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
R363.2; [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W...
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| Published in | Zhongshan da xue xue bao. Zhongshan daxue xuebao yixue kexue ban = Journal of Sun Yat-sen University. Yi xue ke xue ban Vol. 45; no. 2; pp. 196 - 205 |
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| Main Authors | , , , , , , , |
| Format | Journal Article |
| Language | Chinese |
| Published |
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
01.03.2024
Editorial Office of Journal of Sun Yat-sen University |
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| Online Access | Get full text |
| ISSN | 1672-3554 |
| DOI | 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).20240305.004 |
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| Abstract | R363.2; [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. |
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| AbstractList | 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。 R363.2; [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. |
| Abstract_FL | [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. |
| Author | 胡志琴 王娅 伍华燕 方咸宏 单志新 温艺红 梁俣 刘宇鹏 |
| AuthorAffiliation | 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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| Author_FL | SHAN Zhixin WEN Yihong LIU Yupeng HU Zhiqin WNAG Ya LIANG Yu WU Huayan FANG Xianhong |
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| DocumentTitle_FL | CircRNA Circ_0120051 Inhibits the Fibrotic Phenotype of Myocardial Fibroblasts via Targeting miR-144-3p/IDH2 Axis |
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| Keywords | 心肌纤维化 Circ_0120051 cardiac fibrosis circular RNA cardiac fibroblast 微RNA 环状RNA miRNA 心肌成纤维细胞 |
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| Publisher | 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 Editorial Office of Journal of Sun Yat-sen University |
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| Snippet | R363.2; [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... 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... |
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| SubjectTerms | Circ_0120051 微RNA 心肌成纤维细胞 心肌纤维化 环状RNA |
| Title | 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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