The Mechanism Of MiR-26A And Mir-181a In Chicken Marek’s Disease Lymphoma Tumorigenesis

MiRNAs are a class of highly conserved, non-coding endogenous RNAs that emerge as a key post-transcriptional regulator involved in multiple cellular processes including proliferation, differentiation, apoptosis and immune responses. Our previous study revealed many miRNA related to Marek’s disease (MD) tumor formation by Solexa deep sequencing. In this study, we observed the effect of gga-miR-181a and gga-miR-26a, which were significant down-regulation in MDV-infected samples, on the proliferation and migration of MSB-1cells by CCK-8and transwell assay. The study shows that viability and migration ability of MSB-1cells is significantly reduced after the overexpress of gga-miR-26a and gga-miR-181a. These results suggest that gga-miR-26a and gga-miR-181a could inhibit the proliferation and migration of MSB-1cell.In order to further investigate the mechanism of gga-miR-26a and gga-miR-181a in Marek’s disease tumorigenesis, we carried out bioinformatic analysis to identify potential targets of gga-miR-26a and gga-miR-181a by Targetscan and miRDB. Then dual-luciferase reporter gene assay was finished on BTBD3, KIF3B, ANP32A and CNOT2, which were putative targets of gga-miR-26a, and TNRC6B, NEK6, KPNA2, MAPK6, EZH2, which were putative targets of gga-miR-181a. In this study, gga-miR-181a could significantly inhibit the luciferase activity of KIF3B3’-UTR reporter, ANP32A3’-UTR reporter, and BTBD33’-UTR reporter by20%,40%, and15%respectively. Gga-miR-26a could significantly inhibit the luciferase activity of NEK63’-UTR reporter by20%. These results demonstrate significant interaction of gga-miR-181a with BTBD3、KIF3B and ANP32A, and of gga-miR-26a with NEK6. These genes may be involved in tumorigenesis.To further verify experimental validation of miRNA targets and determine the relationship between target gene and miRNA, we identify differential expression of BTBD3、KIF3B、ANP32A and NEK6mRNA and protein in tissue samples and transfected MSB-1cells by qRT-PCR and western blot. The mRNA and protein expression of NEK6were remarkably reduction after gga-miR-26a overexpress in MSB-1, and the mRNA and protein expression of ANP32A were also significantly down-regulation after gga-miR-181a overexpress. Furthermore, the mRNA expression of ANP32A was significantly higher in the MDV-infected samples compared with non-infected spleens. Meanwhile a trend of higher NEK6mRNA expression in the MDV-infected samples compared with non-infected spleens was found, but the difference was not significant. There was a negative correlation between miRNA and corresponding target gene. In conclusion, NEK6and ANP32A is direct target gene of gga-miR-26a and gga-miR-181a, respectively.In summary, his study demonstrated that gga-miR-26a and gga-miR-181a can inhibit the proliferation and migration of MSB-1cells. Meanwhile, gga-miR-26a and gga-miR-181a are involved in MD lymphoma tumorigenesis by regulating their target gene NEK6and ANP32A.