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目的:探讨微小核糖核酸(Micro RNA,Mi RNA)376a/c靶向调控转化生长因子α(transforming growth factor alpha,TGFA)在CD133+U251胶质瘤细胞增殖中作用方式及其机制。方法:构建Mi R376a/c靶向调控TGFA慢病毒克隆体,根据Mi R376a/c与TGFA结合位点分为4组,检测各组酶活性。根据转染体外Mi R376a/c和Mi R376a/c模拟物(Mi R-SCR)不同,将CD133+U251细胞分为4组,通过细胞培养、Gimsa染色,检测TGFA蛋白表达量和细胞生长曲线,观察细胞增殖情况,收集资料并进行统计学分析。结果:CD133+组与CD133-组Mi R376a/c表达比较,差异有统计学意义(P<0.001);wt-TGFA组与mut-TGFA组质粒荧光素酶活性表达差异比较,差异有统计学意义(P=0.000);将Mi R376a/c慢病毒导入CD133+U251细胞中,Mi R376a/c组TGFA蛋白表达相对于Mi R-SCR组和空白对照组明显下调,差异有统计学意义(P=0.000);生长曲线检测示:随时间延长,Mi R376a/c组细胞生长速度慢于对照组,差异有统计学意义(P=0.000)。结论:Mi RNA376a/c靶向调控TGFA抑制CD133+U251胶质瘤细胞增殖、克隆。
OBJECTIVE: To investigate the mode of action of transforming growth factor-alpha (TGF-β) in the proliferation of CD133 + U251 glioma cells and its mechanism by microRNA (Mi RNA) 376a / c. Methods: Mi R376a / c targeting lentiviral vector of TGFA was constructed and divided into 4 groups according to the binding site of Mi R376a / c with TGFA. The enzyme activity of each group was tested. The CD133 + U251 cells were divided into 4 groups according to MiR376a / c and miR-3776a / c mimics (MiR-SCR). The expression of TGFβ1 protein and cell growth curve were detected by cell culture and Gimsa staining. Observation of cell proliferation, data collection and statistical analysis. Results: The expression of Mi R376a / c in CD133 + group was significantly different from that in CD133- group (P <0.001). There was significant difference in luciferase activity between wt-TGFA group and mut-TGFA group (P = 0.000). The miR376a / c lentivirus was transduced into CD133 + U251 cells, and the expression of TGFβ1 in Mi R376a / c group was significantly down-regulated compared with that in Mi R-SCR group and blank control group ). Growth curve test showed that the cell growth rate of Mi R376a / c group was slower than that of control group with time prolongation (P = 0.000). CONCLUSION: Mi RNA376a / c can regulate the proliferation and clone of CD133 + U251 glioma cells through TGFA.