PinlsiRNA对宫颈癌细胞SiHa增殖和凋亡的影响

目的：通过小干扰RNA（siRNA）栽体介导抑制肽基脯氨酰同分异构酶（Pinl）表达，探讨Pinl对宫颈癌细胞SiHa增殖与凋亡的影响。方法：构建pGPU6／GFP／Neo—PinlsiRNA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-PinlsiRNA组）、阴性对照组（SiHa／pGPU6-con组）和空白对照组（Si-Ha）。采用RT—PCR、Western blot法检测Pinl的表达；M1Tr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．PinlsiRNA的SiHa细胞中，PinlmRNA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-PinlsiRNA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〈O．05）。SiHa／pGPU6．PinlsiRNA组的细胞凋亡率为（30．95±16．47）％，显著高于SiHa／pGPU6-COIl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Pinl基因的表达，Pinl基因的下调能抑制宫颈癌细胞增殖并促进细胞凋亡，提示Pinl可能成为治疗宫颈癌的新靶点。

Impact of Pinl inhibition by siRNA on proliferation and apoptosis in human cervicalcancer cell line SiHa

Objective：To investigate the inhibitory effect of Pinl small interference RNA （siRNA） on the expression of Pinl gene in cervical caner cells SiHa and observe the effects of Pinl gene silencing on proliferation and apoptosis in SiHa cell. Methods ： A vector pGPU6/GFP/Neo-Pinl for transcribing siRNA targeting Pinl gene was constructed and intro- duced into cervical cancer ceils SiHa by Lipofectamine 2000. Three groups were designed：the experimental group transfected with pGPU6/GFP/Neo-Pinl （SiHa/pGPU6-Pinl siRNA）, the negative control group transfected with pGPU6/GFP/Neo （SiHa/pGPU6-Con） and the non- transfected group （SiHa）. The Pinl mRNA and protein expression levels of SiHa cells were de- tected by RT-PCR and Western blot respectively. The proliferation activity of SiHa cells was ex- amined by MTT assay. The apoptosis was studied by TUNEL assay and FCM. Results ：The vec- tor of RNA interference was successfully constructed. Pinl was over expressed in SiHa cells andthe expression of Pinl gene was inhibited significantly in SiHa cells after transfection for 48h. The mRNA and protein expression levels of Pinl in SiHa/pGPU6-Pinl siRNA group were all significantly lower than those in other two control groups （ P〈0.05 ）. The interference rate of Pinl mRNA and protein were 68.1% and 54.8% respectively. The proliferation activity of cells in SiHa/pGPU6-Pinl siRNA group was also suppressed obviously than that in SiHa/pGPU6- Con and SiHa groups （P〈0.05）. The apoptosis index were significantly higher in SiHafpG- PU6-Pinl siRNA group [ （29.6± 14.58） % ] than those in other groups （ P〈0.05 ）. The apop- tosis rate detected by FCM was significantly higher in SiHa/pGPU6-Pinl siRNA group [ （30.95 ±16.47）%] than in SiHa/pGPU6-Con group[ （6. 18±6. 10）%] and SiHa group[ （5.95±4.99 ） % ] （P〈0.05）. Conclusions ：The specific small interference RNA targeting Pinl mRNA can specifically and efficiently inhibit Pinl expression, downregulation of Pinl can suppresse proliferation of cervical cancer cells and promote the apoptosis, which suggests Pinl may be- come a new target in the treatment of cervical cancer.