Formation Mechanism And Function Of CircRNA HBV＿circ＿1 Derived From HBV

At present,many eukaryotes have been found to encode circRNAs.Increasing evidence has shown that circRNAs have multiple biological functions.The most classical biological function of circRNA is as a sponge for miRNA,circRNAs can also bind to proteins,and regulate transcription of parental genes,circRNAs can be used as a template for translation.Circ RNAs play an important role in the occurrence and development of many diseases,and may become a new drug therapeutic target.Some recent studies have found that circRNAs can be formed by transcription of some intron-containing viral genes of EB virus(EBV),kaposi sarcoma virus(KSHV),and human papillomavirus type 16(HPV16).Hepatitis B virus(HBV)is one of the important causes of primary liver cancer,a circRNA originating from HBV precursor genome pgRNA has been reported and it was found that this circRNA was not formed by conventional back-splicing,DEx H-box helicase 9(DXH9)is involved in the formation of HBV circRNA,but the formation mechanism and biological functions of circRNA derived from HBV are still unknown.In our previous study,a new circRNA generated from HBV(named HBV＿circ＿1)was found and promoted the cell cycle and inhibited apoptosis,it also can promote the occurrence and/or development of HBV-related HCC through the HBV＿circ＿1-hsa-miRNA-6124-ST7 network and interact with CDK1.On this basis,our study deeply discussed the formation mechanism of HBV＿circ＿1 encoded by HBV,detected the effect of HBV＿circ＿1 on tumor tumorigenesis and progression,explored the inhibition of autophagy by HBV＿circ＿1 and its molecular as well as the possibility of encoding X protein by HBV＿circ＿1.The research results can not only further deepen the understanding of the encoding information of HBV genome,but also provide a new mechanism for the formation of circRNA.It can also help us to elucidate the mechanism of primary liver cancer caused by HBV from a new perspective and provide a new target for the treatment of liver cancer caused by HBV infection at the RNA level.1、Origin and formation mechanism of HBV＿circ＿1In our previous study,a new circRNA encoded by HBV were found by high-throughput sequencing and named HBV＿circ＿1.The length of HBV＿circ＿1 is about 2.5 kb,and its sequence corresponds to the 489-2985 nt region of HBV genome(Gen Bank accession No.KU668446.1).Subsequently,HBV＿circ＿1 were preliminarily verified by PCR,Sanger sequencing and Northern blotting.On this basis,this study further confirms that HBV＿circ＿1 is a circular RNA,we also analyzed the source of HBV＿circ＿1 and explored the possible mechanism of forming HBV＿circ＿1.Sequence alignment and sequence determination of HBV＿circ＿1 confirmed that HBV＿circ＿1 were derived from HBV precursor genomic RNA(pgRNA).There are two Junction sites on HBV＿circ＿1(named Junction site A and Junction site B respectively).Junction site A is formed by the repeat sequence at the end of pgRNA through the homologous recombination.Junction site B were formed by junction of pgRNA sequence corresponding to HBV DNA genome(Gen Bank accession No.KU668446.1)489-3182-2985 nt region.Further studies confirmed that the repeat sequence of pgRNA could promote the formation of circRNA,and it were speculated that proteins were involved in this process.2、Effect of HBV＿circ＿1 on tumorigenesis and progressionOur previous results showed that HBV＿circ＿1 could inhibit the apoptosis of HepG2 cells,promote the proliferation of tumor cells,and improve the HBV replication level,suggesting that HBV＿circ＿1 may play an important role in the formation and development of tumors.In this study,we further determined the effect of HBV＿circ＿1 on the phenotypic characteristics of different cells.The results of flow cytometry showed that transfected with pLCDH-HBV＿circ＿1 expressing HBV＿circ＿1 could promote the proliferation and accelerate the cell cycle process of AML-12,HUVEC,HepG2 and SMMC-7721 cells,while there were no significant change in cells transfected with pLCDH-circ＿GFP or pLCDH-ciR.SMMC-7721 cells transfected with pLCDH-HBV＿circ＿1 or pLCDH-ciR were transplanted to nude mice,the results showed that HBV＿circ＿1 could promote SMMC-7721 cells to form tumors in vivo.PCR detection results showed that HBV＿circ＿1 were found in the tumor tissues formed by SMMC-7721 cells transfected with pLCDH-HBV＿circ＿1.HBV＿circ＿1 were determined in the liver tissue of one tumor mouse and were not determined in other tissues(heart,kidney,intestine,stomach,muscle,and brain).HE staining and immunohistochemistry of the tumor showed that the malignancies and the expression levels of Ki67 and Cyclin D1 in the tumor formed by SMMC-7721 cells transfected with pLCDH-HBV＿circ＿1 were higher than those in the control group.These results suggest that HBV＿circ＿1 can accelerate the progress of cell cycle,promote cell proliferation,and promote tumor formation and development.3.Inhibition of autophagy by HBV＿circ＿1 and its molecular mechanismAutophagy plays an important role in the occurrence and development of tumors.In this study,we explored the mechanism of HBV＿circ＿1 promoting the occurrence and development of tumors from the perspective of autophagy.The results of Western blotting and immunofluorescence showed that the transient expression of HBV＿circ＿1 could promote the accumulation and aggregation of p62,inhibit the formation of LC3-II,and counteract the promoting effect of rapamycin on autophagy,while the knockdown of HBV＿circ＿1 promoted the autophagy of HepG2.2.15 cells,indicating that HBV＿circ＿1 could inhibit autophagy.The results of immunohistochemistry showed that the cumulative amount of p62 in the tumor formed by SMMC-7721 cells transfected with pLCDH-HBV＿circ＿1 were significantly higher than that in the control group,and the inhibition of autophagy by HBV＿circ＿1 were confirmed in the tissue.qPCR results showed that the regulation of HBV＿circ＿1 has no significant effect on the expression of key genes in autophagy initiation complex,suggesting that HBV＿circ＿1 is not involved in the regulation of autophagy initiation.RNA pull down and RIP experiments confirmed that HBV＿circ＿1 interacts with PHB2.The results of bioinformatics show that the binding region of PHB2 and HBV＿circ＿1 contains the binding region of PHB2 and LC3,suggesting that HBV＿circ＿1 may competitively bind PHB2 with LC3.The Co-IP experiment confirmed that HBV＿circ＿1 could inhibit the interaction between PHB2 and LC3,while HBV＿circ＿1 mutant(deletion of PHB2 binding region)could not competitively bind PHB2 with LC3,and it lost its ability to inhibit autophagy.These results suggest that HBV＿circ＿1 inhibits autophagy through competitive binding of PHB2 with LC3 and thus promotes the occurrence and development of tumors.The results provide a new perspective for understanding the carcinogenic mechanism of HBV and a new target for the treatment of HBV-induced liver cancer.4.HBV＿circ＿1 can encode X proteinSome circRNAs have been demonstrated to encode proteins in a cap-independent manner.The sequence of HBV＿circ＿1 were analyzed by bioinformatics,and it were found that HBV＿circ＿1 had the complete open reading frame(ORF)of HBx,internal ribosome entry site(IRES)and m6A modification site,suggesting that HBV＿circ＿1 had the potential to encode X protein.The results of cell immunofluorescence showed that there were a small amount of X protein in HepG2 cells transfected with HBV＿circ＿1 expression vector pLCDH-HBV＿circ＿1.Ds Red were used to replace the ORF of HBx in pLCDH-HBV＿circ＿1 vector to construct the recombinant plasmid pLCDH-HBV＿circ＿1-Ds Red.The expression of Ds Red were also observed in HepG2 cells transfected with pLCDH-HBV＿circ＿1-Ds Red.Furthermore,X protein were determined by immunohistochemistry in the tumor formed by SMMC-7721 cells transfected with pLCDH-HBV＿circ＿1,while no X protein were determined in the control tumor formed by SMMC-7721 cells transfected with pLCDH-ciR.These results show that HBV＿circ＿1 can encode X protein.These results explain the mechanism of HBV＿circ＿1 promoting the development and progression of cancer from the perspective of encoding X protein.