首页|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

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

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[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α 1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circ-SLC8A1_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.
CircSLC8A1_005 Inhibits the Fibrotic Phenotype of Cardiac Fibroblasts by Encoding Protein
[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 mi-gration activities of mCFs were detected by EdU and wound-healing assay,respectively.Dual luciferase reporter gene as-say 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 super-oxide 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 ex-pression of fibrosis-related genes in mCFs.The dual luciferase reporter gene assay revealed the activities of 2 IRES in circ-SLC8A1_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 circ-SLC8A1_005 and SLC8A1-605aa could consistently inhibit the fibrotic phenotype of mCFs.SLC8A1-605aa could up-reg-ulate 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 cardi-ac fibroblasts via translating SLC8A1-605aa protein,and SLC8A1-605aa may be a potential target for the treatment of myocardial fibrosis.

cardiac fibrosiscircular RNAcircSLC8A1_005translationcardiac fibroblast

胡雅婷、高原、伍华燕、梁俣、李晖、徐金东、刘宇鹏、单志新

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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

南方医科大学第二临床医学院,广东 广州 510280

广东省心血管病研究所心内科,广东 广州 510080

广东省人民医院检验科,广东 广州 510080

广东省人民医院麻醉科,广东 广州 510080

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心肌纤维化 环形RNA circSLC8A1_005 翻译 心肌成纤维细胞

国家自然科学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东省自然科学基金广东省自然科学基金广州市科技计划项目广州市科技计划项目

8207025482200325823002772023A15150102012022A15150125222022A15150121752021A1515220122202201011627202102080093

2024

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

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(1)
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