摘要
[目的]研究环状RNA circ_0036167对心肌成纤维细胞纤维化表型的调控作用及可能机制。[方法]对心衰患者及器官捐献者心肌组织进行Masson三色染色以检测心肌胶原水平。实时荧光定量PCR(RT-qPCR)检测circ_0036167及其宿主MYO9A在心衰心肌中的表达。通过放线菌素D和RNase R处理实验鉴定人心肌细胞AC16中circ_0036167的RNA稳定性。利用重组circ_0036167腺病毒感染C57BL/6小鼠心肌成纤维细胞(mCF),在RNA和蛋白水平检测纤维化相关基因COL1A1、COL3A1和ACTA2的表达。利用EdU染色和Transwell细胞迁移实验鉴定circ_0036167对mCF增殖和迁移能力的影响。基于生物信息学预测和RNA结合蛋白免疫沉淀(RIP)实验验证circ_0036167与融合肉瘤蛋白(FUS)的结合作用。检测敲低mCF中FUS表达对circ_0036167调控心肌纤维化相关基因表达的影响。[结果]Masson三色染色显示心衰心肌发生明显的纤维化(P<0.001)。RT-qPCR结果显示circ_0036167在心衰心肌中表达显著增加(P<0.05),而宿主基因MYO9A表达无显著差异。放线菌素D和RNase R消化实验证实circ_0036167具有环状RNA典型的RNA稳定性。过表达circ_0036167可显著抑制mCF的增殖、迁移能力和纤维化相关基因的表达。RIP实验证实circ_0036167可与FUS结合。敲低mCF后FUS的表达可促进mCF中纤维化相关蛋白表达(P<0.05或P<0.01),并可减弱circ_0036167对纤维化相关基因表达的抑制作用(P<0.01或P<0.001)。[结论]FUS可介导circ_0036167发挥抑制mCF心肌纤维化表型的作用。
Aim To investigate the effect of circ_0036167 on the fibrotic phenotype of cardiac fibroblasts and the potential mechanism involved.Methods Masson s trichrome staining was performed in the myocardium of patients with heart failure(HF)and healthy organ donors.Levels of circ_0036167 and its host gene of MYO9A were determined by real-time quantitative polymerase chain reaction(RT-qPCR)in the myocardium of HF patients and healthy organ donors.Actinomycin D treatment and RNase R exonuclease digestion were used to test the stability of circ_0036167 in AC16 cells.The myocardial fibroblasts(mCF)of C57BL/6 mice were infected with recombinant circ_0036167 adenovirus,and the expression of fibrosis related genes COL1A1,COL3A1 and ACTA2 were detected at RNA and protein levels.EdU staining and transwell migration assay were performed to detect the effects of circ_0036167 on mCF proliferation and migration activities.According to the results of bioinformatic prediction,RNA binding protein immunoprecipitation(RIP)assay was performed to confirm the interaction between circ_0036167 and fused in sarcoma(FUS)protein.Effect of FUS knockdown on inhibition of fibrosis-related genes expression by circ_0036167 in mCF was determined.Results Masson s trichrome staining showed that the cardiac fibrosis was significantly increased in the myocardium of HF patients(P<0.001).Circ_0036167 was found markedly increased in the myocardium of HF patients(P<0.05),with no significant difference in its host gene of MYO9A.In response to actinomycin D treatment and RNase R exonuclease digestion,circ_0036167 was more stable than MYO9A.Over-expression of circ_0036167 suppressed proliferation and migration of mCF,and inhibited RNA and protein expression of fibrosis-related genes in mCF.RIP assay revealed the interaction between circ_0036167 and FUS protein.Knock-down of FUS could increase fibrosis-related genes expression(P<0.05 or P<0.01),and significantly attenuated the inhibitory effect of circ_0036167 on fibrosis-related gene expression in mCF(P<0.01 or
作者
陈泽润
郭继深
丰嘉欣
胡雅婷
欧涛
朱杰宁
李晖
徐金东
方咸宏
单志新
CHEN Zerun;GUO Jishen;FENG Jiaxin;HU Yating;OU Tao;ZHU Jiening;LI Hui;XU Jindong;FANG Xianhong;SHAN Zhixin(The Second School of Clinical Medicine,Southern Medical University,Guangzhou,Guangdong 510280,China;School of Biology and Biological Engineering,South China University of Technology,Guangzhou,Guangdong 510006,China;School of Medicine,South China University of Technology,Guangzhou,Guangdong 510006,China;Guangdong Cardiovascular Institute,Guangzhou,Guangdong 510080,China;Research Center of Medical Sciences,Guangdong Provincial People’s Hospital,Guangzhou,Guangdong 510080,China)
出处
《中国动脉硬化杂志》
CAS
2023年第2期101-109,共9页
Chinese Journal of Arteriosclerosis
基金
国家自然科学基金项目(82070254、82200325)
广州市科技计划项目(202102080093)
广东省自然科学基金项目(2022A1515012522、2022A1515012175、2021A1515220122)
广东省卫健委课题(A2021002、A2022334)
广东省人民医院心血管专项(2020XXG003)
广东省人民医院国自然培育项目(KY0120220028)。
