas, miR-10a expression is significantly lower in tumors with 17q gain relative to tumors with diploid 17q copy number status, indicating that a mechanism exists to counteract the gain in dosage. Thus, it appears to be vital for unfavorable tumors, particularly of the 11q-subtype, to have low miR-10a expression levels, presumably because of the positive effects on differentiation. In contrast, miR-10b is expressed at higher levels in less favorable neuroblastoma tumors and is not associated with lower patient survival even though this miRNA causes in vitro biological effects that are indistinguishable from miR-10a. It is tempting to speculate that the single nucleotide difference between miR-10a and 10b which occurs outside of the seed region has led to different secondary effects of these miRNAs which might account for the differences in their distribution in tumor subtypes.

It is also of interest that the role of miR-10a/b in cancer appears to be tumor and cell context dependent, as miR-10a has been reported to be under-expressed in haematological malignancies such as CML [START_REF] Agirre | Down-regulation of hsa-miR-10a in chronic myeloid leukemia CD34+ cells increases USF2-mediated cell growth[END_REF], while both miRNAs have been reported to be over-expressed in a variety of solid tumors [START_REF] Lund | miR-10 in development and cancer[END_REF]. Over-expression of miR-10b has been associated with breast cancer metastasis [START_REF] Ma | Tumour invasion and metastasis initiated by microRNA-10b in breast cancer[END_REF], although this view has been challenged [START_REF] Gee | MicroRNA-10b and breast cancer metastasis[END_REF]. In this regard it is interesting that miR-10b is marginally over-expressed in tumors with either MNA or 11q LOH from patients with metastatic disease, relative to loco-regional tumors lacking these unfavorable genetic alterations.

In conclusion, we have identified two miRNAs which induce differentiation and inhibit cell proliferation in neuroblastoma cell lines which are capable of undergoing ATRA induced differentiation, and validate NCOR2 as a direct target of miR-10a and miR-10b. Interestingly, NCOR2 down regulation leads to the indirect suppression of MYCN, a potent oncoprotein in neuroblastoma. MiR-10a/b could be of potential therapeutic benefit if targeted delivery of these miRNAs is achievable, given their pro-differentiation and anti-proliferative effects.

MATERIAL AND METHODS

ATRA treatment. SK-N-BE cells were treated with 5μmol of all-trans retinoic acid. ATRA (Sigma, St. Louis, MO) was continuously administered by replacing the medium every 24 hrs for 7 days. Cell morphology was monitored using the neuronal specific antibody βIII Tubulin (Abcam, Cambridge, MA) followed by staining with an anti-rabbit Alexa 488 conjugated antibody (Invitrogen, Carlsbad, CA). microRNA and siRNA Transfections. Reverse transfections of microRNAs and siRNAs were carried out at final concentrations of 30nM for miRNAs and 10nM for siRNAs in optiMEM (Gibco TM ). Separate NeoFX and optiMEM solutions were prepared and then mixed together at equal volumes. 200μl or 20μl of this solution was then added to to a well from either a 6 or 96 well plate respectively, which was then over-layed with neuroblastoma cell suspension and incubated at 37º C. A flourescein-labeled double stranded oligonucleotide (Invitrogen) was transfected into cell lines under the conditions stated above in order to determine the transfection efficiencies. Approximately 78% of cells exhibited luciferase activity 48 hours post-transfection.

RNA Extractions.

Total RNA with conservation of small RNAs was extracted using the miRNeasy ® kit (Qiagen) as per manufacturers instructions. RNA quality and quantity was measured using the Nanodrop Spectrophotometer (Thermo Scientific) and RNA dilutions were made up in nuclease free water.

Stem-loop Reverse transcription and Real-time PCR. Reverse transcription of 50 ng of

RNA from ATRA treated SK-N-BE cells at Day 0, Day 3 and Day 7 was carried out using the stem-loop Multiplex primer pools (Applied Biosystems), allowing reverse transcription of 48 different miRNA in each of 8 RT pools. Reverse transcription for individual qPCRs was carried out using 50ng of total RNA and the High Capacity Reverse Transcription Kit (Applied Biosystems), RT specific primers for microRNAs miR-10a and miR-10b (Applied Biosystems)

were used for all microRNA reverse transcription. The microRNA qPCR screen was carried out on the 7900HT Fast Realtime System using TaqMan Low Density Arrays (TLDA) (TaqMan® TLDA MicroRNA Assays AIF v5 containing 364 human microRNAs assays; Applied Biosystems).

Individual qPCRs were carried out on the 7900HT Fast Realtime System (Applied Biosystems) using GAP43, βIII Tubulin, MYCN and NCOR2 specific Taqman gene expression assays (Applied Biosystems) and miR-10a and miR-10b specific Taqman microRNA assays (Applied Biosystems). RPLPO and RNU66 were used for normalization in gene and microRNA expression studies respectively. A relative fold change in expression of the target gene transcript was determined using the comparative cycle threshold method (2 -ΔΔCT ).

Western Blot. Total protein was isolated from cells using a radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma). Cell pellets were washed with PBS and solubilized in RIPA for 30mins. Protein concentration was measured using the BCA assay from Pierce. Proteins were fractionized on 6% or 10% polyacrylamide gels, and blotted onto nitrocellulose membrane. The membrane was probed with the Anti-NCOR2 Antibody (Abcam), Anti-GAP43 (Abcam), Anti-MYCN (Santa Cruz), Anti-βIII Tubulin and anti-GAPDH (Abcam), anti-α-Tubulin, and anti-Actin (used for loading controls). Signal was detected using Immoblion Western (Millipore).

Gene Expression Analysis.

The SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, 11917-020) was used to synthesize cDNA from 10µg total RNA according to manufacturers instructions. Cy3 labelling of cDNA was carried out using the NimbleGen One-Colour DNA Labelling Kit (NimbleGen). Four micrograms of labelled cDNA was hybridised to the Homo Sapiens 4x72K gene expression array (Roche-NimbleGen) for 16 hours. Following washing, arrays were scanned using the GenePix 4000B scanner, and expression values were obtained using the Robust Multi-Chip Average algorithm [START_REF] Irizarry | Exploration, normalization, and summaries of high density oligonucleotide array probe level data[END_REF] in NimbleScan 2.4.

Growth Curve. For cell numbers, cells were set up in 6 well plates. Cells were seeded at equal densities. When carrying out transfections using the microRNA mimics, each time point was set up with a non-transfected (with transfection reagent) and a scrambled oligonucleotide control (negative 1). Cells were trypsinised from 6 well plates at 24, 48, 72 and 96 hour time points. Cells were re-suspended in 1 ml of media. The Scepter cell counter (Millipore) was used to count cell numbers. Counts from triplicate wells were averaged.

Acid Phosphatase Assay. Cells were transfected in 96-well plates (10 3 cells per well). At 96hrs wells were washed twice with PBS and 10mM p-nitrophenol phosphate in 0.1M sodium acetate with 0.1% triton X-100 was added. Plates were incubated at 37°C for two hours and the reaction was stopped with 50µL 1M sodium hydroxide per well. Absorbance was measured at 405nm. Values were normalized to the scrambled oligonucleotide negative control.

Colony Forming Efficiency. A 1% agarose solution was plated on 10cm 2 petri dish until the plate was completely covered. Cells were plated at 1.5 X 10 6 in 10mls of culture media, and media was replaced every 4-5 days, for 21 days. Colonies were stained with 0.1% crystal violet.

Plates were photographed; colonies were counted in replicate studies and counts normalized to the negative scrambled control.

Immunostaining. Cells were fixed in 4% paraformaldhyde 5 days after transfection. Cells were permeabilised using Trition X-100 (Sigma), then blocked for 30mins in BSA. Samples were incubated in the primary β-III Tubulin rabbit antibody (Abcam) overnight at 4ºC, followed by incubated in the secondary Alexa Fluor 488 Anti-Rabbit fluorescent antibody (Invitrogen) for one hour at room temperature. To stain nuclei the cells were incubated for 5min with DAPI (Sigma) and then washed in PBS.

Neuronal Outgrowth Assay. Cells were imaged using Cellomics ArrayScan VTi instrument (Thermofisher) equipped with 10x PlanApo objective lens (NA 0.45), 120W Hg arc illumination source (EXFO) and a monochrome CCD camera (Hamamatsu Orca AG). Images of 49 fields of view were registered from each well with 1024x1024 pixel resolution (645 nm pixel size). The fluorescence of DAPI (nuclei) was excited using 375 -400 nm light and collected in 425 -460 band, whereas the fluorescence of Alexa 488 (whole cells) was excited using 470 -490 nm light and collected in 500 -550 band. The images of nuclear fluorescence were preprocessed using rank leveling (25 pixel window) for background subtraction. The nuclei, cell bodies and neurites were segmented using fixed intensity thresholds. The nuclei were used as seeds for morphological separation of adjacent cell bodies. The neurites were then delineated with skeletonization of their binary masks. The total neurite length (per cell) was used to estimate neurite outgrowth. The length was normalized to the respective value for the non-treated cells.

All image processing operations were executed using Neuronal Profiling BioApplication of the Discovery Toolbox Software (Thermofisher).

Luciferase Reporters A 484 nt long region of the 3'UTR of NCOR2 containing the predicted miR-10a/10b binding site was inserted into the dual luciferase PsiCheck2 reporter vector (Promega), designated Psi/miR-10 (Supplementary Figure 6a). As a negative control, a 3nt mutation was introduced into the miR-10a/10b seed region of this sequence, designated Psi/miR-10mut (Supplementary Figure 6b). SK-N-BE cells were plated in 6 well format, and co-transfected using Lipofectamine 2000 with either 2µg of Psi/miR-10 or Psi/miR-10mut, along with either 30 nm of either the Pre-miR for 10a, 10b, both Pre-miRs together or a scrambled oligonucleotide negative control. Luciferase activity was measured after 72 hours using a Viktor Plate Reader.

  miR-10a/NCOR2 co-transfection. A plasmid containing the coding sequence without the 3'UTR of NCOR2 was obtained from OriGene (Maryland) and co-transfected with miR-10a or a scrambled control oligonucleotide using Lipofectamine 2000 according to manufacturers instructions. Cells were harvested for RNA and protein at 72 hrs post-transfection. Cell viability was carried out using the cell counting technique and acid phosphatase assays at 24, 48, 72 and 96 hrs post transfection. Patient Cohort Tumor material was obtained from either the Children's Oncology Group Tumour Bank (n = 108), or the Tumour Bank at Our Lady's Children's Hospital, Dublin (n = 37). Patients were treated under either the U.S. neuroblastoma treatment protocol or the European treatment protocol between 1998 to 2004. This work was approved by the Research Ethics Committee of the Royal College of Surgeons on 16th October 2007 (application No. REC241) and by the Research Ethics Committee of Our Lady's Children's Hospital on 5th August 2008 (application number GEN/70/07). The tumour cohort came from patients with stage 4 (n = 74), stage 3 (n = 31) and stages 1, 2 and 4s (n = 40) disease (INSS). Tumor RNA was extracted using the miRNeasy kit (Qiagen) according to manufacturers instructions. Array CGH. DNA copy number variation was carried by array CGH on 145 neuroblastoma tumors as previously described by Buckley et al. (25) using a 72,000 feature array (NimbleGen)and protocols originally described by Selzer et al[START_REF] Selzer | Analysis of chromosome breakpoints in neuroblastoma at sub-kilobase resolution using fine-tiling oligonucleotide array CGH[END_REF].

Figure 1

 1 Figure 1 Outgrowth of neurites in neuroblastoma cells. The upper panel shows morphology of

M a i n t e n a n c e o f t r a n s c r i p t i

expected number of binding sites generated by simulation studies based on 1,000,000 iterations of similar numbers of randomly selected mRNAs and miRNAs Table 1 Effect of 10a Transfection on gene expression

				7
	Total down-regulated mRNAs (>2 fold)	977	1915	811 (83.0%)
	Observed 10a Targets	132	286	121 (91.7%)
	Expected 10a Targets*	113.7	223.0	94.3
	Significance of over-representation (P)	0.03	0.00001	0.002
	Total up-regulated mRNAs (>2 fold)	1101	1663	900 (81.7%)
	Observed 10a Targets	116	148	89 (76.7%)
	Expected 10a Targets*	128.2	193.5	104.7
	Significance of under-representation (P)	0.14	0.0001	0.0534
	*

ACKNOWLEDGEMENTS

This work was supported in part by Science Foundation Ireland (07/IN.1/B1776), Children's Medical and Research Foundation, Cancer Research Ireland and the U.S. National Institutes of Health (5R01CA127496). T.B. was supported by a Marie Curie Cofund EU Fellowship (CEMP; jointly funded by the National Biophotonics and Imaging Platform, Higher Education Authority, Ireland).

MicroRNA Target Prediction Targetscan V5.1 microRNA target prediction programme was used throughout this study.

CONFLICT OF INTEREST

The authors have no conflict of interest to declare. measurements are relative to the negative control (NC), a scrambled oligonucleotide (set as 1.0).

All p-values are relative to NC and represent the minimum value for the three sets of cell lines.

Figure 2 Biological effects of miR-10a and miR-10b ectopic over-expression in SK-N-BE.

(A) Growth curve based on cell counts at different time points following transfection of SK-N-BE cells with a negative control (NC) oligonucleotide, or miR-10a, miR-10b or combined miR-10a/b mature mimics. Similar results were obtained by cell counting (Supplementary Figure 3). upper display shows binding of a co-repressor complex consisting of a retinoic acid receptor (RAR), NCOR2, and histone deacetyltransferases (HDAC) to a retinoic acid response element (RARE) upstream to miR-10a. In the middle display, RA causes dissociation and replacement of the co-repressor complex with a co-activator complex that activates miR-10a transcription. MiR-10a transcription is maintained through targeting the NCOR2 3' UTR (lower display).