miR-207促进肝癌侵袭与转移的分子机制研究

MicroRNAs (miRNAs) as a new class of regulators of gene expression have triggered an explosion of research activities. Growing evidence indicates that their deregulation plays an important role in cancer onset and progression as well, where they act as oncogenes or oncosuppressors. Our previous work has demonstrated that miR-207 expression is increased in hepatocellular carcinoma (HCC) cell line and pre-mRNA processing factor 19 (Prp19) may be a potential target of miR-207; however their pathophysiologic role and their relevance to tumorigenesis are still largely unknown. In another study we investigated that Prp19 may be involved in the degradation of Twist in HCC cell lines. Based on these early works, we propose a hypothesis that aberrant expression of miR-207 can contribute to HCC growth and spread by modulating Prp19 expression and Twist-dependent degradation pathway involved in mediating phenotypic characteristics of cancer cells such as cell growth, metastasis and invasion. In this work, we will: 1) investigates the involvement of miR-207 in Prp19 and Twist-dependent pathway in Huh7 and L02 cell lines; 2) investigates the involvement of miR-207 in Prp19 and Twist pathway in HCC cell-implanted nude mice. This study will unravel previous unrecognized insight into the pathogenesis of HCC, and suggest miR-207 pathway as the potential therapeutic target in management of HCC. In addition, this work is helpful to elucidate the collaboration between ubiquitination and epithelial-mesenchymal transition.

近年来，miRNA作为基因表达的调节子，引起了广泛关注。大量研究表明，miRNA作为癌基因或抑癌基因，在肿瘤的发生、发展中发挥重要作用。我们前期的工作表明，肝癌细胞株中miR-207高表达，Prp19可能是其潜在的靶蛋白，然而其在肝癌发生发展中的病理学作用却尚不清楚。在另一项研究，我们发现在肝癌细胞株中，Prp19参与了Twist的降解。由此我们推测HCC中，miR-207通过调控靶蛋白Prp19,影响Twist等EMT主要标志物及其下游基因表达，出现细胞生长、转移和侵袭的显型，进而促进肝癌侵袭转移。本课题拟在体内和体外对miR-207影响Prp19和Twist通路进行研究。本项研究将有助于阐明HCC新的发病原因，并为HCC治疗提供新方向。此外，本工作还有助于阐明泛素化和上皮间质转化的联系。

目的：在肝癌组织和细胞系中检测miR-19a-3p的表达水平；探讨miR-19a-3p对肝癌细胞系生物学功能的影响以及潜在机制和对索拉菲尼敏感性中的作用以及潜在机制。.方法：实时荧光定量PCR检测miR-19a-3p的表达水平；在细胞水平通过CCK-8等实验研究miR-19a-3p对肝癌细胞的增殖、迁移、侵袭、凋亡的影响；3.预测数据库寻找miR-19a-3p的靶基因，并验证其靶点及蛋白表达；CCK-8检测肝癌细胞对索拉菲尼的反应。.结果：miR-19a-3p在肝癌临床标本和肝癌细胞系中高表达；PLC/PRF/5细胞系过表达miR-19a-3p可促进细胞增殖、迁移和侵袭，Hep3B细胞系抑制miR-19a-3p表达抑制增殖、迁移和侵袭；双荧光素酶报告实验显示：过表达miR-19a-3使得野生型荧光素酶报告基因活性明显下降，而对突变型荧光素酶报告基因活性无明显影响；抑制miR-19a-3p使野生型荧光素酶报告基因活性明显增强，而对突变型荧光素酶报告基因活性无明显影响。此外，在PLC/PRF/5细胞系中过表达miR-19a-3p明显下调PTEN的表达，而在Hep3B细胞系中抑制miR-19a-3p表达明显上调PTEN的表达。在PLC/PRF/5和Hep3B细胞系中通过恢复或抑制PTEN的表达可以分别模仿miR-19a-3p低表达或高表达的作用；在PLC/PRF/5细胞系中，过表达miR-19a-3p可上调PI3K/AKT通路中的p-AKT(Ser-473)的表达，在Hep3B细胞系中，抑制miR-19a-3p明显下调PI3K/AKT通路中的p-AKT(Ser473)的表达； PLC/PRF/5细胞系在转染miR-19a-3p后，对索拉菲尼的敏感性下降，而在转染miR-19a-3p抑制剂后，Hep3B细胞系对索拉菲尼的敏感性明显增加。.结论：1. miR-19a-3p在肝细胞癌组织和肝癌细胞系中高表达；2. miR-19a-3p促进肝癌细胞系的增殖、迁移和侵袭，而对其凋亡无明显影响；3. PTEN为miR-19a-3p的直接靶点；4. miR-19a-3p通过激活PI3K/AKT通路促进肝细胞癌的增殖和转移；5. miR-19a-3p可能通过作用于PTEN，调节肝癌细胞系对索拉菲尼的敏感性。