Effect And Mechanism Of MicroRNA Let-7i-5p On The Proliferation Of Cardiomyocyte

BackgroundCurrently,myocardial infarction(MI)is a global disease,and heart failure is the most common complication of MI.The loss of CMs and their insufficient replacement as a major contributor to the pathogenesis of heart failure.At present,the clinical treatment of heart failure can only delay the disease,but can not fundamentally solve the problem of CMs loss.Promoting CMs regeneration is a key step in post-heart damage repair and promoting endogenous CM proliferation is considered to be the most promising pathway for cardiac regeneration.It is well known that cell proliferation is regulated by the cell cycle,and CMs exit the cell cycle shortly after birth,and CM proliferation rapidly stops.Instead,the growth pattern is nuclear replication with only DNA synthesis and nuclear mitosis without cytokinesis(mitotic mitosis),which puts most cardiomyocytes in a dinuclear state.Although limited induction of DNA synthesis in the heart as a response to some factors has been described,there is little evidence for cytoplasmic division or cytokinesis in mammals.The lethal-7(let-7)family of miRNAs,which is highly conserved and the largest of all miRNA family,are ubiquitously expressed in most somatic cells.In CMs,the let-7 family reduces EdU incorporation(a uridine analogue that marks newly synthesized DNA)and ki-67 positivity(a marker of proliferation antigen)in vitro,and let-7i-5p was proposed to be a strong suppressor of CM proliferation in vitro[1].Previous studies in our laboratory showed that inhibition of let-7i-5p promoted DNA synthesis and mitosis of CMs,and promoted the recovery of cardiac function after MI.However,the effect of let-7i-5p on cytokinesis in CM is unclear,and it is not known how he works.In this study,we will observe the effects of let-7i-5p on the growth of cardiomyocytes through cytokinesis,DNA replication and mitosis,and further explore the mechanism.Method1.To observe the proliferative activity of CMs in mice at dififerent ages.A.Collect the mouse heart tissius at E16.5/P1/p56.B.Observing the proportion of pH3 positive CMs in heart tissue by immunofluorescence.2.To observe the expression level of Let-7i-5p in different developmental stages of mouse heart.A.The heart tissues of E16.5,E18-5,P1,P3,P7,and P56 mice were taken,and the CMs of P1,P7,and P10 mice were isolated.B.qPCR was used to detect the expression level of let-7i-5p in heart tissue and CMs of mice at different ages.3.Exploring the effect of interventional expression of let-7i-5p on proliferation of mouse cardiomyocytes in vitro.A.Isolation and culture of P1 mouse primary CMs.B.After overexpression or inhibition of let-7i-5p expression level,the number of CMs and the proportion of EdU-/pH3-/Auroa B positive CMs in cardiomyocytes were observed by immunofluorescence.4.Exploring the mechanism of let-7i-5p regulating the proliferation of CMs.A.To observe the conservation of the binding sequence of let-7i-5P and E2F2/CCND2.B.To observe the effect of let-7i-5p on E2F2 and CCND2 protein levels.C.A model of MI was established using 8-week-old male mice.Immediately after the model was established,adeno-associated virus 9(AAV9)was transfected.The experiment was divided into four groups:AAV9-NC and AAV9-anti-let-7i-5p,AAV9-anti-Iet-7i-5p+AAV9-si-E2F2 and AAV9-anti-let-7i-5p+AAV9-si-CCND2,myocardial tissue was collected 28 days after injection.D.The cardiac function of each group of mice was evaluated by ultrasound.The changes of LVEDd,LVSDd,LVFS and LVEF were observed dynamically.The proportion of EdU positive CMs in myocardial tissue was observed by immunofluorescence.Result1.Proliferative activity of mouse CMs decreases with increasing age.2.The expression level of Let-7i-5p increased with the increase of mouse age,and the expression of let-7i-5p was the highest in adult mice.3.Inhibition of let-7i-5p expression can promote cytokinesis,DNA replication and mitosis of CMs,thereby promoting the proliferation of CMs.Overexpression of let-7i-5p can inhibit the proliferation of CMs.4.The binding sequence of Let-7i-5p and E2F2/CCND2 is highly conserved,and let-7i-5p can inhibit the expression of E2F2 and CCND2 protein.5.Silencing E2F2 or CCND2 reversed the improvement of post-infarction cardiac function and promoted the proliferation of CMs by let-7i-5p inhibition.ConclusionInhibition of Let-7i-5p promotes the proliferation of CMs characterized by cytokinesis,DNA replication and mitosis of CMs and promotes recovery of cardiac function after myocardial infarction by simultaneously binding to mRNAs E2F2 and CCND2.