过表达PDCD4逆转胃癌顺铂耐药的初步机制  

Preliminary mechanism study on overexpression of PDCD4 in reversing cisplatin resistance in gastric cancer

作　　者：柳丹 汤志明 赵红艳[1] 柯镜 白磊 牛成群 金佳晴 武福云[1] 李珊[1] LIU Dan;TANG Zhi-ming;ZHAO Hong-yan;KE Jing;BAI Lei;NIU Cheng-qun;JIN Jia-qing;WU Fu-yun;LI Shan(Institute of Basic Medical Sciences,Hubei University of Medicine,Shiyan 442000,Hubei,China)

机构地区：[1]湖北医药学院基础医学研究所,十堰医学硕士研究生442000

出　　处：《医学研究生学报》2019年第1期51-57,共7页Journal of Medical Postgraduates

基　　金：国家自然科学基金(81502637);湖北省自然科学基金(2018CFB467)

摘　　要：目的程序性细胞死亡因子(PDCD4)是近年来新发现的一类促凋亡基因,文中主要研究PDCD4逆转胃癌顺铂耐药的分子机制,为胃癌的耐药机制研究和治疗提供新的基因靶点。方法通过在人胃癌细胞系SGC7901/DDP中稳定过表达PDCD4基因,实验分为对照组(仅转染p CMV质粒),高表达组(仅转染p CMV-PDCD4质粒),对照加药组(转染p CMV质粒并加入顺铂处理),高表达加药组(转染p CMV-PDCD4质粒并加入顺铂处理),进行细胞免疫荧光﹑流式细胞术、RT-PCR和Western blot实验。实验分为空载加药组(转染p CMV质粒并加入顺铂处理)﹑PDCD4加药组(转染p CMV-PDCD4质粒并加入顺铂处理)和PDCD4加激活剂组(转染p CMV-PDCD4质粒,先加入SC79再加入顺铂处理)进行p Akt回补实验。结果高表达组PDCD4蛋白和mRNA表达较对照组明显升高(P<0.05),高表达加药组PDCD4蛋白和mRNA表达较对照加药组明显升高(P<0.05)。流式细胞术检测细胞早期凋亡水平显示,高表达加药组顺铂诱导的细胞早期凋亡率(25.22%)较对照加药组(21.08%)显著增高(P<0.05)。通过Western blot法表明,相较于对照加药组,高表达加药组中p Akt、p GSK3β和BCL-2的表达量均有明显降低(0.73±0.06 vs 0.42±0.04、0.88±0.05 vs 0.53±0.08、1.15±0.13 vs 0.84±0.14),差异均有统计学意义(P<0.05);而相较于对照加药组,高表达加药组中促凋亡蛋白BAK的表达水平则明显升高(1.28±0.09 vs 1.48±0.09),差异有统计学意义(P<0.05)。PDCD4加药组p Akt、p GSK3β表达较空载加药组、PDCD4加激活剂组明显降低(P<0.05),PARP(C)表达较空载加药组、PDCD4加激活剂组明显升高(P<0.05)。结论过表达PDCD4能够通过p Akt/p GSK3β促进顺铂引起的胃癌细胞凋亡,有利于逆转细胞顺铂耐药的形成。Objective Gastric cancer is the most common cancer in the world.In China,Patients with gastric cancer are mostly treated with platinum-based chemotherapy.Programmed cell death 4(PDCD4)was found as an important proapoptosis recently,the aim of the present study was to investigate the role of PDCD4 reversed the apoptosis induced by cisplatin in gastric cancer cell.The study will provide the target marker for treatment and diagnosis of cisplatin resistance in gastric cancer.Methods Stable transfection with pCMV-PDCD4 vector into human cisplatin resistance gastric cancer cell line-SGC7901/DDP;the cells were divided into control group,over-expression group,control with cisplatin group,over-expression with cisplatin group for following experiments.Hoechst dying with immunofluorescence and flow cytometry were used to measure the cell apoptosis in vitro;Real-time PCR was used to detect the mRNA expression levels of PDCD4,and the protein levels of PDCD4,pAK,pGSK3β,BCL-2 and Bak were detected by Western blot.The cells were divided into vector group,PDCD4 group,PDCD4 with activator group for detect the level of PARP(C)by Western blot.Results Compared with control group,the results of real-time PCR and western blot were showed the level of PDCD4 was augmented in over-expression group(also in the over-expression with cisplatin group),which was indicated stable transfection with PDCD4 was successful.Immunofluorescence(with hoechst dying)and flow cytometry demonstrated that PDCD4 facilitated cell apoptosis exposed to cisplatin.PDCD4 overexpression attenuated the protein levels of pAkt,pGSK3βand BCL-2,but increased the protein levels of BAK.Furthermore,incubation with SC-79(the activator of Akt)reversed cell apoptosis induced by PDCD4.Conclusion Overexpression of PDCD4 promotes the apoptosis induced by cisplatin through pAKT/pGSK3β pathway,which is favorable to reverse cisplatin resistance in gastric cancer.

关 键 词：程序性细胞死亡因子 AKT 糖原合酶激酶3Β 顺铂耐药 

分 类 号：R730.2[医药卫生—肿瘤]