RNA干扰MEX3A基因对膀胱癌细胞增殖和凋亡的影响

Effects of RNA interference targeting MEX3A gene on proliferation and apoptosis of human bladder cancer cells

目的探讨通过慢病毒介导的RNA干扰技术抑制MEX3A基因表达对膀胱癌细胞增殖和凋亡的影响。方法应用GV115载体构建针对MEX3A基因的shRNA慢病毒载体,转染包装细胞293T产生慢病毒颗粒,收集并滴度测定后转染5637膀胱癌细胞,实验组转染MEX3A-shRNA慢病毒,对照组转染阴性对照慢病毒;实时定量PCR(Real-time PCR)检测MEX3A基因敲减效率;慢病毒转染3 d后,使用Celigo连续细胞计数5d检测细胞生长;慢病毒转染5d后,进行膜联蛋白V(Annexin V)染色并用流式细胞术检测细胞凋亡。结果成功构建MEX3AshRNA慢病毒载体以及稳定抑制MEX3A表达的细胞株,MEX3A基因敲减效率达到74%;Celigo细胞计数检测显示,与对照组相比,实验组5637细胞的增殖速率受到显著抑制;流式细胞术检测显示,实验组发生凋亡的5637细胞显著增加。结论 MEX3A基因促进膀胱癌细胞的增殖,抑制膀胱癌细胞的凋亡。

Objective To investigate the effect of inhibition of MEX3A gene expression on proliferation and apoptosis of bladder cancer ceils by lentiviral-mediated RNA interference technology. Methods The shRNA lentiviral vector targeting MEX3A gene was constructed using GV115 vector and transfected into the packaging cells 293T to produce lentivirus particles. The particles were collected and transfeeted into 5637 bladder cancer after titer determination. The experimental group transfected MEX3A-shRNA lentivirus, and the control group transfected negative control lentivirus. Knock down efficiency of MEX3A gene was detected by Real-time PCR. Three days after lentiviral transfeetion, cell growth was measured by cell count for five consecutive days using the Celigo. Five days after lentiviral transfeetion, Annexin V staining was performed and apoptosis was detected by flow eytometry. Results The MEX3A-shRNA lentiviral vector was successfully constructed and a bladder cancer cell line that MEX3A gene stably suppressed was established. The knockout efficiency of MEX3A gene reached 74%. Celigo cell count deteetion showed that the proliferation rate of the experimental group 5637 eells was significantly inhibited compared with the control group. Flow cytometry showed that the apoptosis of 5637 cells increased significantly in the experimental group. Conclusion MEX3A gene can promote the proliferation of bladder cancer cells and inhibit the apoptosis of bladder cancer ceils.