Preliminary Study On The Mechanism Of Circ-Mapk14 And Circ-Klc1 In Cardiac Hypertrophy

Background and purposeCardiac hypertrophy is an important compensatory response of the heart to a variety of pathological and physiological stimuli.Early cardiac hypertrophy is considered to be a beneficial compensatory response to the body,but long-term cardiac hypertrophy will gradually develop into heart failure.Scientists have revealed some of the mechanisms of cardiac hypertrophy,such as signaling pathways,but the in-depth mechanism remains to be elucidated.This subject is dedicated to finding more target molecules that regulate cardiac hypertrophy,to further clarify the molecular mechanism of the occurrence and development of cardiac hypertrophy,and to provide a theoretical basis for the clinical diagnosis and treatment of cardiac hypertrophy.Circular RNA(circRNA)is a type of single-stranded closed-loop RNA widely present in various organisms,formed by reverse splicing of precursor mRNA.It does not contain 5’cap and 3’polyA tail structure,and is connected by covalent bonds to form a circle.Therefore,circRNA is more stable than linear mRNA and is not easily degraded by RNase R.It is a hot spot in the RNA field in recent years.The main functions of circRNA include:affecting the transcription and splicing of linear mRNA through competitive splicing;acting as a miRNA molecular sponge;interacting with proteins;translating functional peptides.Studies have shown that circRNA plays an important role in the occurrence and development of some cardiovascular diseases.However,current research mainly focuses on circRNA as the molecular sponge of miRNA and interacting with proteins to regulate downstream target genes.There are few studies on circRNA encoding proteins,which are only limited to tumor invasion and metastasis.Based on this,we speculate that the protein encoded by circRNA related to cardiac hypertrophy can also participate in the occurrence of cardiac hypertrophy,thereby regulating its occurrence and development.Therefore,we have launched a research on this subject.The purpose of this study is to explore the molecular mechanism of cardiac hypertrophy-related circRNA-encoded protein in the occurrence and development of cardiac hypertrophy.It is expected to provide a new therapeutic target for the clinical treatment of cardiac hypertrophy and provide a theoretical basis for the development of ideal drugs for the treatment of cardiac hypertrophy.Methods1.Screen out circRNAs that are differentially expressed in cardiac hypertrophyBased on the previous circRNA high-throughput sequencing results of our research group,21 circRNAs that were differentially expressed in mouse cardiac hypertrophy induced by isoproterenol hydrochloride(ISO)were selected.2.Verify the above circRNAAccording to the results of circRNA high-throughput sequencing,the divergent primer of circRNA was designed near the reverse splicing site,the circRNA was verified by Real-time PCR experiment,and the product was Sanger sequenced.At the same time,the expression trend in the ISO treatment group and the control group is consistent with the results of circRNA high-throughput sequencing.Then the circRNA was further verified by RNase R digestion experiment and PCR amplification of genomic DNA and cDNA with divergent and convergent primers.3.Get the full length of circRNABy designing two pairs of PCR primers near the splicing site,the two ends of the amplified fragments overlap each other,and the two PCR products were sequenced by Sanger and compared with the mother gene sequence to obtain the full length of circRNA.4.Explore the effect of circRNA on cardiac hypertrophyAccording to the relationship between the mother gene of circRNA and cardiac hypertrophy,and a group of circRNA was selected for functional verification.The overexpression vector of circRNA was constructed and transfected into rat primary cardiomyocytes.At the same time,the primary cardiomyocytes were treated with ISO as a positive control,and the changes in the size of cardiomyocytes were observed by F-actin staining;Real-time PCR was used to detect the expression of cardiac hypertrophy marker genes ANF and BNP;Real-time PCR and Western-blotting were used to detect the expression of cardiac transcription factor GATA4.5.Explore the molecular mechanism of circRNA affecting cardiac hypertrophyUse the bioinformatics software circAtlas to predict the miRNA and protein that bind to circRNA,and verify whether it interacts with circRNA through RNA pull down or RNA-binding protein immunoprecipitation(RIP)experiments.6.Verify the coding ability of circRNAUse the bioinformatics software ORF Finder to analyze the full length of circRNA,and screen out the circRNA with open reading frame(ORF)across the splice site,and the ORF activity was verified by constructing an overexpression vector containing 3×flag tags,and circRNA with active ORF was screened for subsequent experiments.7.Explore the binding of circRNA-encoded protein to GATA4,an important regulatory factor for cardiac hypertrophyCo-immunoprecipitation(Co-IP)experiment was used to verify whether the protein encoded by circRNA binds to the transcription factor GATA4.Results1.Of the 21 circRNAs selected in mouse ventricular tissue,13 were confirmed to exist.Compared with the control group,the expression of circ-Atp2c1,circ-Dcun 1 d2,circ-14346,circ-Rmdn2,circ-Itpr2,circ-Klcl was up-regulated in the ISO treatment group,while the expression of circ-Mapk14 was down-regulated.This is consistent with the trend in high-throughput sequencing results.At the same time,the above 7 circRNAs are resistant to digestion by RNase R;divergent primers can only amplify the target product in cDNA,but cannot amplify the target product in genomic DNA,and the Sanger sequencing showed that the 5’end of the upstream exon and the 3’end of the downstream exon were covalently linked to form a ring with a 3’,5’-phosphodiester bond.2.Sanger sequencing obtained the full-length sequence of circ-Atp2cl,circDcunld2,circ-14346,circ-Rmdn2,circ-Mapk14,and circ-Klc1.3.In ISO-treated primary cardiomyocytes,the expression of circ-Mapk14 was down-regulated and the expression of circ-Klc1 was up-regulated,which is consistent with the results of high-throughput sequencing.Treatment of rat primary cardiomyocytes with overexpression of circ-Mapk14,circ-Klc1 and ISO can increase the size of cardiomyocytes,and up-regulate the mRNA expression of cardiac hypertrophy marker genes ANF and BNP.And the mRNA and protein expressions of cardiomyocyte transcription factor GATA4 were up-regulated after overexpression of circ-Mapk14.4.Bioinformatics predicts that circ-Mapk14 and circ-Klc1 may not bind to myocardial hypertrophy-related miRNAs(such as miR-29a,miR-23a,miR-233,etc.);RIP experiments show that circ-Mapk14 does not work by binding to EZH2 protein.5.The 3×flag fusion overexpression vector of Circ-Mapk14 can express 3×flag protein,but the 3×flag protein is not expressed after the start codon is mutated,and circMapk14 can encode a protein with 136 amino acids.6.The protein encoded by Circ-Mapk14 can bind to GATA4.Conclusions1.CircRNAs is ubiquitous in mouse hearts,in which circ-MAPK14 and circKLC1 promote the occurrence of cardiac hypertrophy.2.Circ-Mapk14 can encode a protein of 136 amino acids and bind to the transcription factor GATA4.