CircSLC8A1_005 Inhibits the Fibrotic Phenotype of Cardiac Fibroblasts by Encoding Protein

HU Yating; GAO Yuan; WU Huayan; LIANG Yu; LI Hui; XU Jindong; LIU Yupeng; SHAN Zhixin

doi:10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2024.0106

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CircSLC8A1_005 Inhibits the Fibrotic Phenotype of Cardiac Fibroblasts by Encoding Protein

Preclinic Research | 更新时间：2024-02-22

- CircSLC8A1_005 Inhibits the Fibrotic Phenotype of Cardiac Fibroblasts by Encoding Protein
- Journal of Sun Yat-sen University(Medical Sciences) Vol. 45, Issue 1, Pages: 35-44(2024)
- 作者机构：
  
  1.华南理工大学医学院，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.广东省人民医院检验科，广东广州 510080
  5.广东省人民医院麻醉科，广东广州 510080
  6.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
- 基金信息：
- DOI：10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2024.0106
  CLC： R363.2
- Published：20 January 2024，
  
  Received：13 September 2023，
  
  Accepted：27 November 2023
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胡雅婷,高原,伍华燕等.CircSLC8A1_005通过编码蛋白抑制心肌成纤维细胞纤维化表型的作用[J].中山大学学报（医学科学版）,2024,45(01):35-44.

HU Yating,GAO Yuan,WU Huayan,et al.CircSLC8A1_005 Inhibits the Fibrotic Phenotype of Cardiac Fibroblasts by Encoding Protein[J].Journal of Sun Yat-sen University(Medical Sciences),2024,45(01):35-44.
胡雅婷,高原,伍华燕等.CircSLC8A1_005通过编码蛋白抑制心肌成纤维细胞纤维化表型的作用[J].中山大学学报（医学科学版）,2024,45(01):35-44. DOI： 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2024.0106.

HU Yating,GAO Yuan,WU Huayan,et al.CircSLC8A1_005 Inhibits the Fibrotic Phenotype of Cardiac Fibroblasts by Encoding Protein[J].Journal of Sun Yat-sen University(Medical Sciences),2024,45(01):35-44. DOI： 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2024.0106.

摘要

目的

探究环形RNA circSLC8A1_005调控心肌成纤维细胞纤维化表型的作用及可能机制。

方法

利用腺病毒介导在小鼠心肌成纤维细胞（mCFs）中过表达circSLC8A1_005，并检测mCFs中纤维化相关因I型胶原α1链（Col1a1）、Ⅲ型胶原α1链（Col3a1）和平滑肌肌动蛋白α2（Acta2）基因表达，通过EdU和划痕实验检测不同干预对mCFs的增殖和迁移能力的影响。双萤光素酶报告基因实验检测circSLC8A1_005包含的潜在核糖体进入序列（IRES）的活性。通过Western blot实验检测circSLC8A1_005翻译蛋白SLC8A1-605aa及其在细胞内分布情况。双萤光素酶报告基因实验检测SLC8A1-605aa对超氧化物歧化酶2（Sod2）的转录激活作用。通过RNA结合蛋白免疫沉淀（RIP）实验检测SLC8A1-605aa与Sod2 mRNA的结合作用。放线菌素D实验检测SLC8A1-605aa对Sod2 mRNA稳定性的影响。

结果

利用腺病毒可在mCFs中有效介导过表达circSLC8A1_005，过表达circSLC8A1_005可显著抑制mCFs中纤维化相关基因表达，抑制mCFs的增殖和迁移能力。双萤光素酶报告基因实验结果提示circSLC8A1_005包含的2个IRES具有活性。Western blot检测结果显示circSLC8A1_005可翻译预期大小为70 ku的SLC8A1-605aa蛋白，并主要分布于细胞核内。过表达SLC8A1-605aa和circSLC8A1_005可一致地抑制mCFs的纤维化表型。SLC8A1-605aa可特异上调超氧化物歧化酶2（Sod2）表达，但并不能转录激活Sod2表达；RIP实验结果显示SLC8A1-605aa与Sod2 mRNA有特异结合作用，而放线菌素D实验结果显示SLC8A1-605aa能够增强Sod2 mRNA的稳定性。

结论

CircSLC8A1_005通过翻译蛋白SLC8A1-605aa发挥抑制心肌成纤维细胞纤维化表型的作用，SLC8A1-605aa可能是潜在的用于心肌纤维化治疗的靶点。

Abstract

Objective

To investigate the effect of circSLC8A1_005 on the fibrotic phenotype of cardiac fibroblasts and the potential mechanism involved.

Methods

The effect of adenovirus-mediated overexpression of circSLC8A1_005 on the expression of fibrosis-related genes， collagen type I alpha 1 chain （Col1a1）， collagen type Ⅲ alpha 1 chain （Col3a1） and smooth muscle actin alpha 2 （Acta2）， in mouse cardiac fibroblasts （mCFs） were detected. The proliferation and migration activities of mCFs were detected by EdU and wound-healing assay， respectively. Dual luciferase reporter gene assay was performed to detect the activity of potential internal ribozyme entry site （IRES） in circSLC8A1_005. CircSLC8A1_005-translated protein， SLC8A1-605aa， and its intracellular distribution was identified by Western blot assay. The effect of SLC8A1-605aa protein on transcription activity of Sod2 gene was detected by the dual luciferase reporter gene assay. RNA binding protein immunoprecipitation （RIP） was utilized to verify the interaction between SLC8A1-605aa and superoxide dismutase 2 （Sod2） mRNA. Actinomycin D treatment was used to detect the effect of SLC8A1-605aa on Sod2 mRNA stability in mCFs.

Results

An efficient adenovirus-mediated overexpression of circSLC8A1_005 was achieved in mCFs. The enforced expression of circSLC8A1_005 suppressed proliferation and migration of mCFs， and inhibited the expression of fibrosis-related genes in mCFs. The dual luciferase reporter gene assay revealed the activities of 2 IRES in circSLC8A1_005. Results of Western blot assay showed that circSLC8A1_005 could translate protein SLC8A1-605aa with the prospected molecular weight of 70 ku， which is predominantly distributed in the nucleus. Overexpression of the circSLC8A1_005 and SLC8A1-605aa could consistently inhibit the fibrotic phenotype of mCFs. SLC8A1-605aa could up-regulate superoxide dismutase 2 （Sod2） expression， but not at the transcriptional level. RIP assay indicated that SLC8A1-605aa could specifically interact with Sod2 mRNA， and the results of actinomycin D assay showed that SLC8A1-605aa could enhance the stability of Sod2 mRNA in mCFs.

Conclusion

CircSLC8A1_005 inhibits the fibrotic phenotype of cardiac fibroblasts via translating SLC8A1-605aa protein， and SLC8A1-605aa may be a potential target for the treatment of myocardial fibrosis.

关键词

心肌纤维化环形RNAcircSLC8A1_005翻译心肌成纤维细胞

Keywords

cardiac fibrosiscircular RNAcircSLC8A1_005translationcardiac fibroblast

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Related Institution

School of Biology and Biological Engineering， South China University of Technology

Guangdong Provincial Key Laboratory of Clinical Pharmacology， Guangdong Provincial People’s Hospital

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