摘要
目的探讨沉默钙连蛋白(Calnexin)基因对胃癌细胞SGC-7901内质网应激凋亡信号通路的影响。方法构建靶向Calnexin基因的慢病毒干扰载体,利用包装细胞293T获得重组慢病毒,转染人胃癌SGC-7901细胞,并分为转染shRNA-Calnexin的实验组,转染shRNA空白质粒的空白载体组,并将未转染的SGC-7901细胞作为对照组。应用RT-PCR技术检测靶向沉默Calnexin的表达,应用四甲基偶氮唑蓝(MTT)检测各组细胞增殖情况;流式细胞术检测各组细胞凋亡情况;Western blot检测GRP94、IRE1、ATF6及CHOP表达水平。结果转染sh RNA沉默Calnexin基因可明显下调Calnexin mRNA的表达,实验组与对照组比较差异有统计学意义(P<0.05);MTT法检测实验组细胞转染后细胞增殖能力明显下降(P<0.05);沉默Calnexin基因可提高细胞凋亡率,转染72h后实验组与对照组比较,差异有统计学意义(P<0.05);沉默Calnexin基因使胃癌细胞中GRP94、IRE1、ATF6及CHOP蛋白表达水平明显升高(均P<0.05)。结论靶向沉默Calnexin基因抑制人胃癌细胞增殖和促进细胞凋亡可能是通过抑制内质网应激信号通路实现的。
Objective To investigate the effects of calnexin gene silencing on endoplasmic reticulum stress-apoptosisand its signaling pathway in gastric cancer SGC-7901cells.Methods Lentiviral vectors for short hairpin RNAs targeting thecoding region of human calnexin gene were constructed.The recombinant lentiviral vectors were harvested from293T cells andwere used to transfect human gastric cancer SGC-7901cells.SGC-7901cells were transfected with shRNA-Calnexin(experimental group)or shRNA blank plasmid(blank vector group),and the non-transfected SGC-7901cells were used ascontrol group.Expression of calnexin mRNA in SGC-7901cells were detected by RT-PCR,cell proliferation was detected byMTT,cell apoptosis was detected by flow cytometry,expression of GRP94,IRE1,ATF6and CHOP were detected by Westernblot.Results The expression of Calnexin mRNA was down-regulated in experimental group compared to control group(P<0.05),and the cell proliferation was significantly decreased(P<0.05).The apoptosis rate was increased72h after transfection,compared with the control group(P<0.05).The expression of GRP94,IRE1,ATF6and CHOP was significantly increased inexperimental group(P<0.05).Conclusion Calnexin silencing can inhibit cell proliferation and induce apoptosis of gastric cancerSGC-7901cells probably by inhibiting endoplasmic reticulumstress signaling pathway.
出处
《浙江医学》
CAS
2017年第4期259-262,276,共5页
Zhejiang Medical Journal
基金
浙江省医药卫生科研项目(2013KYB076)
