CRP基因沉默后心肌细胞系H9c2的miR-21及其靶基因表达、细胞活力观察  

Observation of miR-21 and its target gene expression and cell viability in H9c2 cardiomyocytes after silencing CRP gene

作　　者：程玲慧 余丽霞 叶凤英 权磊[1] 谭文 CHENG Linghui;YU Lixia;YE Fengying;QUAN Lei;TAN Wen(School of Biology and Biological Engineering of South China University of Technology, Guangzhou 510006,China)

机构地区：[1]华南理工大学生物科学与工程学院,广州510006 [2]华南理工大学医学院 [3]广东工业大学生物医药研究院

出　　处：《山东医药》2018年第40期1-5,共5页Shandong Medical Journal

基　　金：广东省自然科学基金资助项目(22016A030313467);广东省社会发展领域新药创制重大项目(2014B020210001)

摘　　要：目的观察沉默炎症因子CRP基因对H9c2心肌细胞活力的影响,并探讨其机制。方法 (1)取H9c2心肌细胞,随机分为CRP沉默组和对照组。CRP沉默组转染对CRP特异性沉默的p LV-shRNA-CRP慢病毒,对照组转染无敲减作用的p LV-shRNA-Scramble慢病毒。筛选与鉴定慢病毒载体成功转染H9c2心肌细胞。转染48 h后,采用实时定量PCR法观察两组H9c2心肌细胞CRP基因的沉默效率、miR-21及PDCD4、PTEN、Spry1 mRNA。(2)取H9c2心肌细胞,随机分为空白组、对照组和CRP沉默组,空白组不转染病毒加入等量的DMEM培养基,对照组转染p LV-shRNA-Scramble慢病毒颗粒,CRP沉默组转染p LV-shRNA-CRP慢病毒颗粒。转染24 h后,采用MTT法检测三组H9c2心肌细胞活力。结果 (1)CRP沉默组和对照组H9c2心肌细胞中CRP mRNA相对表达量分别为0. 320±0. 069、1±0. 090,miR-21相对表达量分别为0. 740±0. 099、0. 280±0. 010,两组相比P均<0. 01。CRP沉默组PDCD4、PTEN和Spry1 mRNA表达均低于对照组(P均<0. 01)。(2)CRP沉默组、对照组和空白组细胞活力分别为0. 78±0. 030、0. 66±0. 010、1. 00±0. 013;与空白组相比,CRP沉默组和对照组细胞活力均下降(P均<0. 01);与对照组相比,CRP沉默组细胞活力上升(P <0. 05)。结论沉默CRP基因可促进H9c2心肌细胞存活,这可能与其可增加H9c2心肌细胞中miR-21表达,抑制PDCD4、PTEN和Spry1的基因表达有关。Objective To observe the effect of silencing C-reactive protein(CRP)gene on the viability of H9c2 cardiomyocytes,and to explore its mechanism.Methods ①H9c2 cardiomyocytes were randomly divided into the CRP silencing group and control group.We screened and identified H9c2 cardiomyocytes with the lentiviral vector.After 48 h of transfection,the silencing efficiency,and the expression levels of miR-21,PDCD4,PTEN and Spry1 mRNA of H9c2 cardiomyocytes were detected by real-time quantitative PCR.②H9c2 cardiomyocytes were randomly divided into the blank group,control group and CRP silence group.The cells in the blank group were not transfected with virus but were added with equal amount of DMEM medium.The cells in the control group were transfected with pLV-shRNA-Scramble lentiviral particles,and the cells in the CRP silencing group were transfected with pLV-shRNA-CRP lentiviral particles.After 24 h of transfection,the viability of three groups was detected by MTT assay.Results ①The relative expression levels of CRP mRNA in the CRP silencing group and the control group were 0.320±0.069 and 1±0.090,respectively.The relative expression levels of miR-21 were 0.740±0.099 and 0.280±0.010,respectively.The expression levels of PDCD4,PTEN,and Spry1 mRNA in the CRP group were lower than those in the control group(all P<0.01).②The cell viability in the CRP silencing group,control group and blank group was 0.78±0.030,0.66±0.010,and 1.00±0.013,respectively.Compared with the blank group,the cell viability decreased in the CRP silencing group and the control group(P<0.01).Compared with the control group,the cell viability in the CRP silencing group increased(P<0.05).Conclusion Silencing CRP gene can promote the survival of H9c2 cardiomyocytes,which may be related to the increase of miR-21 expression in H9c2 cardiomyocytes and the inhibition of PDCD4,PTEN and Spry1 gene expression.

关 键 词：缺血性心脏病 心肌细胞 微小RNA-21 PDCD4基因 PTEN基因 Spry1基因 基因沉默 C反应蛋白 

分 类 号：R541[医药卫生—心血管疾病]