Suppression Of MicroRNA Let-7i Promotes Cardiomyocte Proliferation And Repairs Heart Function Post Injury By Targeting CCND2 And E2F2

BackgroundWith the development of global social economy and improvement of living standards,the average life expectancy of human beings has been significantly prolonged.The incidence and mortality of cardiovascular diseases such as coronary heart disease have also increased year by year,making them one of the major threats to human health.Coronary occlusion,which is caused by intra-or sub-intimal hemorrhage of atherosclerotic lesions,intraluminal thrombosis,or persistent arterial spasm,resulting in complete or incomplete occlusion of the lumen,leads to inadequate cardiac perfusion and ischemia of the heart tissue,resulting in losing a large number of myocardial cells.The loss of cardiomyocytes and their insufficient replacement is the major contributor in the pathogenesis of many cardiovascular diseases(1,10).Myocardial cell proliferation and regeneration is the key to functional recovery.Reentry cell cycle of mature cardiormyocytes is considered as a promising major source of new cardiomyocytes(11).In recent years,the study of cardiomyocyte proliferation has overturned the traditional view that adult cardiomyocytes permanently out of cell cycle activity.Therefore,targeting cardiomyocyte proliferation is one of the potential therapeutic strategies for myocardial regeneration and repair.To do this,there is a need to reveal the differences between cell cycle activities of neonatal and adult cardiomyocytes and make clear of the basic mechanisms of cardiomyocyte cell cycle.MicroRNAs(miRNAs)are a class of single-stranded small-molecule RNAs containing about 22 nucleotides(8).They are widely found in plants,nematodes,flies,and eukaryotes including humans,which are highly conserved,tissue-specific and temporal.They function in RNA silencing and post-transcriptional regeneration of gene expression(12).The previous studies have found that miRNAs play vital roles in the development of heart tissue,in determining the proliferation and maturation of cardiomyocytes.Previous experiments showed that let-7i has a strong inhibitory effect on cardiomyocyte proliferation in vitro(9).However,it remains not yet clear whether let-7i still have the same effect on cardiomyocytes in vivo as well as the specific mechanisms.In previous experiments,we predicted by bioinformatics technology that CCND2 and E2F2 are the potential target genes for let-7i.Previous literature confirmed that CCND2 and E2F2 both are cell cycle regulators,and their abnormal expression leads to abnormal cell proliferation.Therefore,based on these previous studies,we put forward the hypothesis that let-7a(73)could regulate the cell cycle and promote cardiomyocyte proliferation via CCND2 and E2F2.To this end,we verify our hypothesis by the loss-and gain-function of let-7i in cardiomyocytes in vivo and in myocardiac infarction(MI)model of adult mouse.Method1.Differences in expression of let-7i in l-day-old/7-days-old/10-days-old neonatal mouse heartsA.In vitro isolation and culture of l-day-old/7-days-old/10-days-old neonatal mouse ventricular cardiomyocytes.B.Real-time quantitative PCR(qRT-PCR)verified that let-7i differentially expressed in cardiomyocytes of different age.2.Exploring whether ovexpression of let-7i inhibits cardiomyocyte proliferation in vivo.A.Injection of Ad-let-7i/Ad-NC through left ventricular myocardium in neonatal 1 day mice to overexpress the expression of let-7i,and immunofluorescence staining of cell proliferating nuclear antigen to detect cardiomyocyte proliferation(EdU,pH3).B.Injection of Ad-let-7i/Ad-NC through left ventricular myocardium in neonatal 1 day mice to overexpress the expression of let-7i,and immunofluorescence labeled cell boundary to detect whether overexpress let-7i affect myocardial cell size.3.Exploring whether knockdown let-7i promotes cardiomyocyte proliferation in vivo.A.Injection of AAV-anti-let-7i/AAV-NC through left ventricular myocardium in adult mice to overexpress the expression of let-7i,and immunofluorescence staining of cell proliferating nuclear antigen to detect cardiomyocyte proliferation(EdU,pH3).B.Injection of AAV-anti-let-7i/AAV-NC through left ventricular myocardium in adult mice to overexpress the expression of let-7i,and immunofluorescence labeled cell boundary to detect whether suppress let-7i affect myocardial cell size.4.Exploring whether knockdown of let-7i could promote cardiac regeneration after myocardial infarction and improve heart function.A.A myocardial infarction model was established in adult mice.AAV-anti-let-7i/AAV-NC was injected into the marginal zone of myocardial infarction and the proliferation of cardiomyocytes was detected by immunofluorescence staining of cell proliferating nuclear antigen.B.M-mode ultrasound observation of cardiac function changes and masson staining to observe the degree of myocardial fibrosis.4.Exploring the molecular mechanism of regulation of let-7i in cardiac regeneration.A.Bioinformatics Predict the mRNA molecules which let-7i may bind to;B.Experimental Validation of let-7i binding to target mRNA that participate in the regulation of myocardial regeneration.Result1.The expression of let-7i increased with age in mice,and concomitantly,the ability of cardiomyocyte regeneration weakened.2.In vivo we verified that expression of let-7i was able to inhibit cardiomyocyte proliferation.3.In vivo we verified that suppression of let-7i was able to promote the proliferation of cardiomyocytes,repair cardiac function and promote myocardial remodeling after myocardial infarction.4.let-7i regulates the cell cycle of cardiomyocytes by targeting CCND2,and further affects the function of cardiomyocyte regeneration.ConclusionNewborn mouse cardiomyocytes have a certain proliferation potential,however,this proliferation potential gradually losses in 4 weeks after birth.The transformation of cardiomyocyte from proliferation to terminal differentiation is associated with changes in miRNA and mRNAs expression.Some upregulated miRNAs after birth are involved in cell cycle process.let-7i regulates the cardiomyocyte cell cycle through CCND2 and E2F2,further regulates cardiomyocyte proliferation and promote heart function recovery post myocardial infarction.In this study,given that the transformation of cardiormyocyte proliferation and the function of miRNA regulation,and prior lectures upon let-7i inhibition of myocardial cell proliferation in vitro.We found that let-7i inhibits cardiomyocyte proliferation in vivo.The discovery may offer a new clinical strategy for treatment of myocardial ischemia diseases.