癌前病变特异的长链非编码RNA驱动肝癌发生发展的机制研究

Hypermethylation of promoter CpG islands are believed to play an important role in the pathogenesis of hepatocellular carcinoma (HCC). It was thought to be an essential hepatocarcinogenesis in the progress of the disease from chronic hepatitis, cirrhosis, pre-cancerous lesions, and eventually to the HCC. Recently the role of long noncoding RNA (lncRNA) in the development of tumor is getting more and more attention, however, the study of promoter hypermethylation regulated lncRNA and its role in hepatocarcinogenesis are still in the infancy. Using the Arraystar LncRNA Promoter Microarray and LncRNA Microarray to scan the dysplasia nodules, we found 5 candidate lncRNAs (MT1XP1、MT-ATP8、SCARF1、AK126915、LOC113230) with promoter hypermethylation, which have different expression level in dysplasia nodules and hepatitis B cirrhosis tissues. The results were also verified by another 50 cases. In the present study, we are going to examine the regulatory roles of 5 candidate lncRNAs in normal liver cells, HCC cell lines, orthotopic tumor mice models, diethylnitrosamine-induced liver cancer rat models, and specimens from resection or biopsy. It will help us to get a better understanding of specific lncRNA DNA methylation alterations at different stages of HCC, particularly in the earliest steps of hepatocarcinogenesis. It will also provide important molecular insights into the stepwise accumulation of epigenetic alterations during HCC development, and hence be critical for exploring potential new strategies for early diagnosis and therapy of HCC.

在从慢性肝炎、肝硬化、癌前期病变，最终发展到肝癌的转变过程中，表观遗传改变是一种非常重要的肝癌发病机制。近几年，长链非编码RNA（lncRNA）在肿瘤中的作用逐步受到重视，但lncRNA在驱动肝癌发生发展，及其本身表达调控方面的研究尚处于初级阶段。我们从肝癌的癌前期病变-异型增生结节（DN）入手，利用lncRNA启动子芯片联合lncRNA芯片检测，并经扩大病例数验证，发现5个lncRNA（AK091100、MT1XP1、MT-ATP8、SCARF1、AK126915）在DN中表达显著下调且其启动子区存在过甲基化。本研究拟结合前期结果、经体内外以及临床标本的后续验证及功能研究，确定上述5个lncRNA在肝癌发生发展中的地位，并进一步寻找其潜在的调控基因，同时探究其受启动子区过甲基调控的相关机制，为寻找肝癌发生的早期标志物和药物干预靶点提供新的思路。

癌基因与抑癌基因的功能异常能直接导致肿瘤的发生，表观遗传学改变在肿瘤发生发展过程中同样发挥了重要作用。早期研究证实抑癌基因启动子区域高甲基化能直接引起其表达水平的降低，从而导致肿瘤的发生。与之相同的是编码lncRNA启动子区域的甲基化异常也是肿瘤发生的重要原因，同时可能导致肿瘤的转移。然而，目前国内外对于启动子甲基化与lncRNA在肝癌中表达调控的作用以及肝癌发生发展的机制研究仍相对稀少。本研究联合采用 lncRNA 启动子芯片和表达谱芯片实现对由启动子甲基化调控的肝癌特异相关 lncRNA 的高通量筛选，发现了并验证了13个候选 lncRNA作为由启动子甲基化调控的潜在抑癌 lncRNA。同时，将 lncRNA 上游启动子区域甲基化调控与下游蛋白编码基因内在通路调控机制联系起来，首次发现在肝癌细胞中，lncRNA-SCARF1启动子甲基化是导致其表达沉默的重要原因，可能直接干预lncRNA-SCARF1的激活，进一步通过与靶基因CUL9相互作用，显著抑制肝癌细胞的生长活性，促进肝癌细胞凋亡，抑制肝癌体内成瘤。本研究从lncRNA上游启动子区域到下游编码基因分析肝癌发生发展的相关机制，从新的角度阐明肝癌发生发展的分子机制，对发现新的肝癌特异性分子标志物和肝癌治疗的新靶点有重要的理论和实践意义。