Circ_0018478通过编码HERC4-193发挥抑制心肌成纤维细胞纤维化表型的作用  

Circ_0018478 Inhibits the Fibrotic Phenotype of Cardiac Fibroblasts via Encoding Protein HERC4-193

作　　者：丰嘉欣 郭继深 梁俣 姜佳雪 李晖[5] 徐金东[6] 方咸宏[4] 单志新 FENG Jia-xin;GUO Ji-shen;LIANG Yu;JIANG Jia-xue;LI Hui;XU Jin-dong;FANG Xian-hong;SHAN Zhi-xin(School of Biology and Biological Engineering,South China University of Technology,Guangzhou 510006,China;The Second School of Clinical Medicine,Southern Medical University,Guangzhou 510280,China;Department of Cardiology,Guangdong Cardiovascular Institute,Guangzhou 510080,China;Guangdong Provincial Key Laboratory of Clinical Pharmacology,Guangdong Provincial People’s Hospital,Guangzhou 510080,China;Department of Clinical Laboratory,Guangdong Provincial People’s Hospital,Guangzhou 510080,China;Department of Anesthesiology,Guangdong Provincial People’s Hospital,Guangzhou 510080,China)

机构地区：[1]华南理工大学生物科学与工程学院,广东广州510006 [2]南方医科大学第二临床医学院,广东广州510280 [3]广东省心血管病研究所心内科,广东广州510080 [4]广东省人民医院广东省临床药理学重点实验室,广东广州510080 [5]广东省人民医院检验科,广东广州510080 [6]广东省人民医院麻醉科,广东广州510080

出　　处：《中山大学学报（医学科学版）》2022年第6期995-1004,共10页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(82070254);广州市科技计划项目(202102080093);广东省自然科学基金(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)。

摘　　要：【目的】探究环状RNA circ_0018478调控心肌成纤维细胞纤维化表型的作用和可能机制。【方法】RTqPCR检测健康器官捐献者(n=18)和心衰(n=28)患者心肌中circ_0018478及其宿主基因含E3泛素蛋白连接酶4的HECT和RLD结构域(HERC4)的表达水平。荧光原位杂交(FISH)实验和核质RNA定量检测circ_0018478的细胞分布情况,放线菌素D干预实验和核糖核酸外切酶(RNase R)消化实验检测circ_0018478的RNA稳定性。过表达腺病毒介导的circ_0018478时,分别在RNA和蛋白水平检测乳小鼠心肌成纤维细胞(mCFs)中如Ⅰ型和Ⅲ型胶原等纤维化相关基因表达的影响。利用EdU染色和Trans-well细胞迁移实验鉴定circ_0018478对mCFs增殖和迁移能力的影响。质谱shot-gun分析circ_0018478可能翻译蛋白的肽段序列。利用小干扰RNA(siRNA)抑制HERC4-193表达,检测对circ_0018478调控mCFs纤维化表型的影响。【结果】在心衰病人心肌中,相较于无显著表达差异的宿主基因HERC4,环形RNA circ_0018478表达显著增加。FISH和核质分离实验结果证实circ_0018478主要定位于心肌细胞胞质中。放线菌素D和RNase R消化实验证实circ_0018478具有典型的RNA稳定性。过表达circ_0018478可抑制mCFs的增殖、迁移和纤维化相关基因的表达。质谱shot-gun和Western blot检测结果提示circ_0018478可翻译预期的HERC4-193蛋白。过表达circ_0018478和HERC4-193可一致地抑制mCFs的纤维化表型,而抑制HERC4-193表达可有效减弱circ_0018478抑制mCFs中纤维化相关基因表达的作用(P<0.05)。【结论】Circ_0018478通过翻译蛋白HERC4-193发挥抑制心肌成纤维细胞纤维化表型的作用。【Objective】To investigate the effect of circ_0018478 on the fibrotic phenotype of cardiac fibroblasts and the potential mechanism involved.【Methods】The expression of circ_0018478 and its host gene of HECT and RLD domain containing E3 ubiquitin protein ligase 4(HERC4)in the myocardium of patients with heart failure(HF)(n=28)and healthy donors(n=18)was analyzed by real-time quantitative polymerase chain reaction(RT-qPCR)assay.The distribu-tion of circ_0018478 was identified by fluorescence in situ hybridization(FISH)assay and RT-qPCR assay based on nu-cleocytoplasmic RNA in human AC16 cardiomyocytes.Actinomycin D and RNase R exonuclease digestion were used to test the stability of circ_0018478 in AC16 cells.RNA and protein expression of fibrosis-related genes was detected in mouse cardiac fibroblasts(mCFs)with adenovirus-mediated over-expression of circ_0018478.EdU staining and transwell migration assay were performed to detect the effects of circ_0018478 on mCFs proliferation and migration activities.The po-tential circ_0018478-translated protein in mCFs was identified by mass spectrometry(MS)shot-gun assay.HERC4-193 was inhibited by small interfering RNA(siRNA),and the effect of HERC4-193 knock-down on the fibrotic phenotype of mCFs with over-expression of circ_0018478 was studied.【Results】The expression of circ_0018478 was up-regulated in the myocardium of HF patients,with no significant difference in its host gene of HERC4.The results of FISH and RT-qP-CR assay showed that circ_0018478 was mainly in the cytoplasm of AC16 cardiomyocytes.The characteristic RNA stability of circ_0018478 was verified by Actinomycin D and RNase R assay,respectively.The enforced expression of circ_0018478 suppressed proliferation and migration of mCFs,and inhibited the expression of fibrosis-related genes in mCFs.The results of MS shot-gun assay and Western blotting showed that circ_0018478 could translate protein HERC4-193.Overexpression of the circ_0018478 and protein HERC4-193 could consistently inhibit the fibrotic ph

关 键 词：心肌纤维化 circ_0018478 翻译 心肌成纤维细胞 

分 类 号：R363.2[医药卫生—病理学]