Study On Infection Mechanism Of MiR-16 Against Mycoplasma Gallisepticum

Mycoplasma gallisepticum(MG)infection mainly causes chronic respiratory disease(CRD)in chickens.Chicken chronic respiratory disease is a highly contagious respiratory infectious disease characterized by respiratory,cough and runny nose,which brings huge economic losses to the poultry industry.The pathogenesis of mycoplasma infection has not been elucidated yet,and it is important to explore its pathogenesis.Studies have shown that micro RNAs play an important role in the development of many diseases.On the basis of high-throughput sequencing,gga-mi R-16-5p was used as the research object to explore its role and regulation mechanism in MG infection.The relative expression of gga-mi R-16-5p in the lung tissue of infected MG-HS and DF-1 cells was detected by q PCR.The gga-mi R-16-5p expression levels in chicken embryo lung tissues infected MG-HS and DF-1 cells were higher than that in normal group at 6d,7d and 8d(p<0.05 or p<0.01).PIK3R1 was predicted as the target gene of gga-mi R-16-5p by using target gene prediction software and various analysis software,The biluciferase results showed that gga-mi R-16-5p was able to combine with the PIK3R1 3 ’UTR seed sequence to significantly inhibit the report gene activity of the PIK3R1 3’ UTR region.At the same time,Western blot analysis found that the cells after transfection DF-1,gga-mi R-16-5p over-expression can significantly inhibit PIK3R1 m RNA and protein expression,at the same time a significant inhibition of PI3K/AKT pathway downstream protein expression of P-AKT.However,inhibition of gga-mi R-16-5p results in the opposite of overexpression.The relative expression of PIK3R1 was detected by q PCR in the DF-1 cell and the chicken embryo lung tissue after infection with MG-HS,The results showed that the expression levels of PIK3R1 in 7d,8d chicken embryo lung tissues and DF-1 cells were significantly lower than that in normal group after MG-HS infection(p<0.05 or p<0.01).We examined the effects of gga-mi R-16-5p on cell proliferation and cell cycle by CCK-8 and the flow cytometry,and found that after MG infection,the cell proliferation was significantly inhibited,and the cell cycle was stasis in G1 phase.The proliferation of DF-1 cells was significantly inhibited at 48 h and 72 h after Overexpression of gga-mi R-16-5p in the DF-1 cell(p<0.01),which entered the G2 phase were significantly reduced.After inhibition of gga-mi R-16-5p,the proliferation of DF-1 cells increased significantly at 72h(p<0.01),a large number of cells enter the cell cycle of G2+S.This study confirmed that the expression of gga-mi R-16-5p was significantly increased after the chicken embryo and the cells were infected with MG-HS,the target gene of gga-mi R-16-5p was PIK3R1 that could inhibit the activity of the PI3K/AKT/NF-κB signaling pathway,which inhibited cell proliferation and makes a large number of cells stay in G1 phase.