Gga-miR-21-5p Targets The Regulation Of The SATB1 Gene And Its Effect On The Proliferation And Migration Of Marek's Disease Tumor Cells

Marek's disease(MD)is a highly contagious viral tumor disease caused by Marek's disease virus(MDV)which has been cause huge economic losses in the poultry industry.Different varieties(lines)of chicken with different genetic resistance after infect Marek's disease virus,and those with strong genetic resistance chicken may remain in a latent infection stage,after a brief late cytolytic infection after tumor formation,while the susceptible chicken virus proliferation,no tumor early death.However,the molecular regulation mechanism of resistance to chicken and susceptible chicken has not been elucidated so far.In recent years,it has been found that host miRNA plays an important role in antiviral immune response.We used B21(resistance)and B19(susceptible)haplotype chicken as the model to construct the differential expression profiles of miRNAs in bursa of Fabricius of B21 and B19 haplotype chickens in three key stages of MDV virus infection(5,10 and 21dpi)by high-throughput sequencing.Some miRNAs associated with MHC-B haplotype chicken MD resistance were screened out.To explore the molecular mechanism of the difference between the resistant chicken and the susceptible chicken tumor from the perspective of host mi RNA regulation,the gga-miR-21-5p and gga-miR-146b-5p were chosen as the object to study which based on the early study of differential expression profile.We used the real-time fluorescence quantitative PCR to validate the high-throughput sequencing result and further analyzed the expression of gga-mi R-21-5p and gga-miR-146b-5p in other immune organs.It was found that the expression levels of gga-mi R-21-5p andgga-miR-146b-5p in bursa of Fabricius and spleen were significantly higher than those in non-infected group.Through dual luciferase reporter analysis,we found that gga-miR-21-5p could significantly inhibit the reporter gene activity of SATB1 3'-UTR region,which shows that the activity was reduced 33%,the interaction between gga-miR-21-5p and the predicted target gene of SATB1.At the same time,Western blot analysis showed that gga-miR-21-5p overexpression significantly inhibited the expression of SATB1 protein in the transfected MSB-1 cells after 72 hours,but the inhibition of gga-miR-21-5p expression could promote the expression of SATB1 protein.In order to further detect the effect of gga-mi R-21-5p on the proliferation and migration of MSB-1 cells,we used CCK-8 and trans-well to detect the effects of exogenous gga-miR-21 mimics and inhibition the rate of overexpression of MSB-1cells in 24-72 hours showed a significant decrease.At the same time,the ability of MSB-1 cell migration was also significantly decreased after overexpression of ggamiR-21-5p.In summary,this study shows that gga-miR-21-5p can target the regulation of SATB1 gene expression,the proliferation and migration of Marek's disease tumor cells have a certain inhibitory effect.