Construction Of CeRNA Network Regulatory Mechanism Based On Hepatocellular Carcinoma Tissue Sequencing And TCGA Database RNAs

Objective:Primary hepatocellular carcinoma is mainly composed of hepatocellular carcinoma(HCC),intrahepatic cholangiocarcinoma(ICC)and combined hepatocellular-cholangiocarcinoma(CCHCC-CCA).In China,hepatocellular carcinoma(HCC)is the most common primary liver cancer,with the sixth highest incidence and the fourth highest mortality rate in the world.Although early diagnosis and treatment,radical surgery,radiotherapy and immunotherapy have made significant progress in the treatment of HCC,the low survival rate and high recurrence rate of HCC patients are still a major challenge in the treatment of HCC.Therefore,it is clinically important to explore the molecular mechanism of HCC development and to find new biomarkers for HCC diagnosis and treatment.The aim of this study is to screen the differentially expressed RNAs in hepatocellular carcinoma tissues as well as paraneoplastic tissues,construct ceRNA networks,explore the regulatory mechanism of hepatocellular carcinoma tumor progression ceRNA networks,and foundation for the next step of research.Methods:First,mRNA,lncRNA,miRNA,and circRNA libraries of clinical samples from six HCC patients were constructed,and whole transcriptome sequencing was performed to obtain differentially expressed ribonucleic acid(RNA)profiles.Then,bioinformatics was applied to GO-BP function enrichment analysis,KEGG signaling pathway enrichment analysis for differentially expressed mRNAs,and target genes of lncRNAs and miRNAs.The interaction relationships between different proteins were analyzed using STRING,and the PPI networks were analyzed modularly using Cytoscape MCODE plug-in.Competitive endogenous RNA(ceRNA)networks were constructed based on the targeting regulation relationships of dif-miRNA-dif-mRNA and dif-miRNA-dif-lncRNA.Next,the TCGA database of differentially expressed RNAs was screened for integration analysis.Finally,key miRNAs were validated using clinical specimens,and prognostic models were constructed.Results:(1)A total of 1280 differentially expressed mRNAs(dif-mRNAs),99 differentially expressed miRNAs(dif-miRNAs),181 differentially expressed lncRNAs(dif-lncRNAs),and 31 differentially expressed circRNAs(dif-circRNAs)were screened high-throughput sequencing analysis.(2)GO-BP enrichment analysis of differential mRNAs revealed that differential mRNAs were associated with biological processes such as cell division.the target genes of differential lncRNAs were significantly associated with processes such as ubiquitination of protein k63 linkage,and the target genes of differential miRNAs were significantly associated with processes such as axon guidance.KEGG analysis showed that differential mRNAs were significantly associated with fatty acid degradation,complement and coagulation cascade signalling pathways;differential lncRNAs target genes were significantly associated with retinol metabolism and chemical carcinogenesis,and differential miRNAs target genes were associated with pathways of cancer and aldosterone synthesis and secretion.(3)Cytoscape MCODE on four sub-network modules(score R≥5.0)extracted from the PPI network.GO-BP enrichment analysis was performed the four modules,which were mainly enriched for biological processes such as cell cycle and mitosis.(4)Construction of ceRNA network using Cytoscape based on the interregulation of dif-miRNA-dif-mRNA and dif-miRNA-dif-lncRNA.(5)Screening of differentially expressed RNAs in TCGA LIHC with sequencing analysis for integration analysis resulted in screening of 805 dif-mRNAs,6 dif-lncRNAs and 7 dif-miRNAs.(6)qRT-PCR validated7 dif-miRNAs,which were consistent with the preliminary screening results.In the KaplanMeier database survival analysis of these 7 miRNAs showed that patients with HCC with high expression of has-miR-1269 a,has-miR-421 and has-miR-190 b had significantly lower overall survival than those with low expression.Based on qRT-PCR results showed that high expression of has-miR-1269 a,has-miR-421 was associated with a shorter overall survival.Conclusion:In this study,we constructed a ceRNA network based on whole transcriptome sequencing data and analyzed 7 differentially expressed miRNAs.The Kaplan-Meier database as well as clinical specimen validation revealed that hsa-miR-1269 a,hsa-miR-421 were significantly associated with prognosis.It was suggested that hsa-miR-1269 a and hsamiR-421 could be potential targets to predict the prognosis of hepatocellular carcinoma and guide the treatment of hepatocellular carcinoma.