乙型肝炎性肝癌性别差异的假基因活化与长链非编码RNA调控网络及功能研究

The major risk factors for HCC are chronic liver diseases with cirrhosis that include hepatitis B. Epidemiological reports indicate that regardless of etiologies, the incidence of HCC is higher in males than in females with the male: female ratio usually averaging between 3-5:1. In view of this remarkable gender disparity, various in vitro as well as in vivo studies have been initiated from time to time to explore the importance of sex hormones in HCC. Though the studies began as early as in 1980s, the precise role of sex hormones and the significance of their receptors in HCC still remain poorly understood and perhaps contribute to current controversies about the potential use of hormonal therapy in HCC. We need a new perspective to explore this issue. The human transcriptome comprises not only large numbers of protein-coding messenger RNAs (mRNAs) but also a large set of non-protein coding transcripts that have structural, regulatory or unknown functions. A surprisingly wide array of cellular functions has also been associated with long non-coding RNAs (lncRNAs, tentatively defined as non-coding RNAs more than 200 nt in length) and pseudogenes. A handful of studies have implicated lncRNAs and pseudogenes in a variety of disease states, and altered lncRNA and pseudogene levels can result in aberrant expression of gene products that may contribute to cancer biology. However, the overall pathophysiological contributions of lncRNAs and pseudogenes to HCC remain largely unknown. .In this study, we plan to identify non-overlapping signatures of a small number of lncRNAs and pseudogenes that are up-regulated or down-regulated in HBV-related HCC compared with paired peritumoral tissues. Previous studies indicate that lncRNAs are key regulators of chromatin state, yet the nature and sites of RNA-chromatin interaction are mostly unknown. Here we introduce chromatin isolation by RNA purification, where tiling oligonucleotides retrieve with key lncRNAs bound protein and DNA sequences, which are enumerated by mass spectrometry identifying and deep sequencing. The studies presented here were also designed to test whether monitoring the relative expression of key lncRNA and pseudogenes and related pathways in clinic HCC samples will help to predict the prognosis of HCC. Together, our objective here is to highlight the important roles of lncRNA and pseudogene in the development and progress of HCC and test whether it is possible to apply lncRNAs and pseudogenes in HCC therapy and prediction of HCC prognosis.

慢性乙型肝炎相关肝细胞癌（HCC）的发生与预后存在显著性别差异，男女发病约3—5：1。流行病调查和基础实验均证实，雌性激素对肝癌具有保护作用，雄性激素促进男性的肝癌发生。但针对性激素各个环节的干预手段对其防治却没有任何效果。因此我们需要从一个新的角度对这一问题进行重新审视。近年来越来越多的研究表明，曾经一度被人们忽视的长链非编码RNA(long noncoding RNA，lncRNA)、假基因等分子在生理以及各种疾病状态下都有重要的调控功能。本项目计划首先利用新一代测序技术筛选肝癌组织中特征性表达的lncRNA、假基因，对关键性lncRNA、假基因的结构、相互结合的蛋白、DNA分子、参与调控的信号转导通路以及假基因活化、lncRNA作为肝癌早期诊断、疾病进展预测标记分子的可能性等方面进行深入的探讨。为该疾病的早期诊断、预后判断提供一组新的生物标记物，并为其治疗提供新的潜在药物作用靶点。

一直以来，肝癌发生发展的性别差异是肝癌研究的重要课题。本课题研究中，我们针对男女差异最大的遗传物质性染色体间区别进行研究，对性染色体编码的非编码RNA进行了深入探索。.X染色体沉默逃逸使得男女之间基因表达失衡，逃逸基因可能在女性体内发挥重要作用。我们对女性逃逸基因筛选发现了lnc-FTX在肝脏组织中的逃逸，女性肝脏组织中表达明显高于男性。lnc-FTX抑制肝癌的增殖和转移，肝癌组织中lnc-FTX高表达预示肝癌预后更佳。lnc-FTX结合并抑制MCM2蛋白功能，阻碍DNA复制复合物的组装和DNA复制，阻止肝细胞异常快速的增殖，因此女性肝脏组织高水平的lnc-FTX赋予女性对肝癌更强的抵抗力。肝癌进展过程中，lnc-FTX以序列互补方式结合吸附miR-374a，减少游离miR-374a，从而解除miR-374a对下游靶基因Wnt负向调控因子的抑制，降低Wnt通路活性和EMT，抑制肝癌转移。因此，女性高水平lnc-FTX既能抑制肝癌发生，又能减缓肝癌进展速度，是女性肝癌发生、发展过程中发挥“抑癌基因”的角色。.Y染色体转录本在男性肝癌中的筛选中发现，lnc-RBMY2FP在约1/3的男性肝癌组织中特异性活化表达，且阳性病人预后更差。因此，lnc-RBMY2FP作为男性肝癌发生、发展过程中发挥“癌基因”的作用。机制上，lnc-RBMY2FP通过结合DNMT1影响DNA甲基化，激活致癌因子RBMY1A1促进男性肝癌进展。.此外，我研究发现可被转化生长因子-β (transforming growth factor-β, TGF-β)活化的lncRNA-ATB既可通过吸附miR-200家族，促进肝癌细胞发生EMT，进而促进肝癌细胞的侵袭；又可直接结合IL-11的mRNA，促进IL11的分泌，调控肿瘤微环境，促进肝癌细胞于远端转移灶存活、定植。发现癌胚剪切因子MBNL3通过诱导PXN反义转录本1（PXN antisense transcript 1, PXN-AS1）的可变剪接增强肝癌细胞活力，促进肝癌形成，并且抑制MBNL3可显著抑制体内肝癌的形成及进展，揭示剪切因子MBNL3及RNA异常剪接可作为肝癌潜在治疗靶点。.相关研究结果相继发表于Cancer cell、Nature cell biology、Journal of Hepaology、 Hepatology等杂志。