Regulatory Network Analysis Of Rat Liver Regeneration Related LncRNA Based On Biological Network

Liver regeneration is a process of tissue growth after loss or injury of liver tissue.It is a compensatory proliferation rather than a true regeneration,which is mainly dependent on the proliferation of hepatocytes.Because many liver disease treatment strategies,such as liver tumor resection and liver transplantation,depend on the ability of the liver to regenerate,the study of liver regeneration has important clinical significance.A variety of cytokines and growth factors,such as IL-6 and HGF,constitute a complex network involved in the regulation of the hepatocyte cycle to ensure the smooth progression of liver regeneration.Recent studies have shown that miRNAs play important roles in liver regeneration,such as miR-21 and miR-221.Although various cytokines,growth factors,and miRNAs have been shown to regulate hepatocyte proliferation in liver regeneration,it is still urgent to find new therapeutic targets for the treatment of liver diseases.LncRNA is a class of noncoding RNA molecules without protein coding ability.A large number of studies have shown that lncRNA is involved in a variety of cell physiological activities,including cell proliferation,cell differentiation,cell apoptosis,stress response,chromosome modification and carcinogenesis.Although studies have shown that lncRNA is involved in liver regeneration,the overall regulation mechanism of lncRNA in liver regeneration remains unclear.In this study,high-throughput sequencing was used to detect the expression levels of lncRNA and mRNA in rat regenerative liver.It was found that 2446 lncRNA and 4091 mRNA were differentially expressed in rat regenerative liver.Functional enrichment analysis revealed that differentially expressed mRNAs were mainly involved in p53,PPAR,PI3K-Akt,MAPK,metabolic and Fox O pathways.LncRNA could act on adjacent protein-coding genes in a cis-regulatory manner.We searched for the differentially expressed protein-coding genes 10 kb upstream and downstream of differentially expressed lncRNAs,and found 852 lncRNAs that were transcribed closed to 932 mRNAs(<10 kb).Through GO enrichment analysis of lncRNA target genes,it was found that 78 GO terms in biological processes were significantly enriched,including organ regeneration,chromatin silencing,nucleosome assembly,cell cycle regulation,gene expression regulation and stress response.Other genes,including PFKFB1,SHC1,ADH1,AHSG,APOA2,CAST,CCNA2,CDKN1 A,HMGB2,HMBS,LIFR,LPIN1,MKI67,NNMT,SLCO1A5 and SOCS3,were associated with organ regeneration.It suggested that lncRNAs may regulate the process of liver regeneration by acting on adjacent protein-coding genes.We also predicted the trans-regulated target genes of lncRNA by co-expression analysis.The Pearson's correlation coefficients(PCC)were calculated based on the expression levels of differentially expressed lncRNA and mRNA,and an lncRNA-mRNA co-expression network was constructed.The results showed that 59010 pairs of significant lncRNA-mRNA,containing 689 lncRNAs and 1503 mRNAs.Among them,52382 pairs were positively correlated(PCC?0.8)and 6628 pairs were negatively correlated(PCC?-0.8).It suggested that lncRNA mainly regulated gene expression in a positive manner.Subsequently,the screened lncRNA-mRNA pairs were used to construct co-expression network,and it was found that some lncRNAs could interact with multiple mRNAs.According to the correlation degree of nodes,the top two lncRNAs were NONRATT016047.2 and NONRATT008570.2,indicating that they may play an important role during rat liver regeneration.Through functional enrichment analysis of lncRNA target genes,these target genes are mainly involved in biological processes such as cell proliferation,redox,stress response and organogenesis.LncRNAs could be target molecule of miRNA and regulated by miRNA.LncRNA-miRNA interaction analysis could help to analyze the function and mechanism of lncRNAs acting as miRNA sponges.Miranda algorithm was used to predict the interaction between lncRNA and miRNA.Based on miRNA-mRNA interaction analysis and miRNA-lncRNA interaction analysis,miRNA that could interact with mRNA and lncRNA at the same time were screened and the regulation network of lncRNA-miRNA-mRNA was constructed,which consisted of 50 miRNAs,196 lncRNAs and 430 mRNAs.Among them,miR-21,miR-127,miR-34 a,miR-378,miR-125 b,miR-23 b and miR-503 have been reported to play an important role in liver regeneration.According to the interaction between miRNA and lncRNA,20 key lncRNAs were identified,namely NONNATT000367.272,NONNATT001051.2,NONRATT005931.2,NONRATT007218.2,NONRATT007757.2,NONRATT008342.2,NONRATT011758.2,NONRATT012812.2,NONRATT013355.2,NONRATT014979.2,NONRATT015922.2,NONRATT021024.2,NONRATT025666.2,NONRATT026569.2,NONRATT030768.2,TCONS?00008696,TCONS?00008697,TCONS?00008701,TCONS?00009973 and TCONS?00009974.Through the functional enrichment analysis of the lncRNA-mRNA interaction network and the target gene in the lncRNA-miRNA-mRNA regulatory network,a total of 338 cell proliferation-related genes were identified.Then a protein interaction network was constructed using string database.The 15 key genes were screened by the MCC method in the cyto Hubba plugin of Cytoscape v3.6.1,namely CDK1,CCNA2,CDC20,BUB1 B,AURKB,PRC1,KIF11,MAD2L1,TOP2 A,CCNB1,NDC80,BUB1,TTK,KIF20 A and MELK.IPA analysis found that the Cell Cycle: the G1/S Checkpoint Regulation,Acute Phase Response,IL-8,IL-6,JAK/Stat,TGF beta and Aryl Hydrocarbon Receptor,etc.signaling pathways,had been activated during rat liver regeneration.It was suggested that lncRNA may be involved in the regulation of hepatocyte proliferation through these key genes and signaling pathways.