摘要
目的观察炎症环境下微小RNA(miRNA,miR)-613在CHON-001细胞中的变化及其对增殖和凋亡的影响。方法CHON-1细胞购自中国科学院上海细胞库。研究分实验1、2,实验1分为白细胞介素(IL)-1β刺激前0 h组、刺激后3 h组、6 h组、12 h组及24 h组,各组应用实时定量反转录聚合酶链反应(RT-qPCR)检测CHON-001细胞内miR-613的表达。实验2分对照组(不做任何处理)、IL-1β组(仅IL-1β刺激)、IL-1β+miR-613组(IL-1β刺激且转染miR-613激动剂)、miR-613组(仅转染miR-613激动剂);应用细胞计数试剂盒(CCK-8)检测miR-613对CHON-001细胞增殖的影响;应用蛋白质印迹法(Western blot)检测miR-613对CHON-001细胞凋亡的影响。多组间比较采用单因素方差分析(One way-ANOVA)。结果实验1中,0 h组miR-613的相对表达量是0.99±0.06,miR-613在3 h组(0.67±0.12比0.99±0.06,F=8.124,P<0.01),6 h组(0.47±0.07比0.99±0.06,F=12.257,P<0.01),12 h组(0.34±0.10比0.99±0.06,F=17.941,P<0.01)和24 h组(0.19±0.04比0.99±0.06,F=26.899,P<0.01)的相对表达量均较0 h组显著降低。实验2中,CCK-8显示,IL-1β组的细胞增殖率(0.51±0.03)较对照组(1.00±0.01)显著降低(F=16.243,P<0.01);IL-1β+miR-613组的细胞增殖率(0.79±0.02)较IL-1β组(0.51±0.03)显著升高(F=8.257,P<0.01)。Western blot显示,B细胞淋巴瘤/白血病-2相关X蛋白(bax)蛋白在IL-1β组的相对表达量(0.44±0.05)较对照组(0.13±0.02)显著升高(F=11.481,P<0.01);bax蛋白在IL-1β+miR-613组的相对表达量(0.28±0.06)较IL-1β组(0.44±0.05)显著下降(F=7.423,P<0.01)。Active Caspase-3蛋白在IL-1β组的相对表达量(0.41±0.02)较对照组(0.08±0.01)显著升高(F=12.151,P<0.01);Active Caspase-3蛋白在IL-1β+miR-613组的相对表达量(0.23±0.02)较IL-1β组(0.41±0.02)显著下降(F=9.246,P<0.01)。B细胞淋巴瘤/白血病-2(bcl-2)蛋白在IL-1β组的相对表达量(0.22±0.02)较对照组(0.63±0.02)显著下降(F=20.126,P<0.01);bcl-2蛋白在IL-1β+miR-613组的相对表达量(0.45±0.03)较IL-
Objective To investigate the changes of microrRNA(miRNA,miR)-613 in chondrogenic CHON-001 cell in an inflammatory environment and its effects on proliferation and apoptosis of chondrogenic CHON-001 cell.Methods CHON-001 cells were purchased from Chinese Academy of Sciences(Shanghai).The research is divided into experiments 1 and 2,Experiment 1 was divided into five groups:0 h group,3 h group,6 h group,12 h group and 24 h group.Real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)was used to detect the expression of miR-613 in CHON-001 cell.Experiment 2 was divided into four groups:control group(no treatment),interleukin(IL)-1 group(IL-1 stimulation),IL-1+miR-613 group(IL-1 stimulation and transfection with miR-613 agonist),and miR-613 group(transfection with miR-613 agonist).Cell counting kit-8(CCK-8)was used to detect the effect of miR-613 on the proliferation of chondrogenic CHON-001 cells.Western blotting was used to detect the effect of miR-613 on apoptosis of chondrogenic CHON-001 cells.Results In experiment 1,the relative expression of miR-613 in the 0 h group was 0.99±0.06,the relative expression of miR-613 in the 3 h group(0.67±0.12 vs.0.99±0.06,F=8.124,P<0.01),in the 6 h group(0.47±0.07 vs.0.99±0.06,F=12.257,P<0.01),and in the 12 h group(0.34±0.10 vs.0.99±0.06,F=17.941,P<0.01)and in the 24 h group(0.19±0.04 vs.0.99±0.06,F=26.899,P<0.01)was significantly lower than that of 0 h group.In experiment 2,CCK-8 showed that the cell proliferation rate of IL-1 group(0.51±0.03)was significantly lower than that of the control group(1.00±0.01,F=16.243,P<0.01).The cell proliferation rate(0.79±0.02)in the IL-1+miR-613 group was significantly higher than that in the IL-1 group(0.51±0.03,F=8.257,P<0.01).Western blotting showed that the relative expression of bax protein in IL-1 group(0.44±0.05)was significantly higher than that in the control group(0.13±0.02,F=11.481,P<0.01).The relative expression of bax protein in the IL-1+miR-613 group(0.28±0.06)was significantly lower than th
作者
张卫红
曾源
刘振康
朱旭
孙金鹏
李建强
肖鹏
Zhang Weihong;Zeng Yuan;Liu Zhenkang;Zhu Xu;Sun Jinpeng;Li Jianqiang;Xiao Peng(Department of Orthopeadics,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2020年第9期1688-1690,共3页
Chinese Journal of Experimental Surgery
