| BackgroundHeart failure(HF)is the terminal stage of various cardiovascular diseases with high morbidity and mortality.Myocardial infarction is one of the important causes of heart failure.Acute myocardial infarction can cause about 25%of cardiomyocytes to lose in just a few hours.Adult mammalian animals have the weakest heart regeneration capacity,and a large number of lost myocardial cells can only be repaired into scar tissue by fibrosis,which leads to cardiac remodeling.The final irreversible progression is heart failure.Promoting myocardial regeneration after myocardial infarction is the most fundamental measure to prevent heart failure after myocardial infarction,which is the hot point of current research.At present,there are mainly two strategies for inducing cardiac regeneration.One strategy is exogenous transplantation of progenitor/stem cells with self-proliferation and differentiation potential to damaged myocardium.Previous studies have found that although exogenous stem cell transplantation can improve left ventricular function in patients with heart failure to some extent,but did not effectively improve the clinical prognosis of patients with heart failure.The other is to stimulate endogenous cardiomyocytes to regain their regenerative capacity and achieve endogenous cardiac regeneration.Recent studies have also confirmed that pre-existing cardiomyocyte proliferation through division is the main way that mammals maintain normal myocardial homeostasis and post-ischemic or post-injury repair.And it has the advantages of high proliferation of cardiomyocytes and no immune rejection,which may be a more promising method of treatment for myocardial infarction.Therefore,more and more researchers have turned to exploring the latter strategy for heart regeneration,which is how to achieve effective endogenous heart regeneration,however,the underlying mechanism of endogenous cardiomyocytes proliferation is not yet clear.Long non-coding RNA(lncRNA)is an RNA molecule in the ncRNA family that is longer than 200 bp and does not encode any protein.It has been found that lncRNA plays a key role in the biological processes such as cell cycle regulation and proliferation,as well as the developmental regulation process,and has gradually become a research hotspot.At present,the study of lncRNA in the cardiovascular field is still in its infancy.Related studies have found that lncRNA can be specifically expressed in the heart,and it is essential for maintaining the normal development of myocardium.In cardiac pathology,lncRNA regulates the development of cardiac hypertrophy and heart failure and autophagy after myocardial infarction.These advances suggest that lncRNA may be involved in the regulation of endogenous myocardial regeneration.In this study,we screened lncRNA-ROCR that may be involved in human heart regeneration by analyzing and comparing a public database of human fetal heart and adult heart databases and a series of bioinformatics analysis,and confirmed that it has homology in rats.In vitro and in vivo experiments confirmed that knockdown of ROCR can promote myocardial cell proliferation and improve myocardial remodeling after myocardial infarction.Finally,we found that the underlying mechanism of action is that ROCR function as a miRNA sponge,which can bind to miR-214 to release its downstream target gene PTEN and inhibit Akt phosphorylation to exert inhibitory effect on cardiomyocyte proliferation.In addition,ROCR could bind directly to PTEN protein and promote its expression by increasing PTEN mRNA stability.Method1、Screening of Differentially Expressed lncRNA in Human Fetal Heart and Adult HeartA.Comparison of fetal heart rate and adult heart lncRNA and mRNA expression profiles to screen lncRNA-ROCR in adult heart;B.Conservation analysis of ROCR in human and rat;C.Comparison of the expression level of ROCR in different stages of rats after birth and co-expression network analysis showed that it has the potential to regulate cell proliferation.2、In vivo and in vitro experiments confirmed that ROCR regulates myocardial regeneration.A.Cardiomyocytes from neonatal rats at 7 days and 1 day after birth were isolated and ROCR was knocked down or overexpressed.Proliferation of cardiomyocytes and cardiac fibroblasts was detected by immunofluorescence and western blot markers.B.ROCR were knocked down or overexpressed by adenovirus at the apical injection of neonatal rats for 7 days and 1 day.The proliferation of cardiomyocytes was detected by immunofluorescence and western blot markers.;C.ROCR were over-expressed by adenovirus at the apical injection of newborn neonatal rats.Heart weight,fibrosis and hypertrophy were detected after 12 days.3、ROCR regulates myocardial regeneration and ventricular remodeling after myocardial infarction.A.The rat myocardial infarction model was prepared.The shROCR/shNC was injected around the marginal zone of infarction after anterior descending coronary artery occlusion.The evaluation of cardiac function were performed by M-mode ultrasound at different time points.B.At different time points,TTC staining and Masson staining were used to detect area of myocardial infarction and fibrosis.Immunofluorescence was used to detect the proliferation of myocardial cells around the infarct area.C.Immunohistochemistry and immunofluorescence detection of angiogenesis around the infarct area.4、Exploring the potential mechanism of ROCR exerting regulatory effects on cardiac regeneration.A.Subcellular localization of ROCR position and predict the possible binding of miRNA molecules;B.Experiments confirm that ROCR can combine with miRNAs and regulate expression of downstream target gene involved in the regulation of cardiomyocyte proliferation;C.Pulldown assay pulls protein that bind to ROCR.Mass spectrometry determines protein types and screens for downstream proteins that function;Results1.Analyze the lncRNA expression profiles of human fetal heart and adult hearts,and screen out lncRNA-ROCR that may be involved in the regulation of cardiomyocyte proliferation.;2.In vitro and in vivo experiments confirmed that knockdown of ROCR can promote proliferation of myocardiocytes 7 days after birth and overexpression of ROCR can suppress proliferation of myocardiocytes 1 day after birth.Moreover,intervention of ROCR did not cause fibroblast proliferation and myocardial cell hypertrophy;3.In rat myocardial infarction model,knockdown of ROCR can promote the proliferation of myocardial cells around the infarct zone,promote the increase of vascular density around infarction area,reduce the negative remodeling of ventricular after myocardial infarction,and then improve the prognosis of myocardial infarction;4.Mechanistically,ROCR is mainly expressed in the cytoplasm and competitively bind to miR-214 to release its target gene PTEN to inhibit Akt phosphorylation.In addition,ROCR can directly bind PTEN protein to increase its expression by increasing the mRNA stability.In conclusionLncRNA-ROCR can bind to miR-214 to release its downstream target gene PTEN and inhibit Akt phosphorylation to exert inhibitory effect on cardiomyocyte proliferation by function as a ceRNA.In addition,lncRNA-ROCR could bind to PTEN prtein directly to increase its level by increasing the mRNA stability. |
