The Function And Mechanism Of MiR-24 In Cardiomyocyte Phypertrophy

Background:Cardiac hypertrophy plays essential roles in heart failure.Most of pathologic insults would induce cardiac hypertrophy.Cardiac hypertrophy is characterized by an increase of individual cell size of cardiomyocytes and by reactivation of expression of cardiac fetal genes.Functionally,cardiac hypertrophy works as a means to adapt to pathological stimuli.However,prolonged pathological hypertrophy would result in dysfunction of hearts,and ultimately lead to heart failure,and even sudden death.Increasing evidence has shown that miRNAs plays a key role in cardiac hypertrophy.MiRNAs are a class of hightly conserved small non-coding RNAs which can regulate the expression of mRNAs post-transcriptionally either through suppressing translation or promoting mRNAs degradation.Previous studies in our laboratory centre have showed that the expression of miR-24,a broad-spectrum expression,is up-regulated in the hearts with cardiac hypertrophy in patients and rats,suggesting a correlation between miR-24 and cardiac hypertrophy,but the role and mechanism of miR-24 in cardiac hypertrophy is not elucidated.In the present study,we detected the expression of miR-24 and made them compared with the control group by establishing a rat abdominal aorta constriction model and cardiomyocyte hypertrophy model.Then,We constructed miR-24 overexpressing adenovirus and miR-24 inhibitor to transfect into neonatal rat cardiomyocytes,and observed changes in myocardial cell morphology,cell surface area and molecular markers related with myocardial hypertrophy,and clarified the functional role of miR-24 in cardiomyocyte hypertrophy.Finally,we investigated the underlying mechanisms of miR?24 in cardiac hypertrophy by bioinformatics method,dual luciferase reporter gene,and western blotting.Material and methods:1.Rat cardiac hypertrophy modelMale Sprague-Dawley rats(180-200 g)were anesthetized with pentobarbital sodium(30 mg/kg),followed by exposure of the abdomen and isolation of suprarenal abdominal aorta,which was then tightened with a 4-0 nylon suture against 24-gauge needle.After removing the needle,the incision was closed.The sham control group underwent all procedure except aortic constriction.Rats were sacrificed at 2 weeks after abdominal aortic constriction(AAC)or sham operation with routine feeding.2.Histological analysisSham or AAC operated rat hearts were fixed with 4% paraformaldehyde overnight and embedded in paraffin for histological analysis.Paraffin sections were stained with Hematoxylin and Eosin(H&E).3.RNA quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR)analysisTotal RNA of rat hearts,or cultured cells was extracted using TRIzol reagent according to the instruction of the manufacturer.And 1 ug RNA was used to generate cDNA with M-MLV reverse transcriptase.Real-time quantitative PCR was performed with SYBR Green.The amount of target mRNA or miRNAs was normalized to the amount of endogenous control GAPDH or U6 respectively,and expressed as relative level to the control sample.4.Primary cardiomyocytes culturing and adenovirus infectionNeonatal cardiomyocytes were isolated from 2-to 3-day old Sprague–Dawley rats.Briefly,the hearts were isolated and digested with 0.1% collagenase.After dissociation,the cells were subjected to differential pre-plating to remove non-myocytes.Cells were cultured in DMEM with 15% fetal bovine serum.48 h after plating,the cells were infected with recombinant adenovirus at a multiplicity of infection(MOI)50.The cells were harvest after additional 48 h for RNA analysis or for morphology observation.RNA inhibitor was used to knockdown endogenous miR-24 by transfection with Lipofectamine 2000.Phenylephrine(PE,100 uM)was used to establish hypertrophic cardiomyocytes model.5.Cardiomyocytes immunochemistryCardiomyocytes were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 in PBS,followed by blocking with 5% goat serum in PBS for 2 h at room temperature.And then,cardiomyocytes were incubated with anti-actinin antibody overnight.The next day,cardiomyocytes were washed with PBS for three times and incubated with the second antibody coupled with Alexa-555(Molecular Probes).After washing for 3 times,the slides were mounted using fluorescent mounting medium.Cell images were captured with Leica TCS SP5 microscope and the cell surface area was determined with Leica image analysis software.About 100 cells from randomly selected fields were calculated for each experimental group.6.Luciferase assay analysisTargetscan algorithms were used to identify potential targets of miR-24.Potential target sites were cloned into 3' UTR of Renilla luciferase gene of psiCheck2 plasmid.For luciferase assay analysis,the 293 T cells were transfected with recombinant luciferase reporter plasmids and miR-24 expressing plasmid for 24 h.Luciferase activity was measured with a dual luciferase reporter assay kit on a luminometer,as described previously.7.Western blottingMiR-24 overexpressing adenovirus or miR-24 inhibitor were transfected into primary cardiomyocytes.Proteins were extracted with RIPA lysate.The isolated proteins were transferred to the PVDF membrane by SDS-PAGE.The PVDF membrane was blocked with 5% skim milk for 2h and incubated with the primary antibody overnight at 4? in refrigerator.Repeat washed three times with TBST for each 10 min.The secondary antibody was incubated for 1h.Repeat washed three times with TBST for each 10 min again.X-ray film was scanned after development and fixation,and protein bands were analyzed with Image J software to further confirm whether miR-24 regulate target gene expression in cells.8.Statistical analysisAll data were expressed as means±SEM.The mean value of control group is defined as 1.Two-tailed unpaired Student's t test was used for statistical comparison of two groups.Statistical comparisons were carried out using Sigma Plot.P<0.05 were considered to be statistically significant.Results:1.MiR-24 was up-regulated in AAC-induced hypertrophic hearts of ratsA cardiac hypertrophic model of rats was established using abdominal aorta constriction(AAC)to induce hypertrophy by increasing afterload on the heart.The rats subjected to sham operations were used as controls.The rats were sacrificed at 2 weeks after surgery.Hematoxylin and Eosin-stained sections of representative hearts from sham-operated and 2-week AAC rats showed that AAC can successfully establish hypertrophic model of rats.We detected the heart weight/body weight(HW/BW).Compared with sham controls,HW/BW was markedly increased in hearts subjected to AAC for 2 weeks.Detection of these hypertrophic markers showed that compared with hearts from rats subjected to sham operation,the expression level of ANP and myh7 was significantly up-regulated,and the expression of myh6 was markedly down-regulated in hearts of rats subjected to AAC for 2 weeks.These results indicate that cardiac hypertrophic model of rats was successfully established by AAC.Then the expression level of miR-24 during cardiac hypertrophy was analyzed by qRT-PCR,showing that miR-24 was up-regulated in AAC-induced hypertrophic hearts.These results suggest that miR-24 might play significant roles in heart failure or cardiac hypertrophy.2.Over-expression of miR-24 promoted hypertrophic reaction of cardiomyocytesTo investigate the potential function of miR-24 in cardiomyocytes,an adenovirus expressing miR-24(Ad-miR-24)was generated.The adenovirus vector expressing GFP(Ad-GFP)was used as a control.After transfection for 48 h,the expression level of miR-24 was detected by qRT-PCR,demonstrating that compared with Ad-GFP,infection of cardiomyocytes with Ad-miR-24 significantly increased the expression of miR-24 level.To investigate the function of miR-24 in hypertrophy,the expression of several hypertrophic markers were determined by qRT-PCR,showing that the transcriptional level of myh6 was markedly down-regulated by 20% in miR-24 over-expressing cardiomyocytes,while that of myh7 and ANP was markedly up-regulated by 20% and 50% respectively.These results suggested that increased miR-24 expression in cardiomyocytes might influence the contraction of the cardiomyocytes.When cardiomyocytes were infected with Ad-miR-24 or Ad-GFP for 48 h,up-regulation of miR-24 increased the cell size to 1.2-fold as measured by cardiomyocytes immunochemistry and cell surface area(P<0.05).3.MiR-24 was up-regulated in PE-induced hypertrophic cardiomyocytesIn order to investigate the function of endogenous miR-24 in PE-induced cardiomyocyte hypertrophy,we establish a cardiomyocyte model of hypertrophy using PE-stimulation(100 uM).Forty-eight hours later,cardiomyocytes were fixed and analyzed with immuno-fluorescence and cell surface area analysis.100 uM PE was sufficient to increase cell size of cardiomyocytes.Additionally,incubation of PE with cardiomyocytes also resulted abnormal expression of hypertrophic markers in cardiomyocytes.Compared with control group,PE treatment up-regulated the expression of ANP and myh7,while down-regulated the expression of myh6.According to these results,we successfully establish hypertrophic model of cardiomyocytes.Then,qRT-PCR was used to detect the expression of miR-24,showing that miR-24 was significantly up-regulated in PE treated cardiomyocytes by 50%(P<0.05).4.Endogenous miR-24 was partially required for PE-induced hypertrophy of cardiomyocytesTo evaluate the endogenous function of miR-24 in the regulation of PE-induced cardiomyocyte hypertrophy,miR-24 inhibitor was used to knock down the endogenous miR-24.The experiment was divided into three groups: control group(Ctl group),PE+NC inhibitor group and PE+miR-24 inhibitor group.Forty eight hours after miR-24 or NC inhibitor transfected into cardiomyocytes,cardiomyocytes were harvested for further analysis.Compared with PE+NC inhibitor group,the expression level of miR-24 was significantly decreased in PE+miR-24 inhibitor group.To investigate whether miR-24 was essential for PE-induced cardiomyocyte hypertrophy,cultured cardiomyocytes were transfected with miR-24 inhibitor for 12 hours,and then incubated with PE for additional 36 hours.The results showed that knockdown of endogenous miR-24 suppressed the effects of PE on abnormal regulation of hypertrophic markers in cardiomyocytes.Down-regulation of miR-24 can significantly suppress the up-regulation of ANP and myh7,and down-regulation of myh6 induced by PE.Furthermore,reduction of miR-24 levels resulted in smaller cell size than the cells transfected with PE+ NC inhibitor,as measured by relative cell surface areas.Thus down-regulation of miR-24 can markedly suppress the PE-induced increase of cell size of cardiomyocytes.These results indicate that inhibition of miR-24 expression partially reversed PE-induced cardiomyocyte hypertrophy and miR-24 was a prerequisite for PE-induced cardiomyocyte hypertrophy.5.Predict miR-24 target gene and verify itTo explore the mechanism by which miR-24 regulated hypertrophic reaction of cardiomyocytes,we searched for potential targets of miR-24 using Targetscan algorithms.Bioinformatic analysis indicated that ACVR1 B,BCL2L11 and MLEC might be potential targets of miR-24.They were predicted to harbor one to more potential targeting sites of miR-24.Using luciferase assay,we found that over-expression of miR-24 in 293 T cells can markedly suppress the luciferase activity of the reporter fusing with 3'UTR of ACVR1 B and BCL2L11,indicating that they are direct targets of miR-24.Additionally,we showed that miR-24 specifically targets 1568-1574 site,however,did not suppress 438-445 and 953-959 sites of ACVR1 B.In 3' UTR of BCL2L11,miR-24 directly suppressed the luciferase activity of a reporter fused with 473-480 site,but not 2651-2658 sites of BCL2L11.Western blotting results showed that overexpression of miR-24 significantly reduced the expression of ACVR1 B protein,while inhibition of miR-24 expression significantly increased the expression of ACVR1 B protein,indicating that ACVR1 B was the target gene of miR-24.Conclusion:1.Expression of miR-24 was up-regulated in AAC-induced hypertrophic hearts of rats and in PE-induced hypertrophic cardiomyocytes.2.Overexpression of miR-24 promoted cardiomyocytes hypertrophy.Inhibition of miR-24 expression partially reversed cardiomyocyte hypertrophy induced by PE.3.ACVR1 B was the target gene of miR-24.MiR-24 promotes cardiomyocyte hypertrophy by inhibiting the expression of the ACVR1 B.