The Mechanism Of Circ?0008082 Targeting YTHDF2 By Competitively Binding MiR-145-5p To Regulate Cardiac Dysfunction Of Rats With Septic Shock

Objective: Sepsis is associated with a high fatality rate due to multiple organ dysfunction,including cardiac dysfunction.The incidence of sepsis combined with cardiac dysfunction seriously affects the prognosis of patients.Septic shock was developed when metabolic abnormalities and circulatory disturbances occur in sepsis.Septic shock was associated with a higher incidence of cardiac dysfunction and was independently associated with increased mortality.In order to save the lives of patients,there is an urgent need to explore new therapeutic targets and reduce the fatality rate of severe patients.However,the pathogenesis of cardiac dysfunction in septic shock is complex,which is related to cytokine inhibition,cell apoptosis,energy metabolism and other factors.Currently,it is believed that the occurrence and development of cardiac dysfunction in septic shock is the result of the combined action of multiple mechanisms.CircRNAs are a new class of endogenous non-codingRNAs.When the current body mRNA matures,a closed continuous ring formed by reverse splicing of an exon or intron.CircRNAs are widely found in eukaryotic cells,which are both tissue and cell specific and highly stable due to their special circular structure.CircRNA plays a potential role in gene regulation in a variety of life processes.Abnormal expression of circRNA in various cardiovascular diseases is involved in the regulation of various biological functions such as inflammatory response and apoptosis of cells.To investigate whether CircRNA play a role in the heart dysfunction of septic shock,we based on the transcriptome sequencing results of myocardial tissue from septic shock rats and normal control rats,and bioinformatics analysis was used to screen the circRNA with difference.Furthermore,the role and regulatory mechanism of circRNA in cardiac dysfunction in septic shock rats were further investigated.Methods: Based on the total transcriptome sequencing results of myocardial tissue of rats with septic shock(6 rats)and control group(6 rats).Bioinformatics analysis was performed to screen the differentially expressed circRNA,miRNA and mRNA expression profiles,to construct circRNAs mediated ceRNA regulatory networks related to cardiac dysfunction in septic shock.Cardiac dysfunction model of septic shock was established by intraperitoneal injection of LPS(20mg/kg)in rats.Left ventricular myocardial tissue and plasma were collected 12 hours after LPS injection.H&E staining was used to determine the damage of myocardial tissue in rats.Plasma expression levels of pro-inflammatory cytokines(TNF-?,IL-1?,IL-6)and markers of myocardial cell injury(CKMB,c Tn I)were determined by ELISA.Real-time PCR was used to detect the expression levels of pro-inflammatory cytokines(TNF-?,IL-1?,IL-6)and apoptosis-related proteins(Bax,Bcl2,caspase3)in myocardial tissue.The apoptotic level of myocardial tissue was detected by TUNEL and Western blot.The above experiments proved that after the model was successfully established,the accuracy of the sequencing results was verified by qRT-PCR method in rat cardiac tissue.Rat H9c2 cardiomyocytes were induced by LPS(10?g/m L)to establish septic shock model in vitro.Some differentially expressed circRNAs were verified at the cellular level,and circ?0008082 was screened out.Sanger sequencing,RNase R digestion and Agarose gel electrophoresis performed using convergent and divergent primer amplification products were performed,and circ?0008082 was proved to be a real circRNA.The specific probe of circ?0008082 was designed,and the location of circ?0008082 was identified by Fluorescence in situ hybridization(FISH).CeRNA regulatory network was conformed based on bioinformatics,dual luciferase reporter gene analysis was used to verify the miRNA that could bind to circ?0008082 and mRNA that could bind to miRNA.Furthermore,the circ?0008082 mediated circRNAmiRNA-mRNA regulatory axis was obtained through gene knockdown and overexpression,and the function of circRNA-miRNA-mRNA was studied at the cellular level.The inflammatory damage of myocardial cells was evaluated by ELISA assay.Apoptosis levels of cardiomyocytes were analyzed using flow cytometry.Western blotting(WB)was used to analyze the expression levels of apoptosis-related proteins(Bax,Bcl2 and caspase3).Results:1.Through the analysis of transcriptome sequencing results,19 circRNAs with significant differential expression were screened out,of which 12 were up regulated and 7 were down regulated.Among 63 miRNAs with significant differential expression,50 were up regulated and 13 were down regulated.There were 1,951 differentially expressed mRNAs,of which 937 were up regulated and 654 were down regulated.By constructing a ceRNA regulatory network,we screened out circRNA-miRNA-mRNA regulatory network closely related to cardiac dysfunction in rats with septic shock,including 11 circRNAs,21 miRNAs,and 41 mRNAs.2.We successfully induced a rat model of septic shock with LPS.About 2 hours after intraperitoneal injection of LPS,the blood pressure of the rats began to drop,and the decrease level was stable until 12 hours.Compared with the control group,H&E staining showed that the myocardial tissue of rats with septic shock was disordered,with pale cytoplasm,edema,dissolution,necrosis,and infiltration of a large number of inflammatory cells.Myocardial tissue and serum inflammatory biomarkers(tumor necrosis factor-?[TNF-?],interleukin-1? [IL-1?],interleukin-6 [IL-6])were significantly increased.Serum markers of myocardial injury(troponin I[c Tn I] and creatine kinase isoenzyme MB[CKMB])were significantly elevated.TUNEL staining showed that the apoptotic rate of myocardium in rats with septic shock was significantly increased.The mRNA and protein levels of Caspase-3 and Bax,which are related to myocardial tissue apoptosis,were significantly increased,while the mRNA and protein levels of Bcl2 were significantly decreased.The differential expression of circRNA was verified in rat cardiac tissue,and the expression level was consistent with the sequencing results,indicating the accuracy of the sequencing results.3.Partly differentially expressed circRNAs were verified in rat H9c2 cardiomyocytes,the results showed that the expression level of circ?0008082 was significantly increased in LPS-induced H9c2 cardiomyocytes.The ring splicing site of circ?0008082 was confirmed by Sanger sequencing,the results were in good agreement with the predicted sequence of cyclization sites.However,head-to-tail splicing can be produced by trans-splicing or genome rearrangement,in order to eliminate these two possibilities,we further designed convergent and divergent primers for amplification using cDNA and genomicDNA(g DNA)as templates,respectively.The results showed that there were 169 bp and 222 bp amplified products in both convergent and divergent primers in cDNA,and only 222 bp amplified products in convergent primers in g DNA.Under RNase R treatment,circ?0008082 could not be degraded by RNase R due to its covalent closed-loop structure.FISH results showed that circ?0008082 was mainly localized in the cytoplasm.The knockdown and overexpression of circ?0008082 showed that circ?0008082 was involved in the inflammatory damage and apoptosis of cardiomyocytes in septic shock.4.Dual luciferase reporter gene analysis showed that circ?0008082 could bind to miR-145-5p and miR-145-5p could bind to YTHDF2.Circ?0008082 has a negative regulatory relationship with miR-145-5p,miR-145-5p has a negative regulatory relationship with YTHDF2,and circ?0008082 has a positive regulatory relationship with YTHDF2.The expression level of miR-145-5p was significantly decreased in myocardial tissue and LPSinduced H9c2 cardiomyocytes of rats with septic shock,while the expression level of YTHDF2 was high.Functional experiments demonstrated that both miR-145-5p and YTHDF2 were involved in LPS-induced inflammatory injury and apoptosis of H9c2 cardiomyocytes.Conclusion:1.CircRNAs,miRNAs and mRNAs were differentially expressed in cardiac tissues of septic shock rats,circRNA-miRNA-mRNA regulatory network was successfully constructed.It suggested that these differentially expressedRNAs could play an important role in cardiac dysfunction of rats with septic shock.The accuracy of the sequencing results was verified by circRNAs in the myocardium of septic shock rats.2.The expression level of circ?0008082 was increased in myocardial tissue of rats with septic shock and LPS-induced H9c2 cardiomyocytes,it is a real circRNA that promotes inflammatory damage and apoptosis in LPS-induced H9c2 cardiomyocytes.3.The expression level of miR-145-5p was decreased in myocardial tissue of rats with septic shock and LPS-induced H9c2 cardiomyocytes,and inhibits inflammatory damage and apoptosis in LPS-induced H9c2 cardiomyocytes.4.The expression level of YTHDF2 was increased in myocardial tissue of rats with septic shockand LPS induced H9c2 cardiomyocytes,and decrease of YTHDF2 inhibited LPSinduced inflammatory damage and apoptosis of H9c2 cardiomyocytes.5.Circ?0008082 targets YTHDF2 by competitively binding miR-145-5p to promote LPSinduced inflammatory damage and apoptosis of H9c2 cardiomyocytes.In conclusion,We found a new circRNA,circ 0008082,discussed its role and mechanism in rats of septic shock with cardiac dysfunction.This study provides a theoretical basis for its potential role as a molecular marker and therapeutic target for cardiac dysfunction in septic shock.