| Objective:As a common adaptive reaction towards diverse physiologic and pathologic derived stimulus,cardiac hypertrophy involves mechanical,hormonal and hemodynamic factors.The major cellular basis of cardiac hypertrophy includes increased cardiomyocyte size,actin cytoskeletal reorganization and re-expression of fetal genes.As cardiac hypertrophy is highly associated with sudden cardiac death,a greater understanding of the underlying molecular mechanism,especially the roles of hypertrophy mediators,may reveal novel therapeutic targets and thereby improve survival.MicroRNAs?miRNAs?are noncoding single-stranded RNAs made up of 17–25 nucleotides that can control expression of a gene when they bind mRNAs at the 3'untranslated region?UTR?,inhibiting protein synthesis.It is well known that precursor miRNAs can be cleaved from both the 5'and 3'arms of the precursor duplex,becoming the miRNA-5p and-3p strands,respectively.In recent years,increasing evidence suggests significant roles played by miRNAs in cardiac hypertrophy.However,miR-195-3p and miR-195-5p both participate in the process remains unclear.In our research,studies on principles of miR-195-5p/-3p mechanismsin cardiac hypertrophy at the functional and molecular level were performed.Methods:1.In this study,in vitro and in vivo models of cardiac hypertrophy were established by applying angiotensin ??Ang ??to H9c2 cardiomyocytes and infusing chronic Ang ? to mice,respectively.We then evaluated the above models by detecting mRNA levels of the hypertrophic biomarkers?ANP and BNP?using RT-PCR and by observing morphology and the histological changes.And also,we measure the miR-195-3p and miR-195-5p expression in cardiac hypertrophic models.2.To examine the role played in cardiac hypertrophy by miR-195-3p/-5p,we transfected H9c2 cardiomyocytes with miRNA mimics or inhibitors of miR-195-3p/-5p prior to Ang ? stimulation.We then detected mRNA levels of the hypertrophic biomarkers and observed cellular morphology in different groups.3.Because miRNAs exert their functions mainly through inhibiting target genes,a bioinformatics approach?http://www.targetscan.org?had to be employed in deciphering the information of multiple genes that can bind with miR-195-5p.To further investigate how miR-195-5p relate to potential target genes,we studied using qRT-PCR and Western blot analysis respectively how miR-195-5p affects the expression of target genes in H9c2cardiomyocytes.Furthermore,to demonstrate how possible miR-195-5p can directly suppress target genes expression,luciferase reporter assays were used.4.To examine the hypothesis that miR-195-5p might promote hypertrophy by targeting MFN2 and FBXW7,we transfected H9c2 cardiomyocytes with miR-195-5p inhibitor only and a combination of miR-195-5p inhibitor together with siRNA-MFN2 or siRNA-FBXW7 prior to Ang ? stimulation.We then detected mRNA levels of the hypertrophic biomarkers and observed cellular morphology in different groups.5.To examine the hypothesis that miR-195-5p might promote hypertrophy by promoting apoptosis,we transfected H9c2 cardiomyocytes with inhibitors of miR-195-5p and prior to Ang ? stimulation.We then used flow cytometry methods for the quantification of apoptosis.Expression levels of caspase-3,Bcl-2 and Bax were detected by western blot analysis at protein levels.Results:1.In accordance to Ang ? treatment,5 groups of the H9c2 cardiomyocytes were created in the following order:the control group,the 10-77 mol/l Ang ? group,the 10-6mol/l Ang ? group,the 5*10-66 mol/l group and the 10-55 mol/l Ang ? group.Using the control group as a reference,the ANP and BNP mRNAs levels were significantly higher after 10-77 mol/l Ang ? treating for in 48 h.Besides,stimulation for 48h with this concentration induced a substantial increase in the cell size.The Ang?-treated group showed substantial increase in the HW/BW?heart-to-body-weight ratio?in comparison to the sham-infused mice.Furthermore,there was significant expansion in Ang?-treated hearts ventricular tissues cross-sectional area,and ANP and BNP levels of expression were up-regulated.The miR-195-5p levels were considerably up-regulated in hypertrophy of cellular and mouse models that was Ang?-induced.There was upregulation of miR-195-3p expression in vivo condition and down-regulation in vitro,but the change did not reach the significance threshold.2.The overexpressed miR-195-5p led to more significant Ang?-induced cardiomyocytes hypertrophy,which was assessed by hypertrophic genes'?ANP,BNP?mRNA levels.Upon induction of cardiac hypertrophy by Ang ?,miR-195-5p inhibitors effectively suppressed the ANP and BNP mRNA expression levels,decreasing relative cell areas.We also explored whether miR-195-3p had some cardiac hypertrophy regulator roles in vitro.In presence of Ang ?,overexpression of miR-195p-3p induced a modest albeit not statistically significant reduction in the expression of hypertrophic genes.In contrast,knockdown of miR-195-3p can slightly increase the expression levels of hypertrophy-related genes mentioned above,in comparison to the group of NC inhibitor under Ang ? stimulation.3.A bioinformatics approach?http://www.targetscan.org?had to be employed in deciphering the information of multiple genes that can bind with miR-195-5p.Nine genes with high degrees of integration were selected for further screening:BTG2,SESN1,DYRK2,JARID2,SOX6,FBXW7,TXNIP,LATS2 and MFN2.Among these 9 gene candidates,qPCR analyses indicated that FBXW7 and MFN2 were significantly down-regulated in the Ang?-induced cell model,so these two genes were identified as potential target genes.Furthermore,when miR-195-5p was overexpressed,it significantly inhibited mRNA and protein expression level of FBXW7 and MFN2,whereas its inhibitor had opposite effects.And also,the results demonstrated miR-195-5p mimic repressed MFN2 3'-UTR reporter gene luciferase activity,with the reporter gene linked to the 3'-UTR of mutant MFN2 abolishing this inhibitory effects.The luciferase reporter assay for FBXW7 also showed similar results as for MFN2.4.Ang ? caused significant increases in the cell surface area and in the expression of ANP,BNP,but it failed to do so if cells were pretreated with miR-195-5p inhibitor.In addition,in the presence of MFN2 or FBXW7 siRNA,miR-195-5p inhibitor lost its ability to reverse the Ang?-induced hypertrophy.5.Ang ? significantly increased the protein levels of Bax and caspase-3 and decreased the level of Bcl-2.However,H9c2 cardiomyocytes transfected with miR-195-5p inhibitor prior to Ang ? stimulation notably reduced levels of Bax and cleaved Caspase-3,while the level of Bcl-2 was markedly increased.Furthermore,the apoptotic percentage and was increased in H9c2 cells treated with Ang?.In contrast,miR-195-5p inhibitor reversed the Ang?-induced Increased Rate of Apoptosis. |
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