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

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

Preclinic Research | 更新时间：2024-04-15

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- CircRNA Circ_0120051 Inhibits the Fibrotic Phenotype of Myocardial Fibroblasts via Targeting miR-144-3p/IDH2 Axis
- Journal of Sun Yat-sen University(Medical Sciences) Vol. 45, Issue 2, Pages: 196-205(2024)
- 作者机构：
  
  1.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
  2.广州医科大学附属第五医院药学部，广东广州 510799
  3.华南理工大学医学院，广东广州 510006
  4.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
- 作者简介：
  
  SHAN Zhixin， E-mail： shanzhixin@gdph.org.cn
  FANG Xianhong， E-mail： fangxianhong@gdph.org.cn
- 基金信息：
- DOI：10.13471/j.cnki.j.sun.yat-sen.univ（med.sci）.20240305.004
  CLC： R363.2
- Published：20 March 2024，
  
  Received：20 December 2023，
  
  Accepted：22 February 2024
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梁俣,胡志琴,温艺红等.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[J].中山大学学报（医学科学版）,2024,45(02):196-205.

LIANG Yu,HU Zhiqin,WEN Yihong,et al.CircRNA Circ_0120051 Inhibits the Fibrotic Phenotype of Myocardial Fibroblasts via Targeting miR-144-3p/IDH2 Axis[J].Journal of Sun Yat-sen University(Medical Sciences),2024,45(02):196-205.
梁俣,胡志琴,温艺红等.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[J].中山大学学报（医学科学版）,2024,45(02):196-205. DOI： 10.13471/j.cnki.j.sun.yat-sen.univ（med.sci）.20240305.004.

LIANG Yu,HU Zhiqin,WEN Yihong,et al.CircRNA Circ_0120051 Inhibits the Fibrotic Phenotype of Myocardial Fibroblasts via Targeting miR-144-3p/IDH2 Axis[J].Journal of Sun Yat-sen University(Medical Sciences),2024,45(02):196-205. DOI： 10.13471/j.cnki.j.sun.yat-sen.univ（med.sci）.20240305.004.

摘要

目的

2

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

2

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

2

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

2

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。

Abstract

Objective

2

To investigate the regulatory effect of circular RNA circ_0120051 on the fibrotic phenotype of cardiac fibroblasts and the potential mechanism involved.

Methods

2

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 stability 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

2

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 consistently reverse the inhibitory effects of circ_0120051 on fibrosis-related genes expression in mCFs and mCFs migration.

Conclusions

2

Circ_0120051 inhibits the fibrotic phenotype of cardiac fibroblasts via sponging miR-144-3p to enhance the target gene of IDH2 expression.

关键词

心肌纤维化环状RNA微RNACirc_0120051心肌成纤维细胞

Keywords

cardiac fibrosiscircular RNAmiRNACirc_0120051cardiac fibroblast

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Related Author

SHAN Zhixin

HU Yating

GAO Yuan

LIANG Yu

LI Hui

XU Jindong

LIU Yupeng

SHAN Zhixin

Related Institution

Department of Cardiology，Guangdong Cardiovascular Institute

The Second School of Clinical Medicine， Southern Medical University

Department of Clinical Laboratory，Guangdong Provincial People’s Hospital

Department of Anesthesiology，Guangdong Provincial People’s Hospital

Medical Research Institute， Guangdong Provincial People’s Hospital， Guangdong Academy of Medical Sciences， Southern Medical University

Postal code：510080
Tel：020-87331643，87330827 Email：xbmed@mail.sysu.edu.cn
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