亲环素A在胃癌细胞中的表达及与肿瘤细胞增殖的关系

Expression of cyclophilin A in gastric cancer and its relationship with proliferation of gastric cancer cells

目的检测亲环素A(CypA)在胃癌组织和细胞株中的表达情况,采用基因干扰技术抑制胃癌细胞中CypA的表达,探讨CypA对胃癌细胞增殖能力的影响和相关分子机制。方法实时定量PCR和蛋白质印迹法检测CypA在胃癌组织、癌旁组织和细胞株中的表达;合成针对CypA的靶向siRNA并转染胃癌MKN45细胞株,检测CypA-siRNA对胃癌细胞内源性CypA的抑制作用,同时设置非特异性siRNA对照组和阴性对照组。MTT法检测各组细胞增殖情况,流式细胞术检测各组细胞周期;实时定量PCR和蛋白质印迹法检测各组细胞增殖基因PCNA、P21、P16、Cyclin D1的表达。结果胃癌组织中CypA的mRNA和蛋白表达高于癌旁组织,胃癌细胞株CypA的mRNA和蛋白表达高于胃上皮细胞株,且在低分化细胞MKN45中表达最高(P<0.05)。CypA-siRNA可有效抑制内源性CypA的表达;CypA-siRNA转染后MKN45细胞增殖能力下降(P<0.05),G0/G1期细胞比例上升、G2/M期细胞比例下降(P<0.05),PCNA、Cyclin D1的mRNA和蛋白表达下调,P21mRNA和蛋白表达上调(P<0.05)。结论胃癌细胞中CypA的表达增强,CypA基因能够通过调节部分增殖基因的表达而促进胃癌细胞增殖。

Objective To examine the expression of cyclophilin A （CypA） in gastric cancer tissues and cell lines, and to explore the effect and mechanism of CypA on the proliferation of gastric cancer cells. Methods Real-time quantitative PCR and Western blotting analysis were used to detect the expression of CypA in gastric cancer tissues, the corresponding adjacent tissues and gastric cancer cell lines. Small interfering RNAs targeting CypA were synthesized and transfected into the gastric cancer cell line MKN45. The effect of CypA-siRNA on endogenous CypA expression was observed; meanwhile, nonspecific siRNA control group and negative control group were also designed. Cell proliferation was measured with MTT assay in each group; cell cycle was detected with flow cytometry. The expression of PCNA, P21, P16, and Cyclin D1 gene, involved in cell proliferation, were also detected by real-time quantitative PCR and Western blotting analysis. Results CypA expression was significantly up-regulated in gastric cancer tissues compared with para-cancer tissues; and CypA expression in gastric cancer cell lines was also significantly higher than that in the gastric epithelial cell line GES-1, with the highest expression found in the poorly-differentiated gastric cancer cell line MKN45（P〈0.05）. The expression of CypA was significantly inhibited by the CypA-siRNA in MKN45 （P〈0.05）.MTT assay showed that the inhibition of CypA by CypA-siRNA significantly suppressed MKN45 cell proliferation （P〈0.05）. The ratio of G0/G1 phase cells was significantly increased in MKN45 cells after CypA-siRNA transfection, while the ratio of G2/M phase was significantly decreased（P〈0.05）. In addition, the expression of PCNA, Cyclin D1 was significantly decreased at mRNA and protein levels when CypA was inhibited, and the expression of P21 was significantly increased （P〈0.05）. Conclusion CypA is overexpressed in gastric cancer cells; CypA gene can effectively promote gastric cancer cell proliferation by regulating some proliferation related genes.