摘要
目的:探讨microRNA-26b(miR-26b)调控H9C2心肌细胞肥大的作用靶基因及其分子机制。方法:传代培养H9C2心肌细胞,分别转染miR-26b模拟物(miR-26b mimic)和淋巴样增强因子1(LEF1)siRNA(si-LEF1),以增加H9C2心肌细胞中miR-26b水平或抑制LEF1的表达水平。采用FITC-鬼笔环肽染色检测H9C2心肌细胞表面积,双荧光素酶报告基因实验鉴定miR-26b与潜在靶基因LEF13′端非翻译区(3′UTR)的结合作用。RT-qPCR和Western blot法分别检测miR-26b、LEF1及心肌细胞肥大相关基因表达。结果:过表达miR-26b可明显增加H9C2心肌细胞中肥厚相关基因ANP,β-MHC的表达水平,缩小H9C2心肌细胞表面积;双荧光素酶报告基因实验结果提示miR-26b与LEF13′UTR具有相互结合作用,miR-26b在转录水平抑制LEF1的表达;miR-26b mimic和si-LEF1均能抑制H9C2心肌细胞肥大基因的表达。结论:LEF1是miR-26b的作用靶基因,LEF1参与miR-26b抑制H9C2心肌细胞肥大的作用。
Objective:To investigate the potential target gene and molecular mechanisms of microRNA-26b(miR-26b)in H9C2 cardiomyocyte hypertrophy.Methods:miR-26b mimic and Lymphoid Enhancer Factor 1(LEF1)siRNA(si-LEF1)were transfected into H9C2 cell to elevate the level of miR-26b and inhibit LEF1 expression,respectively.H9C2 cell were stained with FITC-phalloidin solution to demonstrate the size of cardiomyocytes.The interaction between miR-26b and the 3′UTR of Lymphoid Enhancer Factor 1(LEF1)was verified by Dual luciferase reporter assay.The expression of miR-26b,LEF1 and hypertrophy-related genes at miRNA and protein levels were determined by RT-qPCR and Western blotting assay,respectively.Results:Overexpression of miR-26b could significantly decrease the expression of cardiac hypertrophy-related genes atrial natriuretic peptide(ANP)andβ-myosin heavy chain(β-MHC)inH9C2 cell Overexpression of miR-26b could effectively inhibit the hypertrophic phenotype of H9C2 cardiomyocytes.Dual-luciferase reporter Assay results verified that miR-26b could interact with the 3 UTR of LEF1,miR-26b could inhibit the expression of LEF1 at the transcriptional level.Moreover,both miR-26b mimic and si-LEF1 could reduce the expression of hypertrophic genes in H9C2 cardiomyocytes.Conclusion:LEF1 is the target gene of miR-26b and it plays a role on the inhibition of miR-26b to H9C2 cardiomyocyte hypertrophy.
作者
唐春梅
赵海霞
随何欢
梁婧
朱丽莎
苏强
TANG Chun-mei;ZHAO Hai-xia;SUI He-huan;LIANG Jing;ZHU Li-sha;SU Qiang(Department of Pharmacy,Nanchong Central Hospital,Second Clinical Medical College of North Sichuan Medical College;Nanchong Key Laboratory of Individualized Drug Therapy,Nanchong 637000,Sichuan,China)
出处
《川北医学院学报》
CAS
2020年第6期943-946,共4页
Journal of North Sichuan Medical College
基金
四川省基层卫生事业发展研究课题(SWFZ18-Z-10)
南充市市校科技战略合作项目(18SXHZ0230)
川北医学院科研发展计划项目(CBY17-A-2D12)。
