Mechanism Of MiRNA-195-3p In Regulating The Development Of Pericardial Fibrosis In Constrictive Pericarditis

OBJECTIVE: Constrictive pericarditis(CP),a rigid pericardium that limits heart filling,is a form of disease that causes diastolic heart failure.Risk factors for the development of CP include tuberculosis infection,cardiac surgery and radiation therapy,but most cases are still considered as idiopathic.Pericardial fibrosis is the most significant pathological feature of CP,but the molecular mechanism of its occurrence remains to be fully elucidated.In the process of pericardial fibrosis,the most important effector cells are pericardial interstitial cells(PICs),and their proliferation and activation are important parts of pericardial fibrosis.Bioinformatics analysis is now widely used to identify disease-specific genes,helping to pinpoint disease-associated biomarkers for clinical diagnosis and prognosis.MicroRNAs(miRNAs)are non-coding RNAs that play an important role in regulating gene expression in almost all biological events.In the past decade,the mechanism of miRNA involvement in various cardiovascular diseases has been explored in many in vitro and in vivo studies.Among them,miR-195 has been shown to promote cell division and apoptosis,and inhibit cell proliferation,but the mechanism of miR-195 in cardiovascular disease is not fully understood.Here,we first sequenced the whole transcriptome of CP and control samples,used bioinformatics analysis to predict the central genes in the potential mechanism of CP pathogenesis,and through tissue level verification and in vitro experiments to explore the regulatory role of miR-195-3p in pericardial fibrosis of CP.Method: This study is divided into three parts.In the first part,non-coding RNA and mRNA expression profiling and ceRNA network construction in constrictive pericarditis: CP samples and normal control samples were collected,and differentially expressed genes were screened from data obtained fromRNA-seq.Perform functional annotation analysis and construct a protein-protein interaction network(PPI)to study potential pathways and central genes that play key roles in CP.In addition,the establishment of ceRNA networks and the implementation of gene enrichment analysis(GSEA)to exploit key ceRNA pathways provide direction and theoretical basis for subsequent experiments.The second part,verification of miR-195-3p/MAPK8 expression in pericardial tissue of CP patients and human pericardial interstitial cells induced by TGF-β1 and its correlation with cell transdifferentiation: real-time PCR Taqman probe was used to detect the expression levels of miR-195-3p and MAPK8 in tissues,and immunohistochemical staining to observe the difference in expression of fibrosis indexes between the experimental group and the control group.At the same time,TGF-β1 was used to induce PICs to construct an in vitro cell model.Real-time PCR were used to detect the expression of α-smooth muscle actin(α-SMA),type Ⅰ collagen(Col-Ⅰ)and type Ⅲ collagen(Col-Ⅲ)in pericardial tissue and in vitro cell model.Statistical correlation between MAPK8 expression and the above three indicators in vitro cell models.The third part,in vitro regulation mechanism of miR-195-3p regulating MAPK8 in CP: culture of human PICs in vitro and divided into 4 groups: miR-195-3p inhibitor group,miR-195-3p mimic group,miR-195-3p inhibitor negative control(NC-inhibitor)group,miR-195-3p mimic negative control(NC-mimics)group.miR-195-3p mimics and inhibitor were transfected into PICs,mRNA expression of MAPK8,Col-Ⅲ and α-SMA was detected by real-time PCR and western blot method was used to detect MAPK8,α-SMA and other protein expression levels.Results: 1.In the present study,we took advantage of RNA-seq and miRNA-seq with CP and matched control samples collected in our hospital.Then 686 DEMs,32 DEMis,33 DELs and 155 DECs were screened for subsequent analysis.GO and KEGG pathway enrichment analysis of DEMs performed subsequently delineated that DEMs were mainly involved in inflammatory response related pathways,such as Fc gamma Rmediated phagocytosis,T cell receptor signaling pathway,B cell receptor signaling pathway and chemokine signaling pathway.It was intriguing that up-regulated DEMs were also enriched in these pathways in the level of KEGG,suggesting that inflammation exerted a significant impact on the CP generation.Then we established PPI network with the DEMs.Among them,POLR2 B,HRAS,JUN,UBC,HSP90AB1,LCK,RPS27 A,HLA-DRB1,MAPK8,HSP90AA1 and RAC2 were the top 11 genes with the highest degree,which was considered as hub genes.Additionally,following the identification of 377 predicted interactions among the differentially expressed genes,the ceRNA network was constructed to obtain a preliminary understanding of the connections between DEMs,DEMis and DECs.Finally,GSEA was conducted on the circRNAs within the ceRNA network to identify biological pathways and functions.We screened the ceRNA network of hsa＿circ＿0008679 and predicted that the regulatory relationship between miR-195-3p and MAPK8 downstream could be used for subsequent studies.2.Real-time quantitative PCR was used to detect the expression levels of miR-195-3p and MAPK8 in tissues.Among them,miR-195-3p was down-regulated in the pericardium of CP patients,and MAPK8 was up-regulated in the pericardium of CP patients,which was consistent with the sequencing results.TGF-β1 was used to induce PICs to establish pericardial fibrosis in vitro cell model.Real-time quantitative PCR were used to detect the time-dependent increase in α-SMA,Col-Ⅰ and Col-Ⅲ expression in PICs,the time-dependent increase in MAPK8 expression,and the mRNA expression of miR-195-3p showed a downward trend.MAPK8 expression is consistent with the mRNA expression trend of α-SMA,ColI,Col-Ⅲ.3.Real-time quantitative PCR detection showed that after transfection of miR-195-3p inhibitor in PICs,miR-195-3p expression was down-regulated and MAPK8,ColI and Col-Ⅲ expression was up-regulated;after transfection of miR-195-3p mimic,miR-195-3p expression was up-regulated,MAPK8,Col-Ⅰ and Col-Ⅲ expression was downregulated.Western blot analysis showed that after transfection with miR-195-3p inhibitor,MAPK8 expression was up-regulated,and α-SMA expression levels were significantly increased;after transfection with miR-195-3p mimic,MAPK8 expression was downregulated,α-SMA expression levels were significantly reduced.Conclusion: 1.Using bioinformatics to analyze the pathogenesis of CP for the first time,11 key genes were obtained and a ceRNA network was constructed,and the predicted targeting relationship of miR-195-3p and MAPK8 was selected for subsequent experiments.Verification showed that the production of CP was closely related to inflammation and fibrosis;2.Constructing an in vitro model of pericardial fibrosis,verifying that MAPK8 is up-regulated in CP tissue and cell model,miR-195-3p is downregulated,the expression of MAPK8 in cell model is consistent with the trend of cell activation and collagen synthesis expression.3.MiR-195-3p can inhibit MAPK8,thereby inhibiting the ability of PICs to synthesize collagen and differentiate into myofibroblasts,suggesting that miR-195-3p may participate in the process of pericardial fibrosis by regulating MAPK8.