CDKN2A在宫颈癌组织中的表达及调控Wnt/β-catenin通路影响SiHa细胞增殖和凋亡的机制

Expression of CDKN2A in cervical cancer tissues and its effects in regulating Wnt/β-catenin pathway on the proliferation and apoptosis of SiHa cells

目的探讨细胞周期蛋白依赖性激酶抑制蛋白2A(CDKN2A)在宫颈癌组织中的表达及对宫颈癌SiHa细胞增殖和凋亡的影响和作用机制。方法采用Western blot检测宫颈癌组织和相应癌旁组织中CDKN2A蛋白表达情况,将人宫颈癌SiHa细胞分为空白对照(Control)组、阴性对照(pcDNA)组和过表达(pc-CDKN2A)组,转染48 h后采用Western blot检测3组SiHa细胞中CDKN2A蛋白表达水平,噻唑蓝(MTT)和AnnexinⅤ-FITC/PI双染色法分别检测SiHa细胞增殖和凋亡情况,Western blot检测SiHa细胞中增殖细胞核抗原(PCNA)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)、活化的含半胱氨酸的天冬氨酸蛋白水解酶9(Cleaved Caspase-9)及Wnt/β-catenin信号通路中β-连环蛋白(β-catenin)和下游关键靶蛋白c-myc的表达情况。结果CDKN2A在宫颈癌组织中的表达明显低于正常癌旁组织(P<0.05);与pcDNA组相比,pc-CDKN2A组SiHa细胞中CDKN2A蛋白表达水平明显升高,细胞增殖能力减弱,凋亡率升高,PCNA、β-catenin和c-myc蛋白表达明显下调,Cleaved Caspase-3和Cleaved Caspase-9的表达明显上调,差异有统计学意义(P<0.05);与pcDNA组相比,Control组中以上各指标差异均无统计学意义(P>0.05)。结论CDKN2A在宫颈癌组织中呈低表达,过表达CDKN2A能够通过抑制Wnt/β-catenin信号通路降低宫颈癌SiHa细胞增殖,促进SiHa细胞凋亡。

Objective To investigate the expression of cyclin-dependent kinase inhibitor 2A(CDKN2A)in cervical cancer,and to explore its effects on the proliferation and apoptosis of cervical cancer SiHa cells.Methods The expression levels of CDKN2A protein in cervical cancer tissues and corresponding paracancerous tissues were detected by Western Blot.The human cervical cancer SiHa cells were divided into blank control group,negative control(pcDNA)group and overexpression(pc-CDKN2A)group.At 48h after transfection,the expression levels of CDKN2A protein in SiHa cells were detected by Western Blot.Tetrazolium blue(MTT)and Annexin V-FITC/PI double staining were used to detect the proliferation and apoptosis of SiHa cells,respectively.The expression levels of proliferating cell nuclear antigen(PCNA),activated cysteine-containing aspartate proteolytic enzyme 3(Cleaved Caspase-3),and activated cysteine-containing aspartate proteolysis in SiHa cells as well as the expression levels ofβ-catenin and downstream key target protein c-myc in Cleaved Caspase-9 and Wnt/β-catenin signaling pathways were detected by Western Blot.Results The expression levels of CDKN2A in cervical cancer tissues were significantly lower than those in normal para-cancerous tissues(P<0.05).As compared with those in pcDNA group,the expression levels of CDKN2A protein in SiHa cells of pc-CDKN2A group were significantly increased,cell proliferation ability was decreased,and apoptosis rate was increased.Moreover the expression levels of PCNA,β-catenin and c-myc were down-regulated,however,the expression levels of Cleaved Caspase-3 and Cleaved Caspase-9 were significantly up-regulated(P<0.05).There were no significant differences in above indexes between pcDNA group and control group(P>0.05).Conclusion CDKN2A is down-regulated in cervical cancer tissues,and the overexpression of CDKN2A can inhibit the proliferation of cervical cancer SiHa cells and promote the apoptosis of SiHa cells by inhibiting Wnt/β-catenin signaling pathway.