RNA干扰maspin基因对胃癌细胞株MKN-28凋亡的影响

Effect of silencing maspin by RNA interference on apoptosis of gastric cancer cell line MKN-28

目的:探讨转染maspin特异性短发夹RNA(short hairpin RNA,shRNA)重组质粒对胃癌细胞株MKN-28凋亡的影响。方法:设计并合成针对maspin基因的shRNA,并将其与真核表达载体pGenesil-1.1连接,构建重组质粒pGenesil-maspin。应用RT-PCR和Western印迹法检测转染重组质粒后MKN-28细胞中maspin、bcl-2和bax mRNA及蛋白的表达变化,应用FCM法检测转染重组质粒对MKN-28细胞的细胞周期和细胞凋亡的影响。结果:成功构建maspin特异性shRNA重组质粒pGenesil-maspin1、pGenesil-maspin2及pGenesil-HK(阴性对照);pGenesil-maspin成功转染后可明显下调胃癌细胞MKN-28中maspin和bax mRNA及蛋白的表达水平(P<0.01),并上调bcl-2 mRNA和蛋白的表达水平(P<0.01);重组质粒转染后,MKN-28细胞的细胞周期呈现G0/G1期细胞减少(P<0.01),细胞凋亡率下降(P<0.01)。结论:外源性maspin shRNA转染MKN-28细胞后能下调maspin的表达,抑制胃癌细胞的凋亡,且使细胞周期分布发生改变;其分子机制可能与上调bcl-2和下调bax的表达有关。

Objective：To construct and identify eukaryotic expression vector containing short hairpin RNA（shRNA） of maspin and investigate its effect on apoptosis of gastric cancer cell line MKN-28.Methods：ShRNA targeting maspin gene was designed,synthesized,conjugated with eukaryotic expression vector pGenesil 1.1 to construct recombinant plasmid pGenesil-maspin.RT-PCR and Western blotting were used to detect the mRNA and protein expressions of maspin,bcl-2,and bax after transfection,respectively.Flow cytometry was used to determine the effects of transfection of pGenesil-maspin on cell cycle distribution and apoptosis.Results：We successfully constructed recombinant plasmids containing maspin specific shRNA（pGenesil-maspin1 and pGenesil-maspin2） and pGenesil-HK（as the blank control）,which were validated by gel electrophoresis and DNA sequence analysis.RT-PCR and Western blotting analysis indicated that maspin and bax mRNA and protein expression was dramatically down-regulated（P0.01） and bcl-2 mRNA and protein expression was up-regulated（P0.01） in gastric cancer cell line MKN-28 after transfection with pGenesil-maspin positive plasmids.Flow cytometric profiles indicated that positive plasmid transfection led to a decrease in the percentage of cells in G0/G1phase（P0.01） and a decrease in apoptotic rate（P0.01）.Conclusion：Transfection of exogenous maspin shRNA effectively down-regulated the expression of maspin,inhibited apoptosis,altered cell cycle distribution of human gastric cancer cell line MKN-28.The molecular mechanism might be associated with altering cell cycle,up-regulating bcl-2 expression and down-regulating bax expression.