Investigating Molecular Mechanisms For Rhamnetin In Oxidative Damaged Cardiomyocytes By RNA-Seq Analysis

Background and objective:Oxidative stress injury is an important pathogenic factor in cardiovascular disease.Ischemia and hypoxia can lead to the production of large amounts of reactive oxygen species（ROS）by cardiac myocytes,which plays an important role in the regulation of vascular endothelial function,vascular tension,cardiac function,inflammation,hypertrophy,proliferation,apoptosis,migration and angiogenesis.Rhamnetin is one of the flavonoids with high biological activity,which has a protective effect on myocardial oxidative stress.However,there are few studies on the molecular biological mechanism of the action of Rhamnetin.Hence,the purpose of this study was to use RNA-seq analysis technology and a variety of bioinformatics methods to reveal the molecular mechanism of Rhamnetin protecting myocardial oxidative stress injury,which will provide a new target for clinical treatment.Methods:Injury model was established by inducing H9C2 myocardial cells with H₂O₂.H9C2cells treated with H₂O₂ were the control group,and cells treated with H₂O₂+Rhamnetin were the treatment group.RNA was extracted for RNA-seq sequencing,and the biological processes were as following:（1）Screening and interaction network construction of differentially expressed genes（DEGs）.The DEGs were calculated using NOISeq data analysis kit with the screening conditions of P value<0.05 and|log2FC|>2.Function and pathway enrichment analysis of DEGs were performed based on Gene ontology and KEGG analysis.According the String database,the protein interaction（PPI）networks were established with the condition of combined score>0.9.On the basis of Cytosape,subnetwork modules were carried out,with threshold setting:Degree Cutoff=2,Node Score Cutoff=0.2,k-core=2,Max.Depth=100.（2）In-depth analysis of data on DEGs.The miRNAs regulated with DEGs was retrieved by miRWalk2.0,and the functional enrichment analysis of miRNA was performed by using R clusterprofiler.MiRNA-lncRNA relationship pairs were screened with score>180based on miRBase and Miranda.GO-BP function and KEGG pathways enrichment of DEGss in ceRNA and miRNA-TF-gene networks were analyzed using DAVID.WebGestalt was used to predict the transcription factors of DEGs.Based on DGIdb,the drug-gene interaction pairs of DEGs regulated by miRNA and TF were predicted.MiRNA-gene,miRNA-lncRNA,ceRNA,TF-gene,miRNA-TF-gene and drug-gene interaction network were constructed according to Cytoscape software.（3）Validation of DEGs with qPCR.Based on the DEGs predicted by bioinformatics,up-regulated expression genes（NOTCH3,NUMBL,BCL2L11）and down-regulated expression genes（PPP6C,MAPK1）were screened,and the differences of DEGs in the control group（H₂O₂ treatment）and the experimental group（H₂O₂ treatment+Rhamnetin administration）were detected by qPCR.Results:（1）A total of 1,452 DEGs between by the control group and experimental group were obtained,with 703 up-regulated and 749 down-regulated genes,respectively.GO functional enrichment analysis showed that the biological processes enriched by DEGs mainly include cellular metabolic or macromolecule catabolic processes,mitotic cell cycle,regulation of metabolic or signaling pathways and morphogenesis.The key signaling pathways involved in the enrichment analysis of KEGG pathway include cellular adhesion,Notch,Oxidative phosphorylation and proteolysis.PPI network was consisted of 609 nodes;in addition,two modules were obtained with score>6,of which module 1 covered 17 nodes and was enriched in functions related to mRNA degradation,translation and protein localization and module 2covered 15 nodes,mainly focusing on the functions related to cell division.（2）The cluster heat map analysis of DEGs showed that there was a significant difference between the experimental group and the control group.For the prediction of miRNAs regulated by DEGs,189 miRNAs and 577 miRNA-gene regulatory pairs were obtained,among which the key miRNAs included miR-349,miR-211-5p and miR-17-5p.KEGG pathway enrichment analysis showed that the predicted miRNA significantly enriched to 75 pathways,among which AMPK and PI3k-Akt signal pathways had the highest number of miRNAs.Next,the network of miRNA-gene regulation was performed,and a total of 120 nodes and 216 interaction relationship pairs were obtained.Based on prediction of lncRNAs regulated by the miRNAs,a total of 475 miRNA-lncRNA relationship pairs were obtained.According to the ceRNA network,there were 74 mirRNAs,90 mRNAs and 60 lncRNA included in the network.GO function and KEGG pathway enrichment analysis showed that the biological processes mainly included the positive regulation of transcription from RNA polymerase II promoter（GO:0045944）,negative regulation of apoptotic process（GO:0043066）and positive regulation of transcription,DNA-templated（GO:0045893）,and pathways mainly included Adrenaline and Notch signaling pathways.In addition,nine transcription factors（PAX4,ELK1,ETS2,NRF1,GABP,E4F1,CACBINDINGPROTEIN,AP2ALPHA and AP2GAMMA）were predicted based on the DEGs.Next,241 regulatory relations were obtained through the integration of miRNA-TF-gene regulatory network,and the functional enrichment and pathway enrichment were respectively enriched to 145 GO-BP results and 52 KEGG pathways,which were consistent with the CeRNA network.Finally,the network of Drug-gene interactions were conducted,and the results showed BCL2L11,DNMT3A,HDAC2,and STAT were important drug small molecule genes when combined with the analysis of genes related to Drug reactions by enriching biological processes.（3）The results of qPCR showed that,compared with the control group,the expression levels of NOTCH3 and MAPK1 were significantly downregulated（P<0.05）and the expression level of BCL2L11 was significantly up-regulated（P<0.05）in the groups treated with rhamnetin.However,while the expression trend of the up-regulated gene BCL2L11 and the down-regulated gene MAPK1 was consistent with the previous prediction results,but the differences were not significant（P>0.05）.Conclusion:In summary,this study was to investigate the molecular mechanism of rhamnetin on myocardial oxidative stress injury based on RNA-seq sequencing.The key DEGs including COX6A2,NDUFA2,UBA52,CDK1,MAPK1,Klf9 and NAA15 were obtained from rhamnetin treatment group and the control group.MiR-211-5p and miR-17-5p were the important miRNAs,and the important transcription factors included ELK1 and PAX4.Moreover,the Notch,AMPK and PI3K/Akt signaling pathways were crucial pathways,and the key drug small molecule genes included BCL2L11,DNMT3A,HDAC2 and STAT3.At the same time,this study constructed a series of regulatory networks,and systematically expounded the interaction between different acting factors in the protective effect of rhamnetin on myocardial oxidative stress injury,which will also provide important basis for future research.