Xie S L, Wang M, Du X H, Zhao Z W, Lv G Y
Department of General Surgery, First Hospital of Jilin University, Changchun, Jilin Province, 130021 China.
Mol Biol (Mosk). 2020 Jan-Feb;54(1):69-77. doi: 10.31857/S0026898420010152.
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.
肝细胞癌（HCC）是一种全球范围内常见的恶性肿瘤，预后较差且死亡率高。微小RNA（miRNA）的异常表达或改变促进癌症的发生和发展。研究表明，miR-455在HCC发生过程中发挥调控作用。因此，在本研究中，采用MTT法和流式细胞术分析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，并使用荧光素酶报告基因检测验证miR-455与RhoC编码基因RHOC之间的靶向关系。之后，通过qPCR和蛋白质免疫印迹法探讨miR-455对RHOC及其产物RhoC的影响。由于PTEN是HCC中的关键抑癌基因，而Bcl-2和Caspase 3是凋亡的重要指标，因此在过表达RhoC的细胞中检测PTEN、Bcl2和Caspase 3蛋白的表达水平。我们发现，miR-455促进HepG2细胞凋亡并抑制其增殖。生物信息学分析和荧光素酶报告基因检测表明，miR-455的特异性识别位点位于RhoC的3'-UTR内。与对照细胞相比，共转染miR-455模拟物和RhoC-WT的细胞中荧光素酶活性显著降低（p < 0.01），表明RHOC基因确实是miR-455的靶基因。miR-455转染HepG2细胞后，RHOC mRNA显著降低。此外，我们发现RhoC可激活HCC细胞的增殖能力并抑制凋亡率（p < 0.01），降低PTEN和Caspase 3的表达（p < 0.01），同时上调Bcl2水平。总之，我们的研究表明，miR-455通过靶向RhoC编码的mRNA在HCC发生中发挥抑制作用。
