Mechanism Of MiR-125b And MiR-221 In Avian Leukosis Virus Subgroup J Infection

Subgroup J of avian leukosis are historically known as neoplastic infectious diseases.It has been demonstrated that subgroup J of avian leukosis virus(ALV-J)can cause myeloid tumors and other multiple organs tumors.The generation of tumor induced by ALV-J was modulated by a lot of factor,not only the virus insertion,but also the host itself.MicroRNAs(miRNAs)are single-stranded noncoding small RNAs of 22 nucleotide(nt)lengths which can post-transcriptionally repress and degrade the expression of targets by base-pairing with targets 3' untranslated region(3'-UTR).Patterns of mi RNAs have been found to be implicated in a large variety of cellular processes,including cell cycle,proliferation,differentiation,apoptosis and so on.Our previous reports showed that gga-miR-221 and mi R-125 b expression had a significantly difference between ALV-J induced tumor tissues and SPF tissues.The two microRNAs have been widely studied in the human as the tumorrigenesis associated molecular.So,we chose the host miR-221 and miR-125 b as the objects to uncover the potential role of these two micro RNAs on ALV-J tumorigenesis.MiR-125 b has been reported that it may be associated with several cellular progression.To uncover the potential role of miR-125 b on ALV-J tumorigenesis,we firstly analyzed the mi R-125 b targeting genes by bioinformatic methods and luciferase assay.We demonstrated that KLF13,PAFAH1B1,PRDM1 and Sema4 D are four targets of miR-125 b.Protein interaction network analysis by STRING database showed that all of KLF13,PAFAH1B1,PRDM1 play great role on tumorassociated progression including cell motility,cytoskeleton and transcriptional regulation.Sema4 D plays an important role in cell-cell signaling,reorganization of the actin cytoskeleton and angiogenesis,which are closely associated with tumor develop.ALV-J infected in vivo and vitro essay showed that ALV-J led a significantly up-regulation of Sema4 D.Functional experiment of cell apoptosis showed that Sema4 D play the great inhibiting role on several cell type.All these data suggested that Sema4 D may be a potential oncogene in chicken.Previously two reports showed that miR-221 played great role on tumorigenesis.To uncover the effect of miR-221 on ALV-J tumorigenesis,we firstly analyzed the miR-221 targeting genes by bioinformatic methods and luciferase assay.Results showed that CDKN1 B is a target gene of miR-221 through bioinformatic methods and the luciferase assay.Further,we established the infected model of ALV-J in vivo and vitro,and the CDKN1 B expression was tested by qRT-PCR in tissues and DF1 cells post ALV-J infection.Our results showed that ALV-J leaded a significantly down-regulation of CDKN1 B in kidney and DF1 cells post ALV-J infection.To uncover the function of miR-221 and its target on ALV-J tumorigenesis,we performed the cell proliferation and cell cycle assay,and we found that ALV-J,miR-221 and CDKN1B-siRNA-846 all promoted DF1 cells proliferation and G1/S transition.While the miR-221 inhibitor and CDKN1 B over-expression played the inhibiting effect on the cellular progression.The mi R-221 knockdown assay and CDKN1 B over-expression assay post ALV-J infection showed that ALV-J enhanced cell proliferation and cell cycle progression required the up-regulation of miR-221 or a down-regulation of CDKN1 B.These studies suggested that ALV-J enhanced the tumor-associated cellular progression via the miR-221 maintaining a low level ofCDKN1 B.Finally,cell cycle pathway analysis showed that ALV-J promoted cell over-progression via mi R-221-CDKN1B-CDK2/CDK6 pathway,which released CDK2,CDK6 expression CDKN1 B suppressed.Above all,we firstly demonstrated four targets of miR-125 b,KLF13,PAFAH1B1,PRDM1 and Sema4 D.And we found that a cell apoptosis inhibitor,Sema4 D,in chicken may be a potential tumorigenic gene.Additionally,we demonstrated a target of miR-221,CDKN1 B,and demonstrated a new pathway of ALV-J tumorigenesis via mi R-221-CDKN1B-CDK2/CDK6 pathway promoting cellular over-progression.Our findings provided a new idea for ALV-J pathogenic mechanism and some potential targets for ALV-J prevention and cure.