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

 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。

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

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 shRNA-mediated 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.