miR-20a调控DNA羟甲基化过程促进肝内胆管癌进展的作用机制研究

Postoperative recurrence has become a bottleneck to improve the prognosis of intrahepatic cholangiocarcinoma. Abnormal DNA methylation at the 5 position of cytosine (5-mC) is a well-known epigenetic feature of cancer. The TET family of proteins are recently discovered DNA demethylases. 5-mC can be converted to 5-hydroxymethylcytosine (5-hmC) by the ten-eleven translocation (TET) family of DNA hydroxylases, and‘‘loss of 5-hmC’’ has been identified as an epigenetic hallmark of malignant tumors including melanoma, with diagnostic and prognostic implications. In our previous study, we found that there was lower 5-hmC level in intrahepatic cholangiocarcinoma tissues, compared to para-tumor tissues, and the level of 5-hmC is closely related with the expression of TET family of genes. However, the mechanism has not yet been elucidated. Here, we found that the expression of miR-20a is significantly increased in intrahepatic cholangiocarcinoma tissues, and miR-20a can regulate the 5-hmC level by targeting TET family of genes and induce the EMT process in intrahepatic cholangiocarcinoma cells. Our further study will attempt to clarify the role of Tet family of genes in miR-20a biological function, and identify the distribution of 5-hmC densities in the region of chromosome by hydroxymethylcytosine DNA Immunoprecipitation Sequene(hMeDIP-Seq) and mRNA sequence technology, and elucidate the key pathways by bioinformatics analysis including KEGG pathway analysis for the associated genes, in order to provides new clues for prevention and treatment of intrahepatic cholangiocarcinoma.

术后高转移复发率已成为进一步提高胆管癌远期疗效的瓶颈。DNA甲基化失调引起基因表达异常是当前表观遗传学研究热点。TET家族基因是近期发现的DNA去甲基化酶。5-羟甲基胞嘧啶(5-hmC)是TET家族蛋白催化DNA去甲基化过程中的重要中间产物，已被鉴定为恶性肿瘤的重要表观遗产学标记。前期研究中，我们发现胆管癌中5-hmC水平显著降低，其降低与TET家族基因的失活密切相关，但其原因尚未被阐明。我们发现miR-20a在胆管癌中的表达明显增高，其可靶向于TET家族基因调控胆管癌细胞5-hmC水平，诱导细胞EMT发生。本研究将在前期研究基础上，进一步明确TET家族基因在miR-20a发挥生物学功能中的关键作用，利用DNA羟甲基化测序(hMeDIP-Seq)结合mRNA-seq技术鉴别其调控的特异性羟甲基化位点，利用生物信息学结合功能研究确定其关键作用蛋白及信号通路，为肝内胆管癌的防治提供新思路。

术后高转移复发率已成为进一步提高肝脏恶性肿瘤远期疗效的瓶颈。我们的研究发现，在肝内胆管癌中，5-hmC水平较癌旁组织显著降低，其降低与TET家族基因的失活密切相关。miR-20a在肝内胆管癌中的表达明显增高，其可靶向于TET家族基因调控胆管癌细胞5-hmC水平，诱导细胞EMT发生，进而促进肝内胆管癌进展。我们的研究为进一步理解肝内胆管癌的发生发展机制提供了新的视角。此外，结合当前肿瘤研究热点，我们亦对肝细胞肝癌血管形成的机制进行了较为系统的研究。我们发现，COX-2/PGE2轴可通过调控缺氧诱导因子2α (HIF-2α) 的表达及核转位，促进肝细胞肝癌的缺氧反应，促进肝细胞癌的进展并诱导其索拉非尼治疗耐受；在机制上，COX-2/PGE2轴可通过调控泛素-蛋白酶体降解途径中的关键蛋白p-VHL表达，抑制HIF-2α蛋白的降解；HIF-2α可对下游信号蛋白TGF-α/EGFR信号通路进行调控，MAPK通路在调控HIF-2α核转移过程中发挥着重要作用。跨膜蛋白TMPRSS4在肝细胞肝癌进展中发挥着重要作用，我们亦对其诱导肝细胞肝癌血管生产的机制进行了研究。我们发现TMPRSS4可通过EGFR/PI3K/AKT/mTOR/HIF-1α通路促进肝细胞肝癌HB-EGF及MMP-9的表达，促进sHB-EGF的产生，进而促进其血管生成。并且HB-EGF抑制剂CRM197可协同多激酶抑制剂索拉非尼抑制肝癌进展。这些研究为COX-2抑制剂及HB-EGF抑制剂应用于肝细胞肝癌的治疗提供了理论基础。