Abstract

Hepatocellular carcinoma (HCC) is a common malignancy worldwide with poor prognosis and high mortality. The aberrant expression or alteration of microRNAs (miRNAs) contributes to the development and progression of cancer. Studies have shown that miR-455 plays a regulatory role in the development of HCC. Therefore, in the present study, the role of miR-455 was analyzed in HepG2 cells proliferation and apoptosis using MTT and flow cytometry methods. Binding sites were predicted by bioinformatics and luciferase assay was used to verify the target relationship between miR-455 and RhoC-encoding gene RHOC. After that, the effects of miR-455 on RHOC and its product RhoC, were explored by qPCR and Western blotting. As PTEN is a key tumor suppressor gene in HCC, and Bcl-2 and Caspase 3 are important indication of apoptosis, expression levels of PTEN, Bcl2 and Caspase 3 proteins were determined in cells overexpressing RhoC. We show that miR-455 promotes HepG2 cells apoptosis and inhibits proliferation. Bioinformatics analysis and luciferase assay indicate that specific recognition sites for miR-455 are within the RhoC 3'-UTR. Luciferase activity was significantly lower in the cells co-transfected with miR-455 mimics and RhoC-WT (p < 0.01) as compared to that in control cells, pointing that RHOC gene is, indeed, targeted by miR-455. RHOC mRNA was significantly reduced after miR-455 transfection in HepG2 cells. In addition, we show that RhoC could activate the HCC cells proliferation ability and inhibit apoptosis rate (p < 0.01), and decrease expression of PTEN and Caspase 3 (p < 0.01), while upregulating levels of Bcl2. In conclusion, our study indicates that miR-455 plays a suppressive role in HCC development by targeting RhoC-encoding mRNA.

中文翻译：

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miR-455通过靶向RhoC抑制HepG2细胞增殖并促进细胞凋亡

摘要

肝细胞癌（HCC）是世界范围内常见的恶性肿瘤，预后差，死亡率高。microRNA（miRNA）的异常表达或改变有助于癌症的发生和发展。研究表明，miR-455在肝癌的发展中起调节作用。因此，在本研究中，使用MTT和流式细胞术分析了miR-455在HepG2细胞增殖和凋亡中的作用。通过生物信息学预测结合位点，并使用荧光素酶测定法验证miR-455和编码RhoC的基因RHOC之间的靶标关系。之后，通过qPCR和Western blotting探索了miR-455对RHOC及其产物RhoC的影响。作为PTENBcl-2和Caspase 3是肝癌中的关键抑癌基因，Bcl-2和Caspase 3是细胞凋亡的重要指标，在过表达RhoC的细胞中测定PTEN，Bcl2和Caspase 3蛋白的表达水平。我们表明，miR-455促进HepG2细胞凋亡并抑制增殖。生物信息学分析和荧光素酶测定表明，miR-455的特异性识别位点位于RhoC 3'-UTR内。与对照细胞相比，在与miR-455模拟物和RhoC-WT共转染的细胞中，萤光素酶活性明显较低（p <0.01），这表明RHOC基因确实是miR-455的靶标。红十字会miR-455转染HepG2细胞后，mRNA显着降低。此外，我们显示RhoC可以激活HCC细胞的增殖能力并抑制细胞凋亡率（p <0.01），并降低PTEN和Caspase 3的表达（p <0.01），同时上调Bcl2的水平。总之，我们的研究表明，miR-455通过靶向RhoC编码的mRNA在HCC的发展中起抑制作用。