siRNA沉默PIM1基因表达对人胃癌MKN-45细胞增殖及凋亡的影响

Effects of silencing PIM1 gene by siRNA on proliferation and apoptosis of human gastric cancer MKN-45 cell line

目的:探讨PIM1基因沉默对人胃癌MKN-45细胞增殖和凋亡的影响。方法:设计2条针对PIM1 mRNA靶序列的干扰RNA正义链和反义链及阴性对照序列,构建重组pSIREN-retroQ质粒载体,并转染胃癌MKN-45细胞。应用实时荧光定量PCR和蛋白质印迹法分别检测PIM1在mRNA水平及蛋白水平的表达。CCK-8法检测胃癌MKN-45细胞的增殖,流式细胞分析胃癌细胞的凋亡,蛋白质印迹法检测凋亡相关蛋白Caspase 3和Caspase 9的表达。结果:重组克隆通过PCR扩增及测序,证明干扰序列插入正确。与正常对照组和阴性对照组相比,两个RNA干扰组胃癌MKN-45细胞PIM1在mRNA及蛋白水平的表达量都显著降低(P<0.05),细胞增殖能力下降(P<0.05)。此外,干扰组细胞凋亡明显增多(P<0.01),Caspase 3和Caspase9蛋白表达上调(P<0.01)。结论:PIM1 siRNA能有效下调靶基因表达,产生特异性基因沉默效应;PIM1基因沉默显著抑制胃癌MKN-45细胞的增殖并促进其凋亡。

Objective:To investigate the effect of siRNA-mediated silencing of PIM1 on proliferation and apoptosis of human gastric cancer MKN-45 cell line.Methods:Two siRNA sequences and negative control targeting PIM1 mRNA were designed.The siRNA sequences were linked with pSIREN-retroQ.Then the recombinant plasmids were transfected into human gastric cancer MKN-45 cell line.The expressions of PIM1 mRNA and protein in MKN-45 cells were detected by real-time fluorescent quantitative PCR and Western blotting,respectively.CCK-8 assay and flow cytometry were used separately to determine the proliferation activity and apoptosis of MKN-45 cells.The expressions levels of Caspase 3 and Caspase 9 were detected by Western blotting.Results:PCR and DNA sequencing indicated that the recombinant plasmids were successfully constructed.Compared with the normal control group and the negative control group,the expression of PIM1 mRNA and protein in the two siRNA groups was significantly reduced(P<0.05),and the cell proliferation ability was decreased(P<0.05).In addition,apoptosis was greatly increased(P<0.01),and Caspase 3 and Caspase 9 protein expression were up-regulated(P<0.01).Conclusion:The silence of PIM-1 gene can result in the suppression of cell proliferation and increasing apoptosis in MKN-45 cell line.