| BackgroundCardiac hypertrophy is an adaptive response of the heart to a variety of inducing factors.Cardiac hypertrophy can help restore normal function of heart,eliminating the influence of external stimuli.But cardiac hypertrophy will develop into heart failure or sudden cardiac death when it lasts too long and it will seriously endanger human health.At present,the incidence and mortality of cardiovascular diseases in China have remained high,which has become a major public health problem.And cardiac hypertrophy is one of the most dangerous factors for cardiovascular disease and sudden death.At present,the clinical treatment of cardiac hypertrophy can only be delayed.Therefore,how to detect and prevent the occurrence of cardiac hypertrophy in the early stage has become the focus of clinical attention.To solve the problems of early diagnosis and new treatment methods in clinical,it is the key to further study on the molecular mechanism of cardiac hypertrophy.Long non-coding RNA(lncRNA)is a class of non-coding RNA whose length is more than 200 nt.It plays an important role in the regulation of cardiac development and cardiovascular diseases.mhrt(myosin heavy chain associated RNA transcript)is a kind of lncRNA encoded by the gene of myosin heavy chain 7 and produced by variable splicing.It is initially thought to inhibit cardiac hypertrophy by Brg1 and play a protective role in the heart.Moreover,the function of mhrt in mice and humans is highly conservative,which indicates that mhrt may become a molecular target for clinical diagnosis and treatment of cardiac hypertrophy.However,the regulatory mechanism of lncRNA is relatively complex,which can regulate the physiological function at many levels.Especially,it can form a molecular regulatory network with miRNAs or transcription factors to exercise the regulatory functions together.Myocardin,as a key transcription factor inducing cardiac hypertrophy,has been extensively concerned and discussed about its regulatory function and molecular mechanism in the process of development and disease occurrence.However,in the process of occurrence and development of cardiac hypertrophy,it is not clear whether there is a correlation between mhrt and myocardin.At present,there are few studies on the molecular mechanism of mhrt regulating cardiac hypertrophy.Especially,it has not been reported at home and abroad about the relationship between mhrt and myocardin in cardiac hypertrophy.Therefore,the study of the interaction and mechanism between mhrt and myocardin is of great significance and value for the complement of the molecular mechanism of the occurrence and development of cardiac hypertrophy.ObjectiveIn this study,molecular biology,cell biology and other technical means were used to reveal the interaction and molecular mechanism between mhrt and myocardin at the molecular level,cell level and animal level,and explore the regulatory role of the relationship between them in the occurrence and development of cardiac hypertrophy.This study can further improve the molecular mechanism of the occurrence and development of cardiac hypertrophy.Meanwhile,it can provide early diagnosis and treatment targets for the prevention and treatment of cardiac hypertrophy in clinic.MethodsPart 1: Myocardin was overexpressed by lentiviral transduction in primary mouse cardiomyocytes,and then overexpressed or knocked down mhrt.RT-PCR,Western Blot and immunofluorescence assay were used to detect the expression of marker genes related to cardiac hypertrophy(ANP,?-MHC,ACTN2),and to explore whether mhrt could affect the regulation function of myocardin on cardiac hypertrophy.At the same time,RT-PCR and Western Blot experiments were used to explore whether mhrt could regulate the expression of myocardin.Part 2: Using bioinformatics and pathway analysis,combining with the experimental techniques on RT-PCR,Western Blot,immunofluorescence,immunohistochemistry and H&E staining,we explored whether mhrt could regulate the expression of myocardin and cardiac hypertrophy through KLF4 at molecular,cellular and animal levels.Then further exploring the molecular mechanism of mhrt regulating the expression of KLF4:(1)Using bioinformatics software such as microcode and mibase to analyze and predict,combining with the experimental techniques on RNA in situ hybridization,luciferase activity analysis,RIP,MS2-RIP and so on,we explored whether mhrt could regulate the expression of KLF4 and exert the function of competitive endogenous RNA by binding to microRNA-145a-5p;(2)Since the phosphorylation of KLF4 protein can be regulated by ERK signaling pathway.Phosphorylated KLF4 protein can be further modified by ubiquitination leading to protein degradation.Therefore,Co-immunoprecipitation experiment was used to explore whether mhrt could regulate the phosphorylation level of KLF4 protein.Then,RPISEQ software was used to explore whether there was a direct binding between mhrt and KLF4 protein,which was confirmed by Co-immunoprecipitation,RNA pull down,RIP and other experiments.Finally,by combining ERK agonists and ERK inhibitors with Co-immunoprecipitation experiments,we explored whether mhrt could reduce the phosphorylation level of KLF4 by competing with ERK,and revealed the molecular mechanism of mhrt regulating the phosphorylation of KLF4 protein.Part 3: The maintenance of normal function of myocardin protein is related to its acetylation level,and HDAC5 can induce deacetylation of myocardin protein.Therefore,Co-immunoprecipitation,luciferase activity analysis and immunofluorescence assay were used to explore whether mhrt could regulate the acetylation level of myocardin protein through HDAC5 and its molecular mechanism.Part 4: Lentiviral transduction was used to overexpress or knock down myocardin in primary mouse cardiomyocytes.Then,RT-PCR and Western Blot experiments were used to explore whether myocardin could reverse the expression of mhrt.Next,the software of UCSC and TargetScan were used to determine the binding site(CarG Box)of myocardin/SRF complex in the promoter region of mhrt.Meanwhile,they were verified by the experiments of luciferase activity and immunoprecipitation of chromatin.ResultsPart 1: RT-PCR,Western Blot and immunofluorescence results confirmed that mhrt could significantly inhibit myocardin's promoting effect on cardiac hypertrophy,and mhrt could inhibit myocardin expression at RNA and protein levels.Part 2: To explore the molecular mechanism of mhrt regulating the expression of myocardin,we found that mhrt could regulate the expression of myocardin and cardiac hypertrophy through KLF4.(1)The results of RT-PCR and Western blot showed that mhrt could promote the expression of KLF4;(2)Further experiments in vivo and in vitro confirmed that mhrt lost its function of regulating myocardin expression and cardiac hypertrophy after knocking down KLF4,which indicated that mhrt could regulate the expression of myocardin and the process of cardiac hypertrophy through KLF4;(3)Subsequently,we further studied the molecular mechanism of mhrt regulating the expression of KLF4 and found that mhrt could regulate the expression of KLF4 in two ways.On the one hand,mhrt could control the expression of KLF4's mRNA by sponging miRNA-145a-5p;on the other hand,mhrt could inhibit the phosphorylation of KLF4 protein by competing with ERK1/2 which prevented further ubiquitination modification and degradation of KLF4 protein,thus up-regulating the level of KLF4 protein.Part 3: The results of Co-immunoprecipitation and luciferase activity analysis confirmed that mhrt could significantly inhibit the acetylation level of myocardin,and this process was achieved by mediating HDAC5.Further immunofluorescence results showed that mhrt could inhibit the acetylation level of myocardin protein by promoting the nucleation of HDAC5.It could inhibit the expression of genes related to the activation of cardiac hypertrophy through reducing the affinity between myocardin protein and downstream target genes.Part 4: RT-PCR results confirmed that myocardin could reverse-activate the expression of mhrt.Through further study results of these experiments on luciferase activity and chromatin immunoprecipitation,we found that the molecular mechanism of myocardin regulating mhrt was that myocardin could bind with CarG Box near the transcription initiation site in the promoter region of mhrt to promote the expression of mhrt.ConclusionThe occurrence of cardiac hypertrophy is regulated by many factors.The interaction between these transcription factors,miRNAs and lncRNAs construct a huge molecular network to regulate the occurrence and development of cardiac hypertrophy.Here,we first discovered the interaction between mhrt and myocardin and its molecular mechanism in the occurrence and development of cardiac hypertrophy,and revealed a new molecular network to regulate cardiac hypertrophy,including mhrt,KLF4,mir-145a-5p,HDAC5 and myocardin.And the molecular mechanism of mhrt regulating KLF4 expression,the molecular mechanism of mhrt regulating myocardin expression and protein activity,and the revelation of "lncRNA-mhrt/miR-145a-5p/KLF4/myocardin" signaling pathway are all innovative points in this study.The results of this study provide a new theoretical basis for elucidating the mechanism of cardiac hypertrophy and also provide a new target for clinical treatment of cardiac hypertrophy. |
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