干扰CTPS基因促进川楝素诱导的人胃癌MKN-45细胞凋亡  

Interference of CTPS gene promotes toosendanin-induced apoptosis of human gastric cancer MKN-45 cells

作　　者：罗海静 潘阳 陈雯 张伟伟[1,2] 邵淑丽 杨清竹[1,2] 李铁 LUO Haijing;PAN Yang;CHEN Wen;ZHANG Weiwei;SHAO Shuli;YANG Qingzhu;LI Tie(College of Life Science and Agroforestry,Qiqihar University,Qiqihar 161006,China;Key Laboratory of Resistant Genetic Engineering and Cold Biodiversity Conservation of Heilongjiang Province,Qiqihar 161006,China)

机构地区：[1]齐齐哈尔大学生命科学与农林学院,黑龙江齐齐哈尔161006 [2]抗性基因工程与寒地生物多样性保护黑龙江省重点实验室,黑龙江齐齐哈尔161006

出　　处：《南方医科大学学报》2022年第8期1126-1133,共8页Journal of Southern Medical University

基　　金：国家青年科学基金(31801148);黑龙江省自然科学基金(LH2021C099);黑龙江省教育厅基本业务专项(135109255);黑龙江省省属高等学校基本科研业务费科研项目(植物性食品加工技术特色学科专项)(YSTSXK201874);2020年齐齐哈尔大学研究生创新科研项目(YJSCX2020045)。

摘　　要：目的探讨干扰CTPS基因对川楝素诱导的胃癌MKN-45细胞凋亡的影响。方法通过生物信息学分析CTPS基因在人胃癌组织中的表达情况以及CTPS基因高表达的胃癌患者的总生存期。以人胃癌MKN-45细胞为实验材料,构建CTPS基因干扰载体,转染MKN-45细胞48 h后,采用qRT-PCR和Western blot检测CTPS基因mRNA和蛋白表达情况。用80 nmol/L的川楝素处理干扰CTPS基因的MKN-45细胞48 h,使用MTT实验法检测细胞存活率,激光共聚焦显微镜观察细胞形态,免疫荧光法检测γH2AX的表达。结果生物信息学分析发现CTPS在人胃癌组织中高表达,CTPS基因高表达的胃癌患者总生存期短。向MKN-45细胞中分别转染Sh-ctrl空载体和Sh-CTPS干扰载体,qRT-PCR和Western blot检测显示,与转染Sh-ctrl空载体相比,转染Sh-CTPS干扰载体的MKN-45细胞中CTPS mRNA和蛋白表达均降低,其中转染ShCTPS-3干扰效果最显著,分别降低了85.21%和53%(P<0.001)。检测细胞存活率及形态变化显示,与Sh-ctrl组相比,ShCTPS-3组细胞存活率降低(P<0.01),细胞皱缩,形态不规则,呈现凋亡特征,细胞凋亡率升高(P<0.05);在此基础上用80 nmol/L的川楝素处理转染Sh-ctrl和ShCTPS-3的MKN-45细胞48 h,与Sh-ctrl+川楝素组相比,ShCTPS-3+川楝素组细胞存活率降低(P<0.001),细胞出现凋亡小体,细胞凋亡率升高(P<0.05)。结论干扰CTPS基因促进川楝素诱导的胃癌MKN-45细胞凋亡。Objective To investigate the effect of interference of CTPS gene on toosendanin-induced apoptosis of gastric cancer MKN-45 cells.Methods Bioinformatic analysis was used to analyze CTPS gene expression in human gastric cancer tissues and the overall survival of gastric cancer patients with high CTPS gene expression.Human gastric cancer MKN-45 cells were transfected with a short hairpin interfering RNA targeting CTPS gene,and 48 h later,qRT-PCR and Western blotting were used to detect cellular expression CTPS at both the m RNA and protein levels.MKN-45 cells with CTPS knockdown were treated with 80 nmol/L toosendanin for 48 h,and the cell viability was assessed with MTT assay;the cell morphology was observed using laser confocal microscope,and the expression ofγH2AX was detected with immunofluorescence assay.Results Bioinformatic analysis suggested that CTPS was highly expressed in human gastric cancer tissues,and gastric cancer patients with high CTPS gene expression had a shorter overall survival.MKN-45 cells transfected with Sh-CTPS interference vector showed significantly lowered cell survival rate(P<0.01)with obvious cell shrinkage,irregular morphology,typical apoptotic changes,and increased cell apoptosis rate(P<0.05).Treatment of the transfected cells with 80 nmol/L toosendanin for 48 h resulted in further reduction of the cell survival rate(P<0.001),and the cells showed an increased apoptotic rate(P<0.05)with appearance of apoptotic bodies.Conclusion Interference of CTPS gene can promote TSN-induced apoptosis of gastric cancer MKN-45 cells.

关 键 词：CTPS基因 川楝素 胃癌 MKN-45细胞 

分 类 号：R285[医药卫生—中药学]