Screening, Expression And Functional Target Genes Of MHC-B Haplotype Marek's Disease Resistance-related MiRNAs

Marek's disease(MD)genetic resistance and/or susceptibility is significant different between species or strains of chicken,but its mechanism has not been clarified.Recent studies showed that host miRNA involved in host-pathogen interaction and may play a key regulatory role in host antiviral immune response and genetic resistance.To screen the host miRNAs that may be associated with MD genetic resistance or susceptibility,we used the high-throughput sequencing technology to construct the differential expression profile between the resistance(B21 haplotype)and susceptible(B19 haplotype)chicken in non-infection and infection bursal tissues at three key stages(5 d,11 d,and 20 d).It was found that miR-200b-3p and miR-200b-5p from the same precursor were up-regulated only in susceptible chickens(B19 haplotype)at 20 days post-infection.We first use fluorescence quantitative PCR technology to verify the results of high-throughput sequencing.The results of fluorescence quantitative PCR results were consistent with high-throughput sequencing,and the expression of gga-miR-200b-3p and gga-miR-200b-5p in the two groups was similar,but the significance of difference was not consistent.To further investigate the role of MD resistance and susceptibility in cancer,we predict and screen the candidate target genes of gga-miR-200b-3p and gga-miR-200b-5p by target genes prediction software,and use the dual luciferase reporter gene method to verify the predicted target genes.The result of wild type vector double luciferase reporter showed that the luciferase activity of JUN(191-422)decreased by 25%(UTR)while mi R-200b-3p was overexpression;and ZEB1(266-530)UTR activity decreased significantly by 29%.After functional site mutation,luciferase activity was restored,and the results showed that JUN(273-279)and ZEB1(445-451)region were the main binding sites of gga-miR-200b-3p.And found by Western blot analysis that overexpression of mi R-200b-3p significantly inhibited the expression of ZEB1 protein after transfection of MSB-1 cells for 72 hours;in contrast,inhibition of mi R-200b-3p expression promotes the expression of ZEB1 protein.To further detect the effect of miR-200b-3p on the proliferation of MSB-1 cells,the CCK-8 method was used to detect.Result showed that the proliferation rate of MDCC-MSB1 cells was increased after inhibit the expression of miR-200b-3p by 24-72 h.In summary,gga-miR-200b-3p can inhibit the expression of ZEB1 protein,which may further promote the abnormal proliferation of Marek's disease.