上调microRNA-150表达对肝癌SMMC7721细胞增殖和凋亡的影响

Effect of microRNA-150 up-regulation on proliferation and apoptosis of hepatocellular carcinoma cell line SMMC-7721

目的探讨上调microRNA-150(miR-150)表达对肝癌SMMC7721细胞增殖和凋亡的影响及其可能机制。方法 SMMC7721细胞分成miR-150感染组(加入pGIPZ-miR-150慢病毒表达载体)、阴性对照组(加入只含GFP的慢病毒载体)和空白对照组(不转染)。采用荧光定量PCR检测miR-150的表达情况,Western blotting检测靶基因c-Myb蛋白的表达,CCK-8法检测细胞增殖能力,流式细胞仪检测细胞凋亡情况。结果荧光定量PCR结果显示,转染miR-150pGIPZ后,SMMC7721细胞miR-150表达量(2.48±0.15)较阴性对照组(0.81±0.09)增加了2倍(P<0.01)。CCK-8法检测结果显示,与阴性对照组和空白对照组比较,miR-150组的细胞增殖率显著降低(P<0.05)。Western blotting检测结果表明,miR-150组细胞c-Myb蛋白表达(0.31±0.07)与空白对照组(0.80±0.09)及阴性对照组(0.81±0.08)相比明显减少(P<0.01)。流式细胞仪检测结果显示,miR-150组细胞凋亡率(19.36%±1.78%)与阴性对照组(5.12%±0.54%)及空白对照组(4.68%±0.35%)相比明显增加(P<0.01)。结论上调miR-150表达可通过降低靶基因c-Myb的表达,抑制细胞增殖,诱导细胞凋亡,miR-150可能成为肝癌靶向治疗新的靶基因。

Objective To investigate the effects ofmicroRNA150 （miR-1$0） up-regulation on proliferation and apoptosis of hepatocellular carcinoma cell line SMMC7721, and the potential mechanism thereof. Methods The SMMC7721 cells were divided into miR-150 modulated group, negative control group, and blank control group. Chemically synthesized hsa-mig-150, according to the maturity sequence ofhsa-miR-150, was inserted into pGIPZ vector. SMMC7721 cells in mig-150 modulated group and negative control were transfected with mig-150pGIPZ and miR-control respectively, while those in blank control group without transfection. The expression ofmiR-150 was determined by real-time fluorescence quantitative PCR. The cell proliferation ability was determined by Cell Counting Kit-8 （CCK-8） assay, and cell apoptosis was assayed by flow cytometry. The protein expression of c-Myb was measured by Western blotting. Results The miR-150 expression increased by 7 folds in cells after being transfected with miR-150 than those in negative control group and blank control group. CCK-8 assay showed that the cellular growth rate of SMMC7721 cells in miR- 150pGIPZ group was significantly lower than those in negative control group and blank control group （P〈0.0S）. The apoptotic rate in miR-150pGIPZ group, negative control group and blank control group was （19.36 ＋ 1.78）96, （5.12 ＋_ 0.54）% and （4.68 ＋ 0.35）%, respectively. The expression level of c-Myb protein in negative control group, blank control group and miR-150pGIPZ group was 0.31 ＋ 0.07, 0.80 ＋ 0.09 and 0.81 ＋ 0.08, respectively. Conclusion The results indicate that up-regulation of miR-150 may suppress the cellular proliferation and induce cell apoptosis in SMMC7721 cells, at least in part, through regulating the expression level of target gene c-Myb, thus implying that miR-150 might serve as a novel molecular target for treatment of HCC.