siRNA靶向沉默胃癌MKN45细胞COX-2基因对Notch基因表达的影响

摘要

Objective:To investigate the effect of silencing COX-2 genes on proliferation and apoptosis and the underlying mechanisms with Notch signaling pathway of human gastric carcer MKN45 cells line by target RNA inter-ference.Methods:The siRNA of COX -2 was transfected into gastric cancer cell line MKN45 by Lipofectamine 2000.MKN45 cells transfected with blank vector was served as negative control group.Proliferation of MKN45 were detected after transfection by methabenzthiazuron assay and apoptosis by flow cytometry.The mRNA expression of COX-2,Notch1,Notch2 and Hes-1 were determined by RT-PCR.The protein expressions of COX-2,N1ICD, N2ICD and Hes-1 were determined by Western blotting.Results:Compared with that in control group and blank group,it can inhibit cell proliferation and induce apoptosis after COX-2 silencing.The mRNA expression of COX-2 and Hes-1 were significantly decreased after transfection,however,did not affect Notch1and Notch2.The protein expressions of COX -2,N1ICD,N2ICD and Hes -1 were obviously inhibited.Conclusion:RNA silence targeting COX-2 can inhibit cell proliferation and induce apoptosis of gastric cell line MKN45 probably by inhibiting Notch signaling pathway.%目的：探讨siRNA靶向沉默COX-2基因表达对人胃癌MKN45细胞增殖和凋亡能力的影响和相关机制研究。方法：通过COX-2小干扰RNA转染胃癌细胞MKN45为实验组，转染siRNA control为阴性对照组，以未转染细胞为空白组，转染48h后，RT-PCR法检测靶向沉默COX-2基因的效果以及Notch信号通路相关基因的表达水平，MTT法检测各组MKN45细胞的增殖能力，流式细胞术分析各组细胞凋亡，Western blot检测COX-2，N1ICD，N2ICD和Hes-1蛋白表达水平。结果：靶向沉默COX-2基因可明显抑制MKN45细胞增殖和提高凋亡率。靶向沉默COX-2基因可明显下调COX-2和Hes-1 mRNA 水平，而对Notch1和Notch2 mRNA水平无明显影响。靶向沉默COX-2基因可明显下调COX-2，N1ICD，N2ICD和Hes-1蛋白表达水平。结论：靶向沉默COX-2基因可抑制胃癌细胞MKN45增殖和促进凋亡，可能是通过抑制Notch信号通路实现的。