miR-150-5p靶向SIRT1提高肝癌细胞系HepG2放疗敏感性

目的探究miR-150-5p靶向SIRT1提高肝癌细胞放射敏感性的作用机制。方法用分次放疗放射递增法诱导建立放射抵抗型细胞株(RR-HepG2);RT-qPCR检测HepG2和RR-HepG2在不同放射剂量下miR-150-5p的表达水平;细胞克隆实验检测相同放射剂量下两种细胞的放疗敏感性;流式细胞计量术和Western blot检测过表达miR-150-5p对HepG2凋亡的影响;双荧光素酶报告基因法检测miR-150-5p与SIRT1的关联;细胞克隆实验和Western blot检测过表达SIRT1后,细胞的放疗敏感性和凋亡蛋白的变化。结果与亲代HepG2比较,相同放射剂量下,RRHepG2组miR-150-5p的表达量均显著降低(P<0. 05);放射处理后,与control、agomiR-NC组比较,agomiR-150-5p组的细胞存活分数显著降低,敏感性高(P<0. 05),Bax、caspase-9蛋白表达水平显著升高,Bcl-2蛋白表达水平显著降低,细胞凋亡率显著升高;双荧光素酶报告基因法验证miR-150-5p靶向调控SIRT1。与agomiR-NC组比较,野生型(WT) agomiR-150-5p荧光素酶活性显著降低,SIRT1蛋白水平显著降低(P<0. 05);与anta-agomiR-NC比较,野生型(WT) anta-agomiR-150-5p荧光素酶活性显著升高,SIRT1蛋白水平显著升高(P<0. 05);放射处理后,与agomiR-NC组比较,agomiR-150-5p组的细胞存活分数显著降低,Bax、caspase-9蛋白表达水平显著升高,Bcl-2蛋白表达水平显著降低(P<0. 05);与agomiR-150-5p+vector组比较,agomiR-150-5p+SIRT1组的细胞存活分数显著升高,Bax、caspase-9蛋白表达水平显著降低,Bcl-2蛋白表达水平显著升高(P<0. 05)。结论 miR-150-5p靶向SIRT1,下调其表达,提高肝癌细胞放射敏感性,可为临床肝癌放射治疗增敏提供靶点。

miR-150-5p enhances radiotherapy sensitivity of hepatocellular carcinoma cells line HepG2 through targeting SIRT1

Objective To investigate the effect on the radiosensitivity enhancement of hepatocellular carcinoma cells by targeting SIRT1 with miR-150-5p. Methods The establishment of radioresistant cell strain RR-HepG2 was induced by fractional incremental radiotherapy. RT-qPCR was used to detect the expression of miR-150-5p in HepG2 and RR-HepG2 at different radiation doses. Cell cloning assay was used to detect the radiosensitivity of the two groups cell at the same radiation dose. Flow cytometry and Western blot were used to detect the effect of overexpression of miR-150-5p on apoptosis of HepG2 cells. Double luciferase assay was used to detect the association between miR-150-5p and SIRT1.Cell cloning and Western blot were used to detect the changes of radiosensitivity and apoptotic protein after over-expression of SIRT1. Results The expression of miR-150-5p in RR-HepG2 group decreased significantly with the same radiation dose( P<0. 05). The survival fraction and sensitivity of cells in agomiR-150-5p group decreased significantly( P<0. 05),the expression of Bax and caspase-9 increased significantly,the expression of Bcl-2 decreased significantly,and the apoptotic rate was significantly increased. The wild type( WT) agomiR-150-5p luciferase activity decreased significantly and SIRT1 protein level decreased significantly( P<0. 05);wild type( WT) anta-agomiR-150-5p luciferase activity increased significantly and SIRT1 protein level increased significantly( P < 0. 05);WT-anta-agomiR-150-5p luciferase activity increased significantly and SIRT1 protein level increased significantly( P < 0. 05). The survival fraction of cells in the agomiR-150-5p group decreased significantly,the expression of Bax and caspase-9 increased significantly,and the expression of Bcl-2 decreased significantly( P<0. 05). The survival fraction of cells in the agomiR-150-5p +SIRT1 group increased significantly,the expression levels of Bax and caspase-9 decreased significantly,and the expression of Bcl-2 increased significantly( P<0. 05).Conclusions The down-regulation of SIRT1 expression by miR-150-5p improves the radiosensitivity of hepatocellular carcinoma cells and provides a potential target for clinical radiotherapy of hepatocellular carcinoma.