摘要
目的观察微小RNA(miR)-1973靶向细胞因子信号转导抑制因子2(SOCS2)对乳腺癌细胞增殖、凋亡及侵袭迁移的影响。方法体外培养BT-483细胞,对其转染并分为空白对照组(NG组)、阴性转染组(NC组)、抑制miR-1973表达组(miR-1973-inhibitor组)。倒置荧光显微镜下观察转染效果;采用实时荧光定量聚合酶链反应(PCR)法检测miR-1973水平;噻唑蓝(MTT)法、流式细胞仪检测分别检测各组细胞增殖、凋亡;划痕实验检测BT-483细胞迁移能力;Transwell实验检测细胞侵袭能力;免疫印迹法检测人增殖细胞核抗原(Ki-67)、凋亡相关蛋白B细胞淋巴瘤/白血病-2(bcl-2)、bcl-2相关X蛋白(bax)、侵袭迁移相关蛋白E-钙黏附蛋白(E-cadherin)、N-钙黏附蛋白(N-cadherin)、波形蛋白(Vimentin)及SOCS2蛋白表达;应用TargetScan数据库预测miR-1973与SOCS2的靶向关系并用双荧光素酶报告基因实验加以验证。结果BT-483细胞转染成功;抑制miR-1973表达后,与NG组[(12.78±2.07)%、(17.62±2.65)%]、NC组[(13.77±2.06)%、(18.53±2.77)%]比较,24、48 h后miR-1973-inhibitor组BT-483细胞增殖抑制率均显著升高[(18.69±2.80)%、(45.55±13.61)%,F=11.018、22.673,P<0.05];与NG组BT-483细胞凋亡率、划痕治愈率及细胞侵袭数[(11.03±1.65)%、(48.25±7.23)%、(237.53±35.63)个]、NC组[(12.12±1.82)%、(46.73±7.01)%、(228.98±34.35)个]比较,miR-1973-inhibitor组BT-483细胞凋亡率[(26.68±4.00)%]显著升高(F=62.370,P<0.05),划痕治愈率[(22.77±3.42)%]、细胞侵袭数[(115.77±17.37)个]显著降低(F=32.507,30.222,P<0.05)。与NG组、NC组比较,miR-1973-inhibitor组BT-483细胞中bax、E-cadherin蛋白表达显著升高(F=32.918、28.574,P<0.05),Ki-67、bcl-2、N-cadherin、Vimentin蛋白表达显著减少或降低(F=8.805、15.085、37.065、15.097,P<0.05)。TargetScan数据库预测显示SOCS2是miR-1973的潜在靶基因,双荧光素酶报告基因实验证实二者存在靶向关系,且miR-1973可负向调控SOCS2表达。结论沉默miR-197
Objective To observe the effect of microRNA(miR)-1973 targeting suppressor of cytokine signaling 2(SOCS2)on proliferation,apoptosis,invasion and migration of breast cancer cells.Methods BT-483 cells were cultured in vitro,transfected and divided into blank control group(NG group),negative transfection group(NC group)and inhibition of miR-1973 expression group(miR-1973 inhibitor)group.The transfection efficacy was observed under inverted fluorescence microscope.The level of miR-1973 was detected by real-time fluorescence quantitative polymerase chain reaction(PCR).The cell proliferation,apoptosis,migration ability and invasion ability were detected by MTT assay,flow cytometry,scratch test and Transwell assay respectively.The expression of human proliferating cell nuclear antigen(Ki-67),B-cell lymphoma-2(bcl-2),bcl-2-associated protein X(bax),E-cadherin,N-cadherin,Vimentin and SOCS2 was detected by Western blotting.The targeting relationship between miR-1973 and SOCS2 was predicted by TargetScan database and verified by double luciferase reporter gene experiment.Results BT-483 cells were successfully transfected.After miR-1973 expression was inhibited,the inhibition rate of BT-483 cell proliferation in miR-1973-inhibitor group[(18.69±2.80)%,(45.55±13.61)%]was significantly higher than that in NG group[(12.78±2.07)%,(17.62±2.65)%]and NC group[(13.77±2.06)%,(18.53±2.77)%](F=11.018,22.673,P<0.05)after 24 h and 48 h.As compared with those in NG group[(11.03±1.65)%,(48.25±7.23)%,(237.53±35.63)]and NC group[(12.12±1.82)%,(46.73±7.01)%,(228.98±34.35)],the apoptosis rate of BT-483 cells in miR-1973-inhibitor group[(26.68±4.00)%]were significantly increased(F=62.370,P<0.05),the wound healing rate[(22.77±3.42)%]was significantly reduced,the number of invasive cells[(115.77±17.37)]was significantly decreased(F=32.507,30.222,P<0.05).As compared with those in NG group and NC group,the protein expression of bax and E-cadherin in BT-483 cells in miR-1973-inhibitor group was significantly increased(F=32.918,28.574,P
作者
仲颖
孙志鹏
何旋
唐葶婷
秦鑫锞
Zhong Ying;Sun Zhipeng;He Xuan;Tang Tingting;Qin Xinke(Department of Operating Room Surgery,Wuhan Children's Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology,Wuhan 430000,China;Department of Anesthesiology,Wuhan Children's Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology,Wuhan 430000,China;Department of Anesthesiology,Renmin Hospital of Wuhan University,Wuhan 430600,China;Department of General Surgery,Wuhan Maternal and Child Health Hospital,Wuhan 430015,China)
出处
《中华实验外科杂志》
CAS
北大核心
2021年第8期1479-1483,共5页
Chinese Journal of Experimental Surgery
基金
湖北省科技计划项目(2018CFC856)。
关键词
微小RNA
乳腺癌
增殖
凋亡
侵袭
迁移
MicroRNA
Breast cancer
Proliferation
Apoptosis
Invasion
Migration
