shRNA介导的hWAPL表达沉默对人宫颈癌CaSki细胞增殖与凋亡的影响  

Effects of shRNA-mediated hWAPL silencing on proliferation and apoptosis of human cervical cancer CaSki cells

作　　者：潘巍巍[1] 徐营[2] 曹利仙[2] 沈忠飞[2] 郭连军[2] 宋方洲[1] 

机构地区：[1]重庆医科大学生物化学与分子生物学教研室,重庆400016 [2]嘉兴学院医学院生物教研室,浙江嘉兴314001

出　　处：《中国病理生理杂志》2013年第7期1213-1218,共6页Chinese Journal of Pathophysiology

基　　金：浙江省自然科学基金资助项目(No.Y2110873;No.Y2110158);嘉兴市科技局科研项目(No.2008AY2039-3;No.2011AY1048-6)

摘　　要：目的:为探讨人半翼(hWAPL)蛋白在细胞增殖与凋亡中的作用及其作为分子靶点用于肿瘤治疗的潜在价值,本研究用RNA干扰技术抑制hWAPL基因的表达,并观察了其对人宫颈癌细胞CaSki细胞增殖和细胞凋亡的影响。方法:实时荧光定量PCR和Western blotting检测hWAPL shRNA的干扰效率;MTT检测细胞增殖;Annexin V-PE和Hoechst 33258染色检测细胞凋亡;蛋白质印迹分析凋亡蛋白cleaved caspase-3和细胞周期抑制蛋白p21、p27的表达。裸鼠荷瘤模型体内研究hWAPL沉默对CaSki细胞增殖的影响。结果:实时荧光定量PCR和Western blotting检测结果表明,筛选到的hWAPL shRNA能有效抑制内源hWAPL的表达。MTT实验表明shRNA介导的hWAPL表达沉默显著抑制CaSki细胞的增殖。Annexin V-PE分析结果表明,hWAPL沉默增加了凋亡细胞数目。细胞核经Hoechst 33258染色出现浓染致密的固缩形态和颗粒状荧光;蛋白质印迹结果显示cleaved caspase-3、p21和p27表达增加;裸鼠成瘤实验表明,hWAPL沉默的肿瘤体积减小,增殖细胞核抗原(PCNA)蛋白表达增加。结论:hWAPL沉默抑制CaSki细胞增殖,促进细胞凋亡,是一个潜在的肿瘤治疗新靶点。AIM： To investigate the role of human wings apart-like（hWAPL） protein in proliferation and apoptosis of human cervical cancer CaSki cells through hWAPL gene silencing by specific short hairpin RNA（shRNA） duplexes.METHODS： The relative hWAPL mRNA and protein expression levels were detected by real-time fluorescence quantitative PCR and Western blotting,respectively.Cell proliferation was detected by MTT assay,and the apoptosis was determined by Annexin V-PE and Hoechst 33258 staining.Western blotting was used to analyze the expression of cleaved caspase-3,p21 and p27.The effect of hWAPL gene silencing on the in vivo tumorigenic capacity of CaSki cells was investigated in a tumor-bearing nude mouse model.RESULTS： Real-time fluorescence quantitative PCR and Western blotting showed that hWAPL mRNA and protein expression in CaSki cells was efficiently inhibited by hWAPL shRNA.The shRNAmediated hWAPL silencing inhibited the proliferation and induced the apoptosis of CaSki cells.Additionally,the expression levels of cleaved caspase-3,p21 and p27 were up-regulated in hWAPL knockdown cells.Knockdown of hWAPL also inhibited the in vivo tumorigenic capacity of CaSki cells.CONCLUSION： hWAPL is involved in the regulation of the proliferation and apoptosis of CaSki cells in vitro and in vivo,and might serve as a therapeutic target in cancer treatment.

关 键 词：hWAPL蛋白 短发夹RNA 细胞凋亡 CASKI细胞 

分 类 号：R373.39[医药卫生—病原生物学]