Circular RNA Expression In Isoproterenol Hydrochloride-induced Cardiac Hypertrophy

BackgroundCardiac hypertrophy is an adaptive response to various physiological and pathologic stimuli,the main pathological features of cardiac hypertrophy include the reactivation of embryonic development marker genes,the obvious increase of myocardial cell volume,the obvious increase of protein synthesis and the corresponding changes of the types of contractile proteins.Although the initial cardiac hypertrophy is a normal physiological change to meet the needs of the heart under overload pressure,it is not infinite.Because the oxygen demand of hypertrophied myocardium will increase gradually,and the blood supply by coronary artery will not be satisfied,which will cause ischemia of hypertrophied myocardium,and then it will lead to the decrease of myocardial contraction function,and eventually induce a series of cardiovascular diseases,so cardiac hypertrophy is a well-recognized risk factor for cardiovascular disease,but no molecular targets for convincingly preventing or treating cardiac hypertrophy have yet been found,most likely because our understanding of the molecular mechanisms underlying cardiac hypertrophy remains incomplete.Therefore,an in-depth study on the molecular mechanism of cardiac hypertrophy is to provide new strategies for clinical prevention and treatment,which is still an important research in the field of cardiovascular biology.As a newly discovered non-coding RNA(ncRNA),circular RNA is a covalently closed endogenous molecule in eukaryotes,which often exhibits cell-type-specifific and tissue-specifific patterns.This high stability is presumably the result of their covalently closed ring structure protecting these molecules from exonuclease-mediated degradation.So it has a longer half-life.It is known that although some circular RNAs are rich in expression,they are relatively conservative in evolution,so they have gradually become a research hotspot of ncRNA.Previous studies have shown that circular RNA plays an important role in biological functions by regulating the expression of target genes,it also can regulate gene transcription and protein production or self-translation.In addition,other studies have shown that it is closely related to cardiovascular diseases,such as cardiac hypertrophy,but the molecular mechanism of its role in the process of cardiac hypertrophy remains to be explored.PurposeIn this project,we explored the expression profile of circRNA in cardiac hypertrophy through RNA sequencing,and in order to study the potential function and molecular mechanism of circRNA in cardiac hypertrophy,which could provide more theoretical support for exploring new therapeutic targets of cardiac hypertrophy.Methods1.Isoproterenol hydrochloride(ISO)solution was used to treat mice for 14 days in abdominal cavity to establish a model of myocardial hypertrophy.The interventricular septum thickness at end-diastole(IVSD),and other parameters,including the end-diastolic left ventricular posterior wall thickness(LVPWd),end-diastolic left ventricular internal diameter(LVIDd),fractional shortening(FS),and end-systolic left ventricular internal diameter(LVIDs),were determined by echocardiography.And then the RNA of heart was extracted and QPCR were used to detect the expression of ANF and miR-23a,which were the indexes of cardiac hypertrophy.2.The mice ventricles which were from the groups of control and isoproterenol,were sent to the company for RNA sequencing by used next-generation sequencing,and the results of sequencing were analyzed by bioinformatics,which was used to determine whether there was difference in the expression of the circular RNA in the two groups.3.We selected 6 circular RNA that were from the results of sequencing,which expressed significant difference.The ventricular RNA from the mice of sequencing was used as samples.Electrophoresis and first-generation sequencing were used to examine the authenticity of circular RNA from sequencing results,and the expression of circular RNA in the sequencing results was verified by QPCR.4.Go and KEGG pathway analysis of the parental genes of differentially expressed circular RNA was carried out,and its potential target miRNA and target mRNA which were united to miRNA,were predicted.Next,the interaction network of miRNA and circRNA was constructed and the target mRNA was annotated through GO.5.Using the RNA which was from liver and human skeletal muscle cell to reverse transcribe into cDNA with random primers,after PCR amplification,the products were detected by electrophoresis to test the conservation and specificity of circular RNA,and then knock down the over expressed circRNA WWP1 in cardiac hypertrophy to detect its effect on the expression of ANF and miR-23a.Results1.The results of echocardiography compared with the contro;group showed that the left ventricular diameter at the end of diastolic and systolic phase increased,the shortening rate of left ventricular short axis,the left ventricular thickness at diastolic phase and the interventricular septum thickness decreased in the isoproterenol treated group;the results of QPCR showed that compared with the control group,ANF and miR-23a in the ISO group increased significantly.2.We got the following results from the sequencing results:in the control group and ISO group,1464 circular RNA were co-expressed,and the expression of 119 circular RNA changed significantly.There was 30 circular RNAs were down-regulated,and 89 circular RNAs were up-regulated.,which was compared with the control group.3.The expression of mmu-circRNA sh3rf3,mmu-circRNA wwp1 and mmu-circRNA ift81 in the isoproterenol treated group significantly decreased,but the expression of mmu-circRNA trappc9,mmu circRNA-klc1 and mmu-circRNA 20702 was up-regulated,which was consistent with the expression trend of circular RNA in next-generation sequencing.The results of electrophoresis and first-generation sequencing also proved the reliability of the sequence of circular RNA that was from RNA-seq.4.(1)GO analysis showed that the parent genes of the differentially expressed circular RNA were involved in cell composition,molecular function and biological process,and most of them were related to metabolic process,biological regulation and stimulation response.KEGG pathway analysis also showed that the parent genes were not only involved in the circulatory system and cardiovascular system,but also involved in the metabolism and signal transduction pathway.(2)The predicted results showed that there were 1974 miRNAs that binded to the differentially expressed circular RNA,120 of which could bind to 7520 mRNA.The subsequent analysis of GO enrichment of 156 mRNA showed that these mRNA were significantly enriched in the processes of multicellular organism development,cell metabolism,protein modification,chromatin covalent modification and intracellular protein transport,and in the cell components,such as cytoplasm,node of Ranvier and neuronal interstitium.The analysis of molecular function showed that these mRNA were only significantly enriched in transferase activity and binding,which includes DNA binding,RNA binding and protein binding.5.(1)The results of PCR electrophoresis showed that circRNA klcl,circRNA 20702,circRNA ift81 and circRNA wwpl were not expressed in HSkMC,while circRNA sh3rf3 and circRNA trappc9 were expressed in HSkMC.In addition,the above six circular RNAs were expressed in the heart and liver of mice.(2)The results of QPCR showed that the expression of circRNA wwp1 was up-regulated,but the expression of ANF and miR-23a was down-regulated.Conclusions1.Through isoproterenol induced cardiac hypertrophy,the expression profile of circRNA was changed,and the differentially expressed circRNA may play a role in cardiac hypertrophy.2.The ANF and miR-23a are downstream targets of circRNA wwp1,suggesting that circRNA wwp1 exerts inhibitory roles of cardiac hypertrophy via down-regulation of ANF and miR-23a.