摘要
目的观察肺癌组织miR-142-3p表达变化,探讨miR-142-3p靶向调控Rho相关结构域BTB蛋白质3(Rho-related BTB domain-containing protein 3,RHOBTB3)基因对肺癌A549细胞增殖能力的影响。方法肺癌患者35例,均行手术治疗,取手术切除癌组织及癌旁组织,采用实时荧光定量PCR法检测miR-142-3p及RHOBTB3 mRNA相对表达量。将对数生长期A549细胞分为miR-142-3p组和对照组,分别转染表达miR-142-3p的慢病毒和阴性对照慢病毒,转染48 h时采用实时荧光定量PCR法检测miR-142-3p相对表达量。筛选出稳定转染的miR-142-3p组A549细胞分为稳转miR-142-3p组和miR-142-3p+RHOBTB3组,稳定转染的对照组A549细胞分为稳转对照组和RHOBTB3组,miR-142-3p+RHOBTB3组、RHOBTB3组采用脂质体转染法转染表达RHOBTB3的质粒,稳转miR-142-3p组、稳转对照组不做处理。转染48 h, 4组采用Western blot法检测RHOBTB3蛋白相对表达量,采用CCK-8法检测培养96 h时细胞增殖率,采用细胞克隆形成实验检测细胞克隆形成数目。采用荧光素酶报告基因实验验证miR-142-3p靶向结合RHOBTB3基因。结果肺癌组织miR-142-3p相对表达量(0.58±0.27)低于癌旁组织(1.06±0.34)(t=-6.508,P<0.001),RHOBTB3 mRNA相对表达量(1.24±0.32)高于癌旁组织(0.51±0.29)(t=10.020,P<0.001)。miR-142-3p组细胞miR-142-3p相对表达量(9.45±1.80)高于对照组(1.05±0.32)(t=11.240,P<0.001)。稳转miR-142-3p组RHOBTB3蛋白相对表达量(0.47±0.05)、细胞增殖率[(48.56±9.97)%]及细胞克隆形成数目[(47.00±8.39)个]均低于稳转对照组[0.79±0.07、(100.00±8.71)%、(165.67±13.92)个]和miR-142-3p+RHOBTB3组[0.77±0.06、(95.82±9.79)%、(141.33±12.88)个](P<0.05),RHOBTB3组RHOBTB3蛋白相对表达量(1.20±0.12)、细胞增殖率[(153.23±18.29)%]及细胞克隆形成数目[(267.83±23.25)个]均高于miR-142-3p+RHOBTB3组(P<0.05)。荧光素酶报告基因实验结果显示,miR-142-3p+pMIR WT组荧光素酶活性(0.41±0.05)低于NC+pMIR WT组(1.01±0.12)(t=11.220,P<0.
Objective To observe the change of miR-142-3 p in lung cancer tissues and to investigate the influence of miR-142-3 p targeting the regulation of Rho-related BTB domain-containing protein 3(RHOBTB3) gene on the proliferation of lung cancer A549 cells. Methods Thirty-five patients with lung cancer were obtained the resected cancer tissues and paracancerous tissues. Real-time fluorescence quantitative PCR was used to detect the relative expressions of miR-142-3 p and RHOBTB3 mRNA in lung cancer tissues and paracancerous tissues. The A549 cells in logarithmic growth phase were transfected with lentivirus expressing miR-142-3 p(miR-142-3 p group) and control virus(control group). The relative expression of miR-142-3 p was detected by real-time fluorescence quantitative PCR 48 h after transfection. A549 cells in miR-142-3 p group stably transfected with lentivirus expressing miR-142-3 p were obtained and then divided into stably transfected miR-142-3 p group and miR-142-3 p+RHOBTB3 group. A549 cells in control group stably transfected with control virus were divided into stably transfected control group and RHOBTB3 group.The plasmid expressing RHOBTB3 was transfected into miR-142-3 p+ RHOBTB3 group and RHOBTB3 group by liposome transfection,and stably transfected miR-142-3 p group and control group were not treated.Western blot was used to detect the relative expression of RHOBTB3 protein in 4 groups,CCK-8 method was used to detect cell proliferation rate,and cell colony formation assay was used to detect the number of cell colonies.Luciferase reporter gene assay was used to verify the targeting binding of miR-142-3 p to the RHOBTB3 gene.Results The relative expression of miR-142-3 p was lower in lung cancer tissues(0.58±0.27)than that in paracancerous tissues(1.06±0.34)(t=-6.508,P<0.001).The relative expression of RHOBTB3 mRNA was higher in lung cancer tissues(1.24±0.32)than that in paracancerous tissues(0.51±0.29)(t=10.020,P<0.001).The relative expression of miR-142-3 p was higher in miR-142-3 p group(9.45±1.80
作者
万启飞
朱晓明
李基伟
姚文健
魏立
WAN Qi-fei;ZHU Xiao-ming;LI Ji-wei;YAO Wen-jian;WEI Li(Department of Thoracic Surgery,Zhengzhou University People’s Hospital,Henan Provincial People’s Hospital,Zhengzhou,Henan 450003,China)
出处
《中华实用诊断与治疗杂志》
2022年第3期237-241,共5页
Journal of Chinese Practical Diagnosis and Therapy
基金
河南省医学科技攻关计划联合共建项目(LHGJ20200021)。
