Mechanism Of CircRNA₂420-Bcl11b-mediated Apoptosis In Regulating ALV-J Infection In Chickens

Avian leukosis virus subgroup J（ALV-J）can cause immunosuppression,growth inhibition,and tumors in multiple organs and tissues,which causes serious economic losses to the breeding industry.Compared with other subtypes,ALV-J has stronger pathogenicity and infectivity,and it is also a subgroup with the highest incidence,serious infection and far-reaching impact in chickens in China.At present,no effective commercial drugs or vaccines have been found to be available,and the mechanism of infection,pathogenesis and tumor remains need to be further studied.Based on the transcriptome data of previous ALV-J infection and normal spleen tissues,and combined with small RNA sequencing data,this study screened transcripts（including mRNAs,circRNAs,lncRNAs,or miRNAs）,possible networks among transcripts,and key signaling pathways for viral infection.Then the role of Bcl11b（B-cell lymphoma/leukemia 11B）in the pathogenic and tumorigenic process of ALV-J virus infection will be revealed,we hope to improve the basic research of the tumorigenic mechanism of ALV-J infection It also provides a theoretical basis for finding effective biomarkers to improve disease-resistant breeding.The main results are as follows:1.In order to explore the interaction network of key genes,circRNAs,lncRNAs,and ceNRA networks in the pathogenesis of ALV-J infection,and to provide basic data for analyzing the molecular regulatory mechanism of ALV-J infection in the chicken,we used transcriptome data of ALV-J-infected and normal spleen tissues,and combined with small RNA sequencing data,and obtained comprehensive and systematic ALV-J infected and uninfected chicken spleen tissue transcriptome data,and a total of 15 candidate genes（including Bbx,Bcl11b,Foxk1,Pag1,Tyro3,Brat1,Ctsb,Scube3,Ulk3,Dock4,Zfhx3,Fmr1,Aoc3,Ralb,and Mll）and multiple related transcripts and possible networks among transcripts,and key signaling pathways of viral infection related to ALV-J infection and tumorigenesis were screened by Venn diagram,functional enrichment analysis,and ceRNA network analysis.Bcl11b expression levels were most significantly different in extra infected tissues,up to 80-fold.Bcl11b signaling pathways were complicated,which may be the key factor in ALV-J infection and tumorigenesis.Therefore,we use the chicken Bcl11b gene as an entry point to study the mechanism of ALV-J infection.2.In order to explore the function of chicken Bcl11b gene,we cloned the CDS sequence of chicken Bcl11b,and found alternative spliceosomes and a new splicing form.Protein structure prediction analysis found that the deletion of exon 3 would affect zinc ion binding sites;qRT-PCR results showed that chicken Bcl11b was expressed in liver,spleen,lung,lymph,and kidney tissues,and was highly expressed in spleen,lung,and lymph and chicken embryo fibroblasts;further we overexpressed Bcl11b in DF-1 interfered its expression in CEFs,and at the same time detected cell proliferation activity,apoptosis and cycle changes.We found that Bcl11b overexpression is significantly reduced cell viability and increased apoptotic levels（P<0.05）,but had little effect on the cell cycle（P>0.05）.After interfering with Bcl11b expression,the apoptosis level was significantly reduced（P<0.05）and had little effect on the cell cycle（P<0.05）,which suggests that chicken Bcl11b reduces cell viability by promoting cell apoptosis,rather than arresting cell cycle.3.In order to further explore the role of chicken Bcl11b gene in the process of ALV-J infection,a model of epithelial-mesenchymal transition（EMT）of ALV-J infected CEF cells in vitro was first constructed,and the trend of Bcl11b gene expression levels was consistent with the level of apoptosis.Simultaneous we detected multiple apoptosis-related proteins revealed that p53 protein levels were highly expressed at 0-2 dpi,and the downstream protein Bax expression levels were high at 0-3 dpi,and the downstream factor cytochrome C（Cyt C）were highly expressed at 2-4 dpi,which basically consistent with the mitochondrial-mediated apoptosis pattern regulated by p53.This also indicates that chicken Bcl11b gene maybe involve in p53-regulated mitochondrial-mediated apoptosis in vitro.4.The molecular mechanism of miRNA targeting Bcl11b to participate in ALV-J infection was further explored.We first cloned and obtained chicken Bcl11b 3’UTR sequence of 1755 nt;Then we used miRDB,Targetscan,PicTar and other online prediction software and combined with sequencing analysis data to predict miRNAs that may target chicken Bcl11b and validated by dual luciferase reporter system and found that gga-miR-1612 and gga-miR-6701-3p can target chicken Bcl11b;Then we detected chicken Bcl11b expression levels as well as the effects of apoptosis and cycle after miRNAs inhibition or overexpression,and we found that gga-miR-1612 and gga-miR-6701-3p can affect the transcription and translation of Bcl11b gene by targeting Bcl11b,which in turn affects the level of apoptosis of the transfected cells;Through suppressing the expression of endogenous gga-miR-1612,gga-miR-6701-3p at inflection point of Bcl11b gene expression（3-5 dpi）during ALV-J infection of CEF,we found that the expression level of Bcl11b was significantly up-regulated,and the level of apoptosis was also significantly up-regulated（P<0.05）.This indicates that gga-miR-1612 and gga-miR-6701-3p mediate apoptosis by targeting the Bcl11b gene.5.In order to explore the regulatory mechanism of circRNA₂420-miRNA-Bcl11b,the characteristics of circRNA₂420 were first analyzed by qRT-PCR,molecular cloning and other methods.We found that circRNA₂420 is a highly stable closed circular transcript,and its biogenesis does not depend on flanking introns repeated sequences.Then through CircRNA₂420 overexpression and inhibition,we found the expression of Bcl11b was significantly increased after over-expression of circRNA₂420（P<0.05）,and the level of apoptosis was also increased accordingly（P<0.05）;Conversely,the expression of Bcl11b was significantly down-regulated after interfering with the expression of circRNA₂420（P<0.05）,and the level of apoptosis also decreased（P<0.05）,but the cell cycle did not change（P>0.05）;The dual luciferase reporter system assay showed that gga-miR-1612 can target circRNA₂420 to affect luciferase activity,and circRNA₂420 also indirectly affects the activity of Bcl11b gene,which suggest that circRNA₂420 can regulate the expression of chicken Bcl11b by sponging miRNAs（including gga-miR-1612）,which in turn affects cell apoptosis.6.In order to explore the mechanism of Bcl11b-mediated apoptosis,we conducted ChIP-seq experiments and found that Bcl11b as a zinc finger protein transcription factor can target multiple genes involved in cell apoptosis and thus participate in apoptosis and other biological processes,which provide clues for revealing the function and mechanism of Bcl11b.In summary,our study found that circRNA₂420 can indirectly promote the expression of chicken Bcl11b gene by sponging miR-1612 during ALV-J infection,and Bcl11b as a zinc finger protein transcription factor regulates the transcription of apoptosis-related genes,and then mediates Cell apoptosis and regulates ALV-J infection process.Our results further improved the basic research on the tumorigenic mechanism of ALV-J infection and also provided new targets and ideas for finding effective biomarkers to improve disease resistance breeding work.