miR-802靶向调控PI3K/Akt信号通路促进肝细胞癌血管新生的机制研究

Mechanism of miR-802 targeting PI3K/Akt signaling pathway in promoting angiogenesis of hepatocellular carcinoma

目的 探究miR-802在肝细胞癌(HCC)细胞中的表达及其对肿瘤血管新生的影响及作用机制。方法 通过转染miR-802 agomir、miR-802 antagomir上调或抑制人HCC细胞系SMMC-7721中mi R-802的表达水平。将SMMC-7721细胞分为NC组(正常培养细胞)、miR-802 agomir组(转染mi R-802 agomir)和miR-802antagomir组(转染miR-802 antagomir)。采用小管形成实验和Transwell实验检测过表达miR-802的SMMC-7721细胞上清液对人脐静脉内皮细胞(HUVEC)成管能力及迁移能力的影响。采用蛋白质印迹法检测mi R-802 agomir组中磷脂酰肌醇3激酶(PI3K)、磷酸化PI3K(p-PI3K)、蛋白激酶B(Akt)、磷酸化Akt(p-Akt)、血管内皮生长因子(VEGF)的表达水平。采用免疫荧光实验检测过表达miR-802对VEGF生成的影响。结果 与NC组相比,mi R-802 agomir组HUVEC细胞的成管能力及迁移能力均显著增强,miR-802 antagomir组HUVEC细胞的成管能力及迁移能力均显著降低,差异均有统计学意义(P均<0.05)。与NC组相比,miR-802 agomir组SMMC-7721细胞中VEGF生成量增加,p-PI3K、p-Akt、Akt蛋白表达水平均显著升高,差异均有统计学意义(P均<0.05)。结论 miR-802可通过调控PI3K/Akt信号通路促进VEGF的释放,参与血管新生,从而促进HCC的发生和进展。

Objective This paper attempts to investigate the expression of miR-802 in hepatocellular carcinoma cells and its effect on tumor angiogenesis and mechanism of action.Methods The expression of miR-802 in human hepatocellular carcinoma cell line SMMC-7721 was up-regulated or inhibited by transfection of miR-802 agomir and miR-802 antagomir.SMMC-7721 cells were divided into the NC group(normal cultured cells),the miR-802 agomir group(transfected with miR-802 agomir),and the miR-802 antagomir group(transfected with miR-802 antagomir).The effects of SMMC-7721 cell supernatant overexpressing miR-802 on the tube-forming and migratory abilities of human umbilical vein endothelial cells(HUVEC)were detected by using tubule formation assay and Transwell assay.The expression of phosphatidylinositol 3-kinase(PI3K),phosphorylated PI3K(p-PI3K),protein kinase B(Akt),phosphorylated Akt(p-Akt),and vascular endothelial growth factor(VEGF)was detected by western blotting in the miR-802 agomir group.Immunofluorescence assay was used to detect the effect of overexpression of miR-802 on VEGF production.Results Compared with the NC group,the tube-forming and migratory abilities of HUVEC cells in the miR-802 agomir group are enhanced,and the tube-forming and migratory abilities of HUVEC cells in the miR-802 antagomir group are reduced,with statistically significant differences(P<0.05).Compared with the NC group,the production of VEGF of SMMC-7721 cells in the miR-802 agomir group is increased,and the protein expression of p-PI3K,p-Akt,and Akt is elevated,with statistically significant differences(P<0.05).Conclusion miR-802 can promote the release of VEGF through regulating the PI3K/Akt signaling pathway,and then participate in angiogenesis,thus promoting the occurrence and progression of hepatocellular carcinoma.