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目的:通过小干扰RNA(siRNA)载体介导抑制肽基脯氨酰同分异构酶(Pin1)表达,探讨Pin1对宫颈癌细胞SiHa增殖与凋亡的影响。方法:构建pGPU6/GFP/Neo-Pin1 siRNA表达载体,脂质体介导将其转染至宫颈癌细胞SiHa中;实验设3组:实验组(SiHa/pGPU6-Pin1 siRNA组)、阴性对照组(SiHa/pGPU6-con组)和空白对照组(SiHa)。采用RT-PCR、Western blot法检测Pin1的表达;MTT法检测细胞增殖活性;原位末端标记法(TUNEL)和流式细胞术(FCM)检测细胞凋亡。结果:转染SiHa/pGPU6-Pin1siRNA的SiHa细胞中,Pin1 mRNA和蛋白表达分别下调68.1%和54.8%,细胞增殖显著抑制。TUNEL显示典型细胞凋亡特征,SiHa/pGPU6-Pin1 siRNA组的凋亡指数(AI)为(29.6±14.58)%,显著高于SiHa/pGPU6-con组[(8.44±5.65)%]和空白对照组[(5.34±4.47)%](P<0.05)。SiHa/pGPU6-Pin1 siRNA组的细胞凋亡率为(30.95±16.47)%,显著高于SiHa/pGPU6-con组[(6.18±6.10)%]和空白对照组[(5.95±4.99)%](P<0.05)。结论:RNA干扰技术能特异高效的抑制Pin1基因的表达,Pin1基因的下调能抑制宫颈癌细胞增殖并促进细胞凋亡,提示Pin1可能成为治疗宫颈癌的新靶点。
OBJECTIVE: To investigate the effect of Pin1 on proliferation and apoptosis of cervical cancer cells SiHa mediated by small interfering RNA (siRNA) -mediated inhibition of Pin1 prolyl isoformis (Pin1) expression. METHODS: The pGPU6 / GFP / Neo-Pin1 siRNA expression vector was constructed and transfected into SiHa cells by lipofectamine. The experimental groups were divided into experimental group (SiHa / pGPU6-Pin1 siRNA group), negative control group (SiHa / pGPU6-con group) and blank control group (SiHa). The expression of Pin1 was detected by RT-PCR and Western blot. Cell proliferation was detected by MTT assay. Cell apoptosis was detected by TUNEL and flow cytometry (FCM). Results: SiHa / pGPU6-Pin1 siRNA transfected SiHa cells, Pin1 mRNA and protein expression were down-regulated 68.1% and 54.8%, cell proliferation was significantly inhibited. The apoptotic index (Si) of SiHa / pGPU6-Pin1 siRNA group was (29.6 ± 14.58)%, which was significantly higher than that of SiHa / pGPU6-con group [(8.44 ± 5.65)%] Group [(5.34 ± 4.47)%] (P <0.05). The apoptosis rate of SiHa / pGPU6-Pin1 siRNA group was (30.95 ± 16.47)%, which was significantly higher than that of SiHa / pGPU6-con group (6.18 ± 6.10%) and blank control group (5.95 ± 4.99)% P <0.05). Conclusion: RNAi can inhibit the expression of Pin1 gene specifically and efficiently. The down-regulation of Pin1 gene can inhibit the proliferation and promote the apoptosis of cervical cancer cells, suggesting that Pin1 may be a new therapeutic target for cervical cancer.