摘要
目的:研究核内微小RNA-199b-5p(miR-199b-5p)对心肌细胞肥大的调控作用及其机制。方法:利用RT-qPCR检测健康人与心衰患者心肌组织中miR-199b-5p的表达水平。通过核质分离及RT-qPCR技术确定miR-199b-5p在人心肌AC16细胞中的核质分布。原代分离培养C57BL/6乳小鼠心室肌细胞(NMVCs),转染miR-199b-5p mimic,利用RT-qPCR和Western blot检测心房钠尿肽(ANP)、MYH7/β-肌球蛋白重链(β-MHC)和细胞周期蛋白依赖性激酶9(CDK9)表达。敲减NMVCs中CDK9基因表达,检测其对miR-199b-5p诱导的心肌细胞肥大相关基因表达的影响。双萤光素酶报告基因实验确定miR-199b-5p对CDK9基因的转录激活作用及与其启动子区的结合作用。通过ChIP-PCR实验验证miR-199b-5p通过募集RNA聚合酶Ⅱ(PolⅡ)和p300参与CDK9基因的转录激活。结果:RT-qPCR结果显示心衰患者心肌中miR-199b-5p表达增加。核质分离和RT-qPCR结果显示人心肌AC16细胞中miR-199b-5p在胞核中富集存在。转染miR-199b-5p mimic能显著提高NMVCs中ANP、MYH7/β-MHC和CDK9表达。抑制CDK9表达可逆转miR-199b-5p的促心肌细胞肥大作用。双萤光素酶报告基因实验显示miR-199b-5p可增加CDK9基因的转录激活,miR-199b-5p与CDK9基因启动子区存在特异的结合位点。ChIP-PCR实验显示miR-199b-5p能募集PolⅡ和p300到CDK9基因启动子区,参与CDK9基因的转录激活。结论:核内miR-199b-5p通过转录激活CDK9基因发挥促进心肌细胞肥大作用。
AIM:To investigate the effect of nuclear microRNA-199b-5p(miR-199b-5p)on myocardial hypertrophy and the potential mechanism.METHODS:RT-qPCR was used to detect the expression of miR-199b-5p in the myocardium of heart failure(HF)patients and the healthy controls.The nucleocytoplasmic distribution of miR-199b-5p in human cardiac AC16 cells was determined by nucleocytoplasmic separation and RT-qPCR.miR-199b-5p mimic was transfected into neonatal mouse ventricular cardiomyocytes(NMVCs),and the expression of atrial natriuretic peptide(ANP),MYH7/β-myosin heavy chain(β-MHC)and cyclin-dependent kinase 9(CDK9)was detected by RT-qPCR and Western blot.The expression of CDK9 gene in the NMVCs was knocked down to detect its effect on the expression of hypertrophyrelated genes induced by miR-199b-5p.The transcriptional activation of CDK9 gene by miR-199b-5p and the binding of miR-199b-5p on the promoter of CDK9 gene were determined by dual-luciferase reporter assay.ChIP-PCR was performed to confirm the effect of miR-199b-5p on recruitment of RNA polymerase Ⅱ(Pol Ⅱ)and p300 to participate in the transcription activation of CDK9 gene.RESULTS:The expression of miR-199b-5p was increased in the myocardium of HF patients.The results of nucleocytoplasmic separation and RT-qPCR showed that miR-199b-5p was enriched in the nucleus.The expression of ANP,MYH7/β-MHC and CDK9 was significantly increased in the NMVCs transfected with miR-199b-5p mimic.Knock-down of CDK9 gene expression reversed the effect of miR-199b-5p on cardiomyocyte hypertrophy.Dualluciferase reporter assay showed that miR-199b-5p increased the transcription activation of CDK9 gene and there was a specific binding site for miR-199b-5p in CDK9 gene promoter.The results of ChIP-PCR showed that miR-199b-5p recruited Pol Ⅱ and p300 to CDK9 gene promoter region to enhance the transcription activation of CDK9 gene.CONCLUSION:miR-199b-5p promotes cardiomyocyte hypertrophy by up-regulating CDK9 expression.
作者
易芷瑶
赵安职
张铭
曾妮
朱杰宁
杨莹
严钰敏
郭惠明
单志新
YI Zhi-yao;ZHAO An-zhi;ZHANG Ming;ZENG Ni;ZHU Jie-ning;YANG Ying;YAN Yu-min;GUO Hui-ming;SHAN Zhi-xin(School of Biology and Biological Engineering,South China University of Technology,Guangzhou 510006,China;School of Pharmacy,Southern Medical University,Guangzhou 510515,China;Guangdong Provincial Key Laboratory of Clinical Pharmacology,Guangdong Provincial People's Hospital,Guangdong Academy of Medical Sciences,Guangzhou 510080,China;School of Medicine,South China University of Technology,Guangzhou 510006,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2021年第2期193-201,共9页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81770264,No.82070254)
广东省医学科研基金资助项目(No.B2020215)
广州市科技计划项目(No.202002030039)。
