MicroRNA-379调控FAK在肝癌侵袭转移中的作用及机制研究

We have demonstrated that focal adhesion kinase (FAK) plays an important role in invasion and metastasis of hepatocellular carcinoma (HCC). However, the roles of microRNA (miR) targeting FAK remain unclear in HCC. In preliminary studies, we found that miR-379 expression was significantly downregulated in HCC tissues by microRNA microarray. Furthermore, FAK is predicted to be the direct target of miR-379 by bioinformatics methods. Moreover, our studies showed that forced expression of miR-379 significantly downregulated FAK protein expression and suppressed cell migration and invasion in HCC cells. Thus, we propose that miR-379 suppresses invasion and metastasis of HCC by targeting FAK. This study aims to confirm that miR-379 suppresses invasion and metastasis of HCC by targeting FAK and to elucidate its underlying molecular mechanisms through gene transfection, quantitative reverse transcription polymerase chain reaction, western-blot, luciferase reporter gene assay, animal model, in situ hybridization, immunohistochemistry, etc. Taken together, this study will help elucidate the mechanisms underlying invasion and metastasis of HCC, provide a novel biomarker for predicting metastasis and recurrence of HCC, and provide enough scientific evidence to establish miR-379 as a novel target for antimetastatic therapy of HCC.

我们已证实FAK(局部粘着斑激酶)在肝癌侵袭转移中发挥重要作用。但是，肝癌中靶向调控FAK的microRNA（miR）及作用尚不清楚。前期，我们通过microRNA芯片发现miR-379 在肝癌组织中表达明显下调，且生物信息学方法预测FAK是miR-379的直接靶基因；进一步研究显示在肝癌细胞中过表达miR-379可显著下调FAK蛋白表达，同时细胞的迁移侵袭能力明显下降。由此，我们提出miR-379通过靶向调控FAK抑制肝癌侵袭转移。本项目拟采用基因转染、定量PCR、Western-blot、报告基因、动物模型、原位杂交、免疫组化等方法，旨在获得miR-379通过调控FAK抑制肝癌侵袭转移的可靠证据并阐明其分子机制。本项目不仅有助于阐明肝癌的侵袭转移机制，为预测肝癌转移复发提供新的生物标志物，且为确立miR-379作为抗肝癌转移治疗的新靶点提供充分的科学依据。

肝癌是人类最常见的恶性肿瘤之一，预后差。一些微小RNA（miRNA）已证实在肝癌的发生发展中起重要作用。但尚有较多miRNA在肝癌中的作用和机制不明。在本课题执行期间我们研究了miRNA （miR）-379在肝癌侵袭转移中的作用及其相关分子机制。我们发现miR-379-5p在人肝癌组织和细胞株中均明显下调，且与肝癌TNM分期及转移密切相关；外源性过表达miR-379-5p能显著抑制肝癌细胞的迁移、侵袭和上皮间质转化（EMT），而抑制miR-379-5p表达则明显促进肝癌细胞的迁移侵、袭和EMT。通过软件预测和分子生物学实验确证FAK (局部粘着斑激酶)是miR-379-5p的直接靶基因。过表达miR-379-5p能抑制FAK的表达进而抑制其下游的AKT信号通路，而抑制miR-379-5p表达则增加FAK表达进而促进AKT信号通路的转导；过表达FAK 或激活AKT信号通路可逆转miR-379-5p介导的对肝癌的抑制作用，而阻断FAK 表达或抑制AKT信号通路则可逆转miR-379-5p抑制剂的作用。裸鼠体内实验进一步证实过表达miR-379-5p能显著抑制肝癌的转移和EMT及FAK的表达。进一步研究发现在人肝癌组织中miR-379-5p表达与FAK表达存在负相关性。这些结果表明miR-379-5p在肝癌发生发展中发挥重要作用，其通过抑制FAK/AKT信号通路来抑制肝癌的侵袭和转移，提示miR-379-5p可作为肝癌治疗的潜在的新靶点和可作为预测肝癌转移复发的生物学标志物。因此，本项目不仅提供了肝癌侵袭转移的新机制，且为确立miR-379作为抗肝癌转移治疗的新靶点提供充分的科学依据。