环状RNA circ_0120051通过miR-144-3p/IDH2轴抑制心肌成纤维细胞的纤维化表型研究

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中文摘要：[目的]研究环形 RNA circ_0120051对心肌成纤维细胞的纤维化表型的调控作用和机制.[方法]通过实时萤光定量PCR(RT-qPCR)检测健康器官捐献者(n=24)与心力衰竭(HF)病人(n=21)心肌标本中 circ_0120051及其宿主基因溶质载体家族8成员A1(SLC8A1)的表达水平.核糖核酸外切酶(RNase R)消化实验鉴定circ_0120051的RNA稳定性.RT-qPCR检测circ_0120051在人心肌细胞AC16中的核质分布情况.利用腺病毒在C57BL/6乳小鼠心肌成纤维细胞(mCFs)中过表达circ_0120051,通过RT-qPCR和Western blot检测过表达circ_0120051对mCFs中纤维化相关基因表达的影响,利用细胞划痕实验检测对mCFs迁移能力的影响.通过RNA免疫共沉淀技术(RIP)验证circ_0120051与miR-144-3p间的结合作用,利用双萤光素酶报告基因实验鉴定miR-144-3p与靶基因异柠檬酸脱氢酶2(Idh2)3'-UTR的结合位点.[结果]Circ_0120051在心衰病人心肌中表达显著增加,而其宿主基因SLC8A1表达无显著差异.Circ_0120051主要定位于人心肌细胞的胞质中.RNase R消化实验证实circ_0120051相对于线性SLC8A1 mRNA具有典型的环状RNA稳定性.过表达circ_0120051可抑制mCFs中纤维化相关基因表达和mCFs的迁移能力.RIP实验证实circ_0120051与miR-144-3p之间具有明显结合作用.在mCFs中转染miR-144-3p可促进纤维化相关基因的表达,并有效逆转circ_0120051对mCFs纤维化表型的抑制作用.双萤光素酶报告基因实验证实miR-144-3p与Idh2的3'-UTR存在结合作用.miR-144-3p可在转录水平抑制mCFs中Idh2表达,而过表达circ_0120051可增加mCFs中IDH2表达.在mCFs中转染miR-144-3p和Idh2的小干扰RNA(siRNA),可一致性地逆转circ_0120051对纤维化相关基因表达和mCFs迁移的抑制作用.[结论]Circ_0120051通过特异结合miR-144-3p并增加其靶基因IDH2表达来发挥抑制mCFs纤维化表型的作用.

外文标题：CircRNA Circ_0120051 Inhibits the Fibrotic Phenotype of Myocardial Fibroblasts via Targeting miR-144-3p/IDH2 Axis

外文摘要：[Objective]To investigate the regulatory effect of circular RNA circ_0120051 on the fibrotic phenotype of cardiac fibroblasts and the potential mechanism involved.[Methods]The expression of circ_0120051 and its host gene of solute carrier family 8 member A1(SLC8A1)mRNA in the myocardium of healthy organ donors(n=24)and heart failure(HF)patients(n=21)were assessed by real-time quantitative polymerase chain reaction(RT-qPCR)assay.RNA stabili-ty of circ_0120051 was identified by RNase R exonuclease digestion assay.The cytoplasmic and nuclear distribution of circ_0120051 in human cardiomyocyte AC16 was detected by RT-qPCR assay.The expression of fibrosis-related genes in mouse cardiac fibroblasts(mCFs)with adenovirus-mediated overexpression of circ_0120051 was detected by RT-qPCR and Western blot assay,respectively.The effect of overexpression of circ_0120051 on the migration activity of mCFs was evaluated by wound-healing assay.RNA co-immunoprecipitation(RIP)was conducted to detect the interaction between circ_0120051 and miR-144-3p.The binding site of miR-144-3p in the 3'-UTR of isocitrate dehydrogenase 2(Idh2)mRNA was identified by the dual luciferase reporter gene assay.[Results]Circ_0120051 was significantly up-regulated in the myocardium of HF patients,while the mRNA expression of its host gene SLC8A1 was not changed.Circ_0120051 was mainly located in the cytoplasm of human AC16 cells.Results of RNase R exonuclease digestion revealed that circ_0120051 possesses the characteristic stability of circular RNA compared to the linear SLC8A1 mRNA.Overexpression of circ_0120051 could inhibit the expression of fibrosis-related gene in mCFs and mCFs migration.RIP assay confirmed the specific interaction between circ_0120051 and miR-144-3p.Transfection of miR-144-3p mimic could efficiently promote the expression of fibrosis-related genes in mCFs and reverse the inhibitory effect of circ_0120051 on the fibrotic phenotype of mCFs.Results of the dual luciferase reporter gene assay confirmed the interaction between miR-144-3p and the 3'-UTR of Idh2.Transfection of miR-144-3p transcriptionally inhibited Idh2 expression,and overexpression of circ_0120051 enhanced IDH2 expression in mCFs.MiR-144-3p mimic and Idh2 small interfering RNA(siRNA)could consis-tently reverse the inhibitory effects of circ_0120051 on fibrosis-related genes expression in mCFs and mCFs migration.[Conclusions]Circ_0120051 inhibits the fibrotic phenotype of cardiac fibroblasts via sponging miR-144-3p to enhance the target gene of IDH2 expression.

外文关键词：

cardiac fibrosiscircular RNAmiRNACirc_0120051cardiac fibroblast

作者：

梁俣、胡志琴、温艺红、伍华燕、王娅、刘宇鹏、单志新、方咸宏

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作者单位：

广东省心血管病研究所//南方医科大学附属广东省人民医院//广东省医学科学院广东广州 510080

广州医科大学附属第五医院药学部, 广东广州 510799

华南理工大学医学院,广东广州 510006

广东省临床药理学重点实验室//南方医科大学附属广东省人民医院//广东省医学科学院广东广州 510080

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关键词：

心肌纤维化环状RNA 微RNA Circ_0120051 心肌成纤维细胞

基金：

国家自然科学基金国家自然科学基金广东省自然科学基金广东省自然科学基金广州市科技计划广州市科技计划广东省医院药师青年托举研究基金(晴粤药学基金)

项目编号：

82070254822003252023A15150102012021A15152201222022010116272021020800932023QNTJ16

出版年：

2024

DOI：

10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).20240305.004

中山大学学报(医学科学版)

中山大学

中山大学学报(医学科学版)

CSTPCD北大核心

影响因子：1.608

ISSN：1672-3554

年,卷(期)：2024.45(2)

参考文献量27