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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 detect the expression of CypA in gastric cancer cells and cell lines, and to study the effect of CypA on the proliferation of gastric cancer cells and the related molecular mechanisms by using gene interference technology to inhibit the expression of CypA in gastric cancer cells. Methods The expression of CypA in gastric cancer tissues, paracancerous tissues and cell lines was detected by real-time quantitative PCR and Western blotting. Targeting siRNA targeting CypA was synthesized and transfected into gastric cancer MKN45 cells to detect the effect of CypA-siRNA on the expression of CypA Inhibition, while setting non-specific siRNA control group and negative control group. Cell proliferation was measured by MTT assay. Cell cycle was detected by flow cytometry. PCNA, P21, P16 and Cyclin D1 were detected by real-time quantitative PCR and Western blotting. Results The mRNA and protein expression of CypA in gastric cancer tissue were higher than those in paracancerous tissues. The mRNA and protein expression of CypA in gastric cancer cell line was higher than that in gastric epithelial cell line and was highest in poorly differentiated MKN45 cells (P <0.05). CypA-siRNA could inhibit the expression of endogenous CypA. The proliferation of MKN45 cells was decreased after CypA-siRNA transfection (P <0.05), the proportion of cells in G0 / G1 phase increased and the proportion of cells in G2 / M phase decreased (P <0.05) , The mRNA and protein expressions of PCNA and Cyclin D1 were down-regulated and the expression of P21 mRNA and protein were up-regulated (P <0.05). Conclusion The expression of CypA in gastric cancer cells is enhanced. CypA gene can promote the proliferation of gastric cancer cells by regulating the expression of some proliferative genes.