An Integrated Analysis Of MRNA-miRNA-circRNA Expression Profiling In HepG2 Cell Line Induced By TNF-?

Circular RNA?circular RNA?is a special type of non-coding RNA molecule.Unlike traditional linear RNA?containing 5' and 3' ends?,circRNA molecules have a closed circular structure.Therefore,they are not affected by exonuclease,not easily degraded and more stable expression in cells.Recent studies have shown that circRNA molecules contain microRNA?miRNA?and RNA binding protein?RBP?binding sites,act as miRNA sponges and trans-acting factors in cells and play an important role in the development of diseases.At present,the research of circRNA is in the ascendant and its function needs to be further excavated.It is an in-depth research direction.Tumor necrosis factor TNF-? is mainly secreted by lipopolysaccharide-stimulated macrophages,has a variety of biological activity of inflammatory cytokines,can bind to TNFR on the target cell membrane,and then informs the corresponding signal pathway through receptors.Transfer to the nucleus,promote the expression of the target gene and then synthesize the protein,and together with other cytokines,activate the immune response,leading to tumor necrosis.In this study,normal human hepatocytes HL-7702,liver cancer cells HepG2,and hepatoma cells induced by tumor necrosis factor TNF-??HepG2₃h?were used as experimental samples and divided into two experimental groups.All the cultured cell samples were performed rRNA depletion based RNA-seq and small RNA-seq using next generation sequencing method separately.Different bioinformatics methods were applied to obtain their mRNA/ circRNA/microRNA expression profiles with or without TNF-? treatment,explore the impact of TNF-? on gene expression in hepatoma cells.Furthermore,the integrated analysis of expression profiles was used to screen out the relationship between circRNA-miRNA-mRNA regulatory networks that may be related to TNF-? mediation.This topic is mainly divided into the following three parts:1.The RNA-seq data of HL-7702 and HepG2 cell lines before and after TNF-? treatment was evaluated in terms of base quality and base distribution.The results showed that sequencing data was quality reliable and can be used furtherly.Then,we predicted the circRNAs in the three samples,and performed the statistical analysis on the number of predicted circRNA,the distribution of circRNAs in different genomic regions,the reads mapped to each chromosome of genome,the back-splicing reads,and the lengths of circRNAs.The results is basically consistent with the other human circRNA studies,so the prediction results is credible.2.In this part,we performed the differential expression analysis of the circRNA in HL-7702 and HepG2 cell lines before and after TNF-? treatment and functional annotation of their host genes,including GO functional enrichment analysis and KEGG Pathway analysis.Firstly,the transcript levels of the three samples were quantified and normalized.Then we compared the different expression in three samples,and picked out the differently expressed circRNAs.Finally,through the functional annotation and pathway analysis of the selected circRNA,we found that in the down-regulated circRNA,the corresponding GO which host genes enrich were highly correlated with the tumor,such as cell cycle and cell migration.At the same time,by signal pathway analysis,we found two cancer-related signaling pathways: hsa04120: Ubiquitin mediated proteolysis,hsa04012: ErbB signaling pathway.The study revealed that many circRNA whose host gene related to metabolic apoptosis have a significant change after TNF-? treatment,suggesting that the apoptosis pathway which TNF-? induced may be related to circRNA.3.In the last part of research,we constructed the mRNA-miRNA-circRNA regulatory network by the integrated analysis of the whole gene expression profiling.We found there were 17 circRNAs differentially expressed in the two groups.By analyzing the kegg pathway of miRNAs which interacted with the 17 circRNAs,we found that 10 mRNAs which were targeted by three miRNAs were enriched in the NF-kappa B pathway.Combining with mRNA expression profile,we speculated that the expression of the 6 circRNAs: hsa_circ₀112428,hsa_circ₀004027,hsa_circ₀107593,hsa_circ₀005524,hsa_circ₀003511 and hsa_circ₀008077 were decreased after hepatocellular carcinogenesis.The hsa-miR-3911 almost bound to the mRNA transcribed from TNFSF14 gene in HepG2,thus the expression of TNFSF14 protein were negatively regulated.However,in the HepG2 cells stimulated by TNF-? for 3 hours,the expression of the selected 6 circRNAs were increased,they might adsorb the hsa-miR-3911 molecules competitively like a sponge,so that the most TNFSF14 mRNA could be translated and the TNFSF14 protein expression were up-regulated,which could induce tumor cell apoptosis.