摘要
目的:研究环状RNA MYO9A_043(circular RNA MY09A_043, circMY09A_043)对小鼠心肌纤维化的调控作用和机制。方法:利用重组腺病毒circMYO9A_043介导其在C57BL/6乳小鼠心脏成纤维细胞(mouse cardiac fibroblasts, mCFs)中过表达,通过RT-qPCR和Western blot实验检测纤维化相关基因Ⅰ型胶原α1链(collagen type Ⅰ alpha 1 chain, Col1a1)、Ⅲ型胶原α1链(collagen type Ⅲ alpha 1 chain, Col3a1)和平滑肌肌动蛋白α2(smooth muscle actin alpha 2, Acta2)的表达。利用划痕实验检测mCFs的迁移能力。利用RNA测序分析和RT-qPCR验证circMYO9A_043对mCFs中胞质多腺苷酸化元件结合蛋白4(cytoplasmic polyadenylation element binding protein 4,CPEB4)等基因的下调作用。利用腺病毒介导mCFs过表达CPEB4,检测CPEB4对纤维化相关基因表达的影响。通过RNA结合蛋白免疫沉淀(RNA binding protein immunoprecipitation, RIP)实验检测CPEB4与纤维化相关基因的结合作用。进一步通过放线菌素D处理实验,检测mCFs中CPEB4对Col1a1、Col3a1和Acta2 mRNA的稳定作用。结果:利用腺病毒可以有效介导circMYO9A_043在mCFs中过表达。circMYO9A_043可显著下调Col1a1、Col3a1和Acta2表达,抑制mCFs的迁移能力(P<0.05)。circMYO9A_043可显著降低mCFs中CPEB4的表达(P<0.01),过表达CPEB4可逆转circMYO9A_043对纤维化相关基因表达的抑制作用。RIP实验显示CPEB4与mCFs中纤维化相关基因的mRNA有显著结合作用(P<0.01),而放线菌素D处理实验结果显示CPEB4可显著增强上述基因mRNA的稳定性(P<0.05)。结论:circMYO9A_043通过下调CPEB4的表达而抑制mCFs纤维化表型。
AIM:To study the effect of circular RNA MYO9A-043(circMYO9A_043)on the fibrotic phenotype of mouse cardiac fibroblasts(mCFs),and to explore the potential mechanism.METHODS:The recombinant circMYO9A_043 adenovirus was prepared for overexpressing circMYO9A_043 in neonatal C57BL/6 mCFs.The expression levels of myocardial fibrosis-related genes,collagen type Ⅰ alpha 1 chain(Col1a1),collagen type Ⅲ alpha 1 chain(Col3a1)and smooth muscle actin alpha 2(Acta2),were determined by RT-qPCR and Western blot.Wound-healing assay was conducted to evaluate the migration ability of mCFs.The down-regulation of cytoplasmic polyadenylation element binding protein 4(CPEB4)by circMYO9A_043 in mCFs was identified through RNA sequencing analysis and RT-qPCR.After the overexpression of CPEB4,the mRNA and protein expression levels of Col1a1,Col3a1 and Acta2 were determined in mCFs.The interactions between CPEB4 and Col1a1/Col3a1/Acta2 mRNA were detected by RNA binding protein immunoprecipitation(RIP)assay and RT-qPCR.Additionally,an actinomycin D treatment was performed to identify the effect of CPEB4 on the stability of Col1a1,Col3a1 and Acta2 mRNA.RESULTS:Efficient adenovirus-mediated overexpression of circMYO9A_043 was achieved in mCFs.The overexpression of circMYO9A_043 significantly inhibited the expression of Col1a1,Col3a1 and Acta2 genes in mCFs and the migration ability of mCFs(P<0.05).However,overexpression of CPEB4 could reverse the inhibitory effect of circMYO9A_043 on the expression of fibrosis-related genes in mCFs.The RIP assay revealed specific interactions between CPEB4 and Col1a1/Col3a1/Acta2 mRNA(P<0.01).Results from the actinomycin D treatment showed that CPEB4 could enhance the mRNA stability of Col1a1,Col3a1 and Acta2 in mCFs(P<0.05).CONCLUSION:The circMYO9A_043 inhibits the fibrotic phenotype of mCFs by down-regulating the expression of CPEB4.
作者
陈泽润
李艺
梁俣
苏金凤
高原
欧涛
李晖
徐金东
方咸宏
单志新
CHEN Zerun;LI Yi;LIANG Yu;SU Jinfeng;GAO Yuan;OU Tao;LI Hui;XU Jindong;FANG Xianhong;SHAN Zhixin(The Second School of Clinical Medicine,Southern Medical University,Guangzhou 510280,China;Department of Car-diology,Guangdong Provincial People's Hospital,Guangdong Academy of Medical Sciences,Southern Medical University,Guangzhou 510080,China;School of Medicine,South China University of Technology,Guangzhou 510006,China;Medical Research Institute,Guangdong Provincial People's Hospital,Guangdong Academy of Medical Sciences,Southern Medical University,Guangzhou 510080,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2023年第7期1225-1232,共8页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.82070254,No.82200325)
广东省自然科学基金资助项目(No.2022A1515012522,No.2022A1515012175,No.2021A1515220122,No.2023A1515010201,No.2021A1515111173)
广州市基础与应用基础研究项目(No.202201011627)
广东省人民医院心血管专项(No.2020XXG003)
广东省人民医院国自然培育项目(No.KY0120220028)。
