| BackgroundMyocardial hypertrophy is a compensatory response of the heart to increased volume load and pressure load,which can reduce ventricular wall tension and myocardial oxygen consumption,and is an important mechanism to maintain normal cardiac function,as well as a common pathological process in the occurrence and development of many cardiovasc?lar diseases[1].Studies have confirmed that pathological myocardial hypertrophy is an essential risk factor for ischemic heart disease,arrhythmia and heart failure[2],often leading to severe clinical events such as cardiogenic shock and sudden death.However,after long-term exploration,it is still unable to effectively control and solve the clinical problem of pathological myocardial hypertrophy.Therefore,it is of great medical value and social significance to explore the unique mechanism of the disease and find new drug targets.In numerous factors affecting myocardial hypertrophy,the mechanical stress is one of several signaling molecules that trigger cellular hypertrophy caused by mechanical molecules.In 2006,heinecke and colleagues discovered that z-disc and its related proteins could drive mechanical stress-stimulated signal transduction,a process also known as"mechanical transduction"[3]Calsarcins,a well-known z-disc protein cluster,were shown to couple the cardiac skeletal apparatus to components that directly regulates gene transcription[4,5].In addition,many of the molecular signals such as insulin-like growth factor 1?IGF-1?,transforming growth factor beta?TGF-beta?,angiotensin?and endothelin-1 can induce different signaling cascades mediated by different cell receptors,eventually lead to cellular-level hypertrophic response and transcriptional level changes[6-8].More importantly,inflammatory signaling pathways and oxidative stress also play important roles in the pathogenesis of myocardial hypertrophy[9,10].Long non-coding RNAs?lncrnas?are a cluster of RNA nucleotides longer than 200nt that scarcely have ability to encode proteins.Although lncRNAs do not encode proteins,they can still act as signaling molecules or"sponges"to produce sequence-specific RNAs by binding to regulate gene expression[11].In recent years,emerging evidences have shown that a variety of lncRNAs,such as Chaer,Chast,Mhrt,CHRF,H19,MIAT,and Tincr,play important roles in the development of myocardial hypertrophy[12-14][15-17].Among these lncRNAs Tincr was reported to epigenetically regulate the expression of CaMK?by directly bind to PRC2[17].However,it is unclear whether Tincr has other targets or mechanisms.Therefore,in our present study,the expression of Tincr in myocardial hypertrophy was observed downregulated using a overload-pressure mouse model of myocardial hypertrophy,and overexpression of Tincr was confirmed to attenuate myocardial hypertrophy by TAC.The H9c2 myocardial cell culture technique was used to explore the new possible direct targets of Tincr,and to explore the new signaling mechanism of Tincr to improve myocardial hypertrophy,so as to provide an important basis for the development of drugs to intervene myocardial hypertrophy in the future.Part 1:Long-non-coding RNA Tincr inhibits stress-load-induced cardiac hypertrophyObjective:To observe the expression of Tincr in cardiac hypertrophy caused by thoracic aortic constriction.Tincr was shown to improve cardiac hypertrophy in mice.Methods:Adult C57bl/6 J male mice were randomly numbered and divided into sham operation group?control group?and thoracic aortic constriction model group?TAC model group?,overexpressed Tincr+TAC group and overexpressed viral vector+TAC group.The expression of Tincr,ANP,BNP,and?-MHC in left ventricular myocardial tissue were detected at 6w after TAC modeling by using AAV9 technique,and Tincr was overexpressed by tail vein injection or negative control 2 weeks before TAC.?1?Observed each group's survival,diet,drinking water,activity and other conditions;the expression of Tincr in each group was detected by RT-qPCR.?3?H&E staining and masson staining were used to detect the pathomorphologic changes of myocardial tissues in each group.?4?echocardiography was used to detect the changes of heart function in each group;RT-qPCR and Western Blot were used to detect the expression changes of ANP,BNP and?-MHC in myocardial tissues of each group.Results:?1?compared with the control group,the myocardium of the TAC model group significantly increased in thickness,heart/body weight ratio and myocardial cell area,with statistically significant differences.The mRNA expression of Tincr was significantly decreased.The mRNA and protein expressions of ANP,BNP,and?-MHC in mouse myocardial tissue were significantly increased.?2?compared with the no-load virus+TAC group,the mRNA level of Tincr overexpressed in the Tincr+TAC group was significantly increased,the myocardial cell area was significantly decreased,and the heart-to-body weight ratio was decreased.The protein levels of ANP,BNP and?-MHC were significantly reduced,and the difference was statistically significant.?3?six weeks after TAC surgery,color doppler echocardiography indicated that LVPWs,LVPWd,IVSs,IVSd and EF of the four groups of mice were significantly different,and LVPWs,LVPWd,IVSs and IVSd of the TAC model group were significantly superior than that of the sham operation group,while EF was significantly lower.LVPWs,LVPWd,IVSs and IVSd were significantly decreased in the Tincr overexpression group and the viral vector group,while EF was increased.?4?H&E staining and masson staining indicated that the area of myocardial cells in TAC group was significantly larger than that in Sham group,and there was a little fibrosis.The area of cardiomyocytes in the Tincr overexpression+TAC group was significantly smaller than that in the viral vector+TAC group,but there was no significant statistical difference in fibrosis.Conclusion:?1?there was obvious pathological hypertrophy in the cardiac hypertrophy model of pressure overload mice established by thoracic aortic constriction,and the mRNA level of Tincr was significantly decreased.Overexpression of Tincr could lessen the degree of pathological cardiac hypertrophy caused by TAC,improve the function of the hypertrophic myocardium,and reduce the protein expression of ANP,BNP and?-MHC.Tincr is a target for intervention in the process of cardiac hypertrophy,but the mechanism of Tincr to attenuate cardiac hypertrophy needs to be clarified.Part 2: Tincr alleviates the effects of pressure overload-induced hypertrophy through mir-31-5pObjective: To identify the downstream target of Tincr and further discuss the role of Tincr in cardiac hypertrophy.Methods:?1?Cultivate H9c2 cells treated by Ang ? to observe Tincr mRNA expression and the changes of myocardial cells.The expression of ANP BNP ?-MHC mRNA and protein in cultured H9c2 cells were also detected.?2?Through the bioinformatics prediction of Starbase database,it was found that lnc RNA-Tincr may bind to miR-31-5p and miR-544.Dualluciferase experiment confirmed that mir-31-5p and mir-544 were direct targets of Tincr.The mRNA expressions of mir-31-5p and mir-544 were observed by RT-q PCR after transfection of H9c2 cardiomyocytes with the overexpressed plasmid pc DNA-Tincr by 0ug,2ug,5ug and 10 ug.?3?mRNA expressions of mir-31-5p and mir-544 in the two groups of the first part of the mouse stress load model were detected by RT-q PCR.?4?Bioinformatics analysis and luciferase assay were applied to detect the potential downstream target of miR-31-5p,PKC epsilon.?5?H9c2 cells treated with Ang?and followed by overexpression of Tincr were used to observe myocardial cell area changes by immunofluorescence and PKC epsilon mRNA expression by RT-q PCR.Results:?1?In the Ang?treared H9c2 cell line,compared with saline control group,Tincr mRNA expression levels were decreased in myocyte hypertrophy group,while the mRNA and protein expressions of ANP,BNP,and ?-mhc were elevated;?2?Double luciferase assay confirmed that miR-544 was bound to lnc RNA-Tincr in H9c2 cardiomyocytes,while,miR-31-5p was also bound to lnc RNA-Tincr in H9c2 cardiomyocytes.However,the enzymatic activity of the mutated miR-544 and miR-31-5p showed no significant difference from that of the cell vector.The mRNA expressions of miR-31-5p and miR-544 were inhibited in H9c2 cardiomyocytes overexpressed with Tincr,respectively,in a dose-dependent manner.As the Tincr dose increased,miR-31-5p and miR-544 decreased in a concentration-dependent manner.?3?in the mouse model of myocardial hypertrophy induced by pressure overload,only the mRNA expression of miR-31-5p was increased,while miR-544 did not change.?4?the potential target of miR-31-5p in bioinformatics analysis was PKC epsilon.The double luciferase experiment showed that compared with the miR-NC group,the luciferase activity in the WT group significantly decreased,while the luciferase activity in the mut-PKC? group showed no difference.?5?After Ang?treatment,the area of cardiomyocytes increased compared with that of the control group,and the area of overexpressed Tincr decreased.Compared to the control group,PKC epsilon's mRNA level was reduced by Ang ? treated H9c2 group,but overexpression of Tincr raised PKC epsilon mRNA level after combined treat.Conclusion:?1?miR-31-5p was confirmed to be the direct target of Tincr in vivo or vitro model.Tincr may attenuate cardiac hypertrophy by inhibiting miR-31-5p and affecting the expression of PKC epsilon.The potential target of mir-31-5p is PKC epsilon.Part 3 Study on the signal transduction pathway of Tincr to attenuate cardiomyocyte hypertrophyObjective: To further investigate whether Tincr can inhibit cardiac hypertrophy through miR-31-5p,and to elucidate the signaling pathway of Tincr to attenuate cardiac hypertrophy.Methods: H9c2 cardiomyocytes were cultured by miR-31-5p-mimic intervention and the expression of PKC epsilon was detected by RT-q PCR and western blot.?2?lnc RNA Tincr overexpressed plasmids were transfected and co-transfected with miR-31-5p/miR-544 inhibitor to observe their effects on mRNA and protein expression of PKC epsilon.?3?lentivirus was used to interfere with lnc RNA Tincr or sh-tincr + mir-31-5p inhibitor to inhibit or overexpress sh-tincr +PKC?,and then mRNA expression of mir-31-5p and PKC were observed.Protein expression of PKC??ANP? BNP and ?-MHC were detected by western blot.?4?the effect of Tincr overexpression on miR-31-5p and PKC?in mouse myocardium was verified by western blot and immunohistochemistry.Results :?1?In H9c2 cardiomyocytes,overexpression of mir-31-5p inhibited the mRNA expression and protein expression of PKC? in a concentration-dependent manner.?2?after inhibition of miR-31-5p or miR-544 in H9c2 cardiomyocytes,the mRNA expression of PKC? in the miR-31-5p inhibitor group was found to be statistically significant higher,and the mRNA and protein expressions of the inhibition of miR-31-5p + overexpression of Tincr group were not statistically significant compared with those of the inhibition miR-31-5p group.The differences of mRNA and protein expression in the group with overexpression of Tincr+ miR-544 inhibitor were statistically significant when compared with the group with miR-544 inhibitor alone.?3?interference with Tincr caused the increase of H9c2 cardiomyocyte area,and inhibition of mir-31-5p or overexpression of PKC epsilon could inhibit the hypertrophy effect of cardiomyocytes.Interference with Tincr can increase the mRNA level of miR-31-5p.When Tincr was inhibited and miR-31-5p was inhibited,the mRNA and protein expressions of PKC? were increased.Interference with Tincr can increase the protein expression of ANP,BNP and ?-MHC in H9c2 cardiomyocytes,while H9c2 cells treated with sh Tincr and mir-31-5p inhibitor or sh Tincr and overexpression of PKC?can be observed down-regulate the protein expression of ANP,BNP and?-MHC.?4?the inhibition of mir-31-5p was enhanced in the model of cardiac hypertrophy in mice with overexpression of Tincr,so as to up-regulate the protein expression of PKC?.Conclusion: Inhibition of the expression of miR-31-5p rather than miR-544 could block the regulation of Tincr on the expression of PKC?,which was downstream of mir-31-5p.Inhibition of miR-31-5p or overexpression of PKC? can reverse the hypertrophy effect of cardiomyocytes induced by Tincr interference.Tincr regulated the mRNA and protein expression of PKC? by down-regulating mir-31-5p. |
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