miR-637调控RING1表达抑制口腔鳞癌Tac-8113细胞增殖实验研究

Experiment study on miR-637 in regulating RING1 expression and inhibiting the proliferation of oral squamous cell carcinoma Tca-8113 cells

目的:探讨口腔鳞癌细胞Tca-8113中miR-637靶向RING1对其细胞增殖活性的影响。方法:培养人口腔鳞癌细胞Tca-8113作为对照组,应用Lipofectamine 2000试剂将转染miR-637 NC、miR-637 mimics的Tca-8113细胞分别作为miR-637 NC组、miR-637 mimics组,倒置荧光显微镜检测转染效率,实时荧光定量聚合酶链式反应法(qRT-PCR)法检测转染后各组miR-637表达情况,通过细胞计数试剂盒(CCK-8)法以及平板克隆法检测各组miR-637细胞增殖情况,通过双荧光素酶报告基因检测miR-637与RING1间的靶向关系,应用免疫印迹法(WB)检测各组RING1蛋白以及增殖相关蛋白PCNA表达水平。结果:qRT-PCR显示,与对照组、miR-637 NC组相比,miR-637 mimics组miR-637表达显著升高(P<0.05);CCK-8实验显示,与对照组、miR-637 NC组相比,miR-637 mimics组转染后48、72、96 h后OD值显著降低(P<0.05);平板克隆法显示,与对照组、miR-637 NC组相比,miR-637 mimics组转染后克隆形成率显著降低(P<0.05);双荧光素酶报告基因检测显示共同转染miR-637+WT-RING1后,荧光素酶活性表达受抑制,而转染Neg-miR637+WT-RING1、Neg-miR637+MT-RING1、miR-637+MT-RING1组荧光素酶活性无明显变化;WB结果显示,与对照组相比、miR-637 NC组相比,miR-637 mimics组RING1蛋白显著降低(P<0.05);与对照组相比、miR-637 NC组相比,miR-637 mimics组PCNA蛋白表达显著降低(P<0.05)。结论:miR-637可能通过下调RING1表达抑制口腔鳞癌Tac-8113细胞增殖。

Objective:To investigate the effect of miR-637 targeting RING1 on the proliferation of oral squamous cell carcinoma Tca-8113 cells.Methods:Human oral squamous cell carcinoma Tca-8113 cells were cultured as control group.The Tca-8113 cells transfected with miR-637 NC and miR-637 mimics by Lipofectamine 2000 reagent were used as miR-637 NC group and miR-637 mimics group,respectively.Inverted fluorescence microscopy was used to detect the transfection efficiency.Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression of miR-637 in each group after transfection.Cell counting kit(CCK-8)and plate cloning were used to detect the proliferation of miR-637 cells in each group.The targeting relationship between miR-637 and RING1 was detected by double luciferase reporter gene.The expression levels of RING1 protein and PCNA were detected by Western blotting(WB).Results:The qRT-PCR showed that the expression level of miR-637 in the miR-637 mimics group was significantly higher than that in the control group and the miR-637 NC group(P<0.05).The CCK-8 experiment showed that the OD value of miR-637 mimics group decreased significantly at 48,72 and 96 hours after transfection compared with control group and miR-637 NC group(P<0.05).The plate cloning method showed that the cloning rate of miR-637 mimics group was significantly lower compared with control group and miR-637 NC group(P<0.05).The double luciferase reporter gene assay showed that,after co-transfection of miR-637+WT-RING1,the expression of luciferase activity was inhibited,but there was no significant change in the transfection of Neg-miR 637+WT-RING1,Neg-miR 637+MT-RING1,and miR-637+MT-RING1 groups.The WB results showed that the expression levels of RING1 and PCNA protein in the miR-637 mimics group were significantly lower than those in the control group and the miR-637 NC group(P<0.05).Conclusion:miR-637 may inhibit the proliferation of oral squamous cell carcinoma Tca-8113 cells by down-regulating RING1 expression.