Identification And Functon Of CircRNA Encoded By HBV

In order to understand the cellular response to HBV infection,and to further investigate the mechanism of HBV infection in HBV-associated HCC at whole transcriptome?mRNAs,miRNAs and circRNAs?level,whole transcriptome sequencing was conducted to characterize the expression profiles of RNAs in HepG2?n=2?and HBV-positive HepG2.2.15?n=2?hepatocelluar carcinoma cell lines,and correlated expression networks of circRNAs-miRNA-mRNA were constructed.Compare to HepG2 cell line,10114?6452?gene expression were up?down?-regulated,37?33?miRNAs were up?down?-regulated in HepG2.2.15.Moreover,5672 and 13026 circRNAs were found in HepG2 and HepG2.2.15,respectively,186?65?up?down?-regulated?P<0.05 and FDR<0.05?in HepG2.2.15.GO analysis showed that the hosting genes of dysregulated circRNAs were mainly enriched into cellular processes,biological regulation,metabolic regulation and catalytic activity.KEGG analysis revealed that the hosting genes of dysregulated circRNAs were mainly involved in p53 signaling pathway,pathways in cancer,viral carcinogenic,Ras signaling pathway,mTOR signaling pathway etc.Based on the correlation analysis between the differentially expressed circRNAs,miRNAs and mRNAs,86 and 22 mRNAs could be regulated by interaction of circRNA?circ_FIRRE 1,chrX:130870155-130928494?with NovelmiRNA-63 1 and NovelmiRNA-969,22 mRNAs could be regulated by interaction of circRNA?circ_FIRRE2,chr11:22696396-22777499?-NovelmiRNA-323,these co-expressed genes in circRNA-miRNA-mRNA network were mainly enriched in positive regulation of transcription from RNA polymerase ? promoter and cytoplasm and protein binding GO terms,and carcinogenic and antiviral signaling pathways KEGG pathways.These findings suggest that HBV infection changes the expression pattern of RNA hepatocelluar carcinoma cell,and provided a novel clue to understand the HBV-related HCC.At present,it has been found that transcripts of some DNA viruses can form circRNA by back-splicing.In order to verify whether HBV can form circRNA,the circRNA sequencing of HBV-positive HepG 2.2.15 hepatocarcinoma cells and HBV virus genome data were compared with HBV genome.It was found that there is a possible circRNA encoded by HBV,and its sequence matches the 489-2985 nt region of HBV genome.The circRNA is about 2.5 kb and was maned HBV_circ₁.To further identify the authenticity of circRNA encoded by HBV,reverse PCR was performed with divergent primers.The results of Sanger sequencing of PCR products show that there is indeed a spliced junction site in HBV circ 1 and its paralateral sequence is consistent with that of high throughput sequencing.The presence of HBV-circ l in HepG2.2.15 cells was further confirmed by Northern blotting with oligonucleotide probe targeting the junction site.Tissue in situ hybridization showed that HBV_circ₁ was also present in clinical samples of HBV related HCC.Comparing HBV pgRNA and HBV_circ₁ sequence,it is suggested that HBV_circ₁ originated from pgRNA,as a new RNA molecule encoded by HBV.Moreover,the results of tissue microarray analysis showed that the expression level of HBV_circ₁ in HCC tissues was significantly higher than that in paracancerous tissues,suggesting that HBV_circ₁ might be involved in the carcinogenesis and/or development of HCC.To investigate the function of HBV_circ₁,the effects of HBV_circ₁ on the phenotypic characteristics of HCC cells were detected by flow cytometry.The results showed that HBV_circ₁ promoted the proliferation,cell cycle progression and cell migration of HepG2 cells,and the apoptosis was inhibited.RNA pull-down and dual-Luciferase reporter assay system showed that HBV_circ₁ could interact with hsa-miRNA-6124 to regulate the expression of ST7?Suppression of tumorigenicity 7,GenBank:AK297510.1?,suggesting that the occurrence and/or development of HCC is regulated by HBV_circ₁-hsa-miRNA-6124-ST7 network.Furthermore,the binding proteins of HBV_circ₁ were identified by RNA pull-down,RNA binding protein immunoprecipitation assay and mass spectrometry.It was found that HBV_circ₁ could interact with CDK1?cyclin-dependent kinases 1,CDK1?,eukaryotic translation extension factor?Elongation factor 1-alpha 1,EEF1A1?and other proteins.Cellular immunofluorescence and western blotting further confirmed that HBV_circ₁ could interact with CDK1 and EEF1A1.These results suggested that HBV_circ₁ may regulate the phenotypic characteristics of HCC cells by interacting with proteins.In addition,we also found that HBV_circ₁ could promote HBV replication and increase the level of CDK1 protein.These results suggest that HBV can produce functional HBV_circ₁ which plays an important role in the development and development of HBV-related HCC through HBV_circ_l-hsa-miRNA-6124-ST7 network and interacting with CDK1 and EEF1A.HBV_circ₁ may be a molecular target for the treatment of HBV patients.To further understand the function of HBV_circ₁,we studied the effect of HBV_circ₁ on the expression pattern of cellular genes at the whole genome level.The results showed that a total of 147 differentially expressed genes were identified in HepG 2 cells over-expressing HBV_circ₁.Compared with the control cells,57 genes were up-regulated and 90 genes were down-regulated.Annotation of differentially expressed genes showed that differentially expressed genes were enriched in tumor-related signaling pathways,such as PIK-AKT,MAPK and Hippo.In addition,gene fusion and alternative splice events were both changed in HepG 2 cells over-expressing HBV_circ₁.These results provide a clue to understand the role of HBV_circ₁ in HBV-related hepatocellular carcinoma.