| Background:Heart failure refers to a pathological process in which the cardiac output is not enough to meet the metabolic needs of the whole body due to the dysfunction of the heart pump.It is often accompanied by maladaptive myocardial hypertrophy,myocardial fibrosis and myocardial cell apoptosis.With the application of high-throughput RNA sequence technology,many current studies have explored the role of nc RNA in heart failure.Micro RNA(micro RNA,miRNA)is a class of small molecules with clear functions of nc RNA.Previous literature reports that miRNAs mainly target the 3'UTR region of the gene to inhibit the protein expression of target genes.In recent years,researchers have separated and sequence the nucleus/cytoplasm of different types of cells,and found that a large number of miRNAs are highly expressed in the nucleus.The function of these miRNAs is still not very clear.This article studies the effect of miRNA in the nucleus of the process of myocardial fibrosis and its mechanism.Objectives:1.To clarify the role of miR-199b-5p located in the nucleus of myocardial fibrosis.2.To clarify the mechanism of miR-199b-5p in the nucleus to regulate myocardial fibrosis and detect the downstream target genes of miR-199b-5p.Methods:1.To explore the expression of miR-199b-5p in the process of myocardial fibrosis1.1 Western-blot and Masson staining to detect the degree of myocardial tissue fibrosis in patients with heart failure and healthy controls.1.2 The miRNA expression profile chip detects the differential expression of miRNA in the myocardial tissue of patients with heart failure and healthy controls,and RT-q PCR verifies the expression of miRNA and identifies the nuclear/cytoplasmic distribution.1.3 Primary isolation of human atrial fibroblasts(Human atrial fibroblast,HAFs),and verify it by ACTA2 immunofluorescence.1.4 Angiotensin II(Ang-II)induces HAFs fibrosis,RT-q PCR and Western-blot detect the expression of fibrosis-related genes,miR-199b-5p,FISH experiment and nuclear/cytoplasmic separation RT-q PCR to detect the effect of Ang-II on the nuclear localization changes of miR-199b-5p.1.5 Primary isolation of neonatal mouse atrial myofibroblasts(m CFs),and verify it by ?-SMA immunofluorescence.1.6 Ang-II induces fibrosis of m CFs,RT-q PCR and Western-blot detect the expression of fibrosis-related genes and miR-199b-5p,FISH experiment and nuclear/cytoplasmic separation RT-q PCR to detect the effect of Ang-II on the nuclear localization changes of miR-199b-5p.2.To explore the effect of miR-199b-5p in the nucleus on the fibrotic phenotype of cardiac fibroblasts2.1 Overexpression of miR-199b-5p in HAFs and m CFs respectively,Ed U,flow cytometry and Trans-well were used to detect its effects on cell proliferation and migration.2.2 Overexpression of miR-199b-5p in HAFs and m CFs respectively,Western-blot to detect its effects on fibrosis-related gene expression.FISH experiment detects the effect of miR-199b-5p mimic transfection on the expression level of miR-199b-5p in the nucleus.3.To explore the molecular mechanism of miR-199b-5p regulating myocardial fibrosis in the nucleus3.1 UCSC data browsing shows that miR-199 b locus has strong enhancer histone modification markers H3K27 ac and H3K4me1 levels.3.2 ChIP-qPCR detects the levels of histone modification markers H3K27 ac and H3K4 mel in the enhancer of miR-199 b locus.3.3 The recombinant adenovirus mediated m CFs up-regulate the expression of CDK9,and use Ed U,flow cytometry and Trans-well experiments to detect its effect on cell proliferation and migration.3.4 Recombinant adenovirus mediated m CFs up-regulate the expression of CDK9.Western-blot was used to detect its effect on the expression of fibrosis-related genes,and the nuclear localization changes of CDK9 and p-Smad3 were further detected by immunofluorescence and Western-blot.3.5 The dual luciferase reporter gene experiment detects the binding effect of miR-199b-5p with the promoter region of human and mouse CDK9.3.6 The dual luciferase reporter gene experiment detects whether miR-199b-5p has a binding effect with human CDK9 3'UTR region.3.7 After silencing the nuclear transporters IPO8(importin 8,IPO8)and Xpo5(Exportin5,Xpo5)respectively.Nuclear and cytoplasmic separation RT-q PCR detection of miR-199b-5p nuclear and cytoplasmic distribution.4.To explore the effect of inhibiting the expression of CDK9 on reversing miR-199b-5p in promoting myocardial fibrosisIsoprenaline(ISO)administration at the whole animal level induces myocardial fibrosis in mice.On this basis,ago-miR-199b-5p(cholesterol-modified miR-199b-5p mimic)or ago-miR-199b-5p combined with CDK9 si RNA was injected to detect the effects of inhibiting the expression of CDK9 at the overall survival ratio,cardiac function and myocardial fibrosis of mice.Results:1.The expression of miR-199b-5p increases in myocardium of patients with heart failure and Ang-II-induced myocardial fibrosis1.1 The expression level of miR-199b-5p in the myocardial tissue of patients with heart failure was significantly increased.1.2 Ang-II treatment caused a further increase in the level of miR-199b-5p in the nucleus of HAFs.1.3 Ang-II perfusion induces a significant increase in the expression of miR-199b-5p in the mouse myocardial tissue in the mouse myocardial fibrosis model.1.4 Ang-II treatment can further increase the level of miR-199b-5p in the nucleus of m CFs.2.miR-199b-5p promotes the proliferation,migration and fibrosis-related gene expression of human and mouse cardiac fibroblasts2.1 miR-199b-5p mimic transfections of HAFs can significantly increase the expression of miR-199b-5p in the nucleus of HAFs,and promote cell proliferation,migration and fibrosisrelated genes COL1A1,COL3A1,ACTA2 expression increase and Smad3 signal activation.2.2 miR-199b-5p mimic transfection of m CFs can significantly increase the expression of miR-199b-5p in the nucleus of m CFs,and promote cell proliferation,migration and fibrosisrelated genes COL1A1,COL3A1,?-SMA expression increase and Smad3 signal activation.3.MiR-199b-5p in the nucleus promotes myocardial fibrosis by promoting CDK9-mediated Smad3 activation3.1 miR-199 b gene locus has enhancer histone modification markers due to the.3.2 miR-199b-5p can activate the expression of adjacent gene CDK9.3.3 Adenovirus-mediated overexpression of CDK9 can promote the proliferation,migration and fibrosis-related gene expression.3.4 Knockdown of CDK9 can inhibit the proliferation,migration and fibrosis-related gene expression.3.5 CDK9 mediates the effect of miR-199b-5p in promoting the expression of fibrosisrelated genes.3.6 miR-199b-5p can specifically bind to the CDK9 promoter region and recruit pol II to activate CDK9 transcriptional expression.3.7 The miR-199b-5p in the cytoplasm does not bind to the CDK9 3'UTR.3.8 The nuclear transporter Ipo8 can mediate the transfer of miR-199b-5p into the nucleus.4.Inhibition of CDK9 expression can reverse the effect of miR-199b-5p in promoting myocardial fibrosis4.1 The overall survival of mice injected with ISO or ISO combined with ago-miR-199b-5p mimic(cholesterol-modified miR-199b-5p mimic)was significantly shorter than that of the Sham group.Adding Cdk9 si RNA can significantly improve overall survival period.4.2 Significant reductions in ejection fraction(EF)and Fraction shortening(FS)were observed in ISO-treated mice that had received ago-miR-199b-5p mimic injection,but they had no reduction in EF and FS in ISO-treated mice injected with ago-miR-199b-5p and Cdk9 si RNA.4.3 In ISO-treated mice injected with ago-miR-199b-5p mimics,left ventricular wall thickness during end diastole and left ventricular wall thickness during end systole was significantly reduced,but the left ventricular posterior wall thickness during end diastole in ISO-treated mice injected with ago-miR-199b-5p mimic wasn't significant decrease in left ventricular posterior wall thickness during end systolic.4.4 Sirius red staining results showed that miR-199b-5p aggravated ISO-induced myocardial fibrosis,which can be reversed by adding Cdk9 si RNA.4.5 The protein level test results showed that miR-199b-5p increased the expression of COL1A1,COL3A1,?-SMA and CDK9 and the activation of Smad3,while the injection of Cdk9 si RNA into the tail vein of ISO-induced mice reversed the fibrosis process.Conclusion:1.The nuclear transporter Ipo8 mediates the intranuclear transport of miR-199b-5p.2.The nuclear miR-199b-5p combines with the CDK9 promoter region to recruit pol II and promote transcriptional activation of downstream genes.3.MiR-199b-5p in the nucleus promotes myocardial fibrosis by activating CDK9/Smad3 signals. |
PDF Full Text Request