| Chicken chronic respiratory disease(CRD)caused by Mycoplasma gallisepticum(MG)is one of the major diseases to reduce the economic benefits of poultry industry.The disease is widespread in the flocks around the world,there is still no ideal means of prevention and control,the pathogenesis remains to be further elucidated.Studies have shown that mi RNA plays an important role in the development and progression of the disease.The aim of this study is to investigate the effect of gga-mi R-130b-3p obtained from Illumina deep sequencing on MG infection,and to provide a basis theoretical understanding of the pathogenesis of MG.In this study,gga-mi R-130b-3p was used as the research object.The potential target gene of gga-mi R-130b-3p was predicted and analyzed by bioinformatics technique and validated by double luciferase reporter gene vector method.Furthermore,overexpression and inhibition of gga-mi R-130b-3p confirmed its negative relationship with the target gene.The expression of gga-mi R-130b-3p and target gene in MG-HS infected and non-infected SPF chicken embryo lung tissue and DF-1 cells was analyzed by q PCR method,and the accuracy of depth sequencing and target gene was confirmed.The results showed that gga-mi R-130b-3p could play a role in the PTEN / PI3 K / AKT signaling pathway by using the online software Ami GO prediction and then the expression of PI3 K,AKT and NF-κB was detected by q PCR method.To explain the mechanism of gga-mi R-130b-3p in MG infection,the proliferation and cycle of DF-1cells were analyzed by CCK-8 and flow cytometry.The main results are as follows:1.The target gene of gga-mi R-130b-3p was predicted to be PTEN by using mi RDB,Target Scan software and Pathway correlation analysis.Further,the RNAhybrid software was used to analyze the relationship between the gga-mi R-130b-3p and PTEN 3’untranslated region(UTR).The secondary structure of RNA and the conserved energy of RNA double strand and the conservativeness of target sequences between different species were analyzed.At the same time,the function of target gene was analyzed by Ami GO software.PTEN was identified as gga-mi R-130b-3p target gene.2.The double-luciferase reporter gene analysis showed that overexpression of gga-mi R-130b-3p in DF-1 cells significantly inhibited the activity of PTEN 3’UTR double luciferase reporter gene,whereas gga-mi R-130b-3p inhibition,PTEN 3’UTRdouble luciferase reporter gene activity was significantly improved(P<0.01).Preliminary validation of PTEN may be the target gene of gga-mi R-130b-3p.3.gga-mi R-130b-3p overexpression and inhibiting experiment shows:overexpression of gga-mi R-130b-3p in DF-1 cells significantly down-regulated the expression of PTEN(p<0.01),while the inhibition of gga-mi R-130b-3p can significantly increased the expression of PTEN(p<0.01),indicating that gga-mi R-130b-3p negatively regulates PTEN expression.It proved that PTEN may be the target gene of gga-mi R-130b-3p.3.Animal and cell experiments showed that the expression of gga-mi R-130b-3p was significantly increased in MG-HS-infected cells and chick embryo lung tissue(chicken embryo hatch on the 14 th,16th and 18 th day)(p <0.05).Consistent with the depth of sequencing results.In contrast to the expression of gga-mi R-130b-3p,the expression of PTEN was significantly down-regulated(p<0.05).Indicating that gga-mi R-130b-3p negatively regulates PTEN expression.It is further confirmed that PTEN is the target gene of gga-mi R-130b-3p.5.Gga-mi R-130b-3p functional studies show that the expression of PI3 K,AKT and NF-κB genes was significantly up-regulated by gga-mi R-130b-3p overexpression(p<0.01).In contrast,the expression of PI3 K,AKT and NF-κB genes was significantly down-regulated after gga-mi R-130b-3p inhibition(p <0.05).6.Cell proliferation and cycle experiments showed that MG-HS could significantly decrease the proliferation of DF-1 cells and block the cell cycle progression.Gga-mi R-130b-3p mimics significantly promoted the proliferation of DF-1 cells infected with MG-HS at 24 h,48h and 72 h,while gga-mi R-130b-3p inhibitor significantly inhibited the proliferation of infected cells.At the same time,the cell cycle progression of DF-1 cells infected with MG-HS was significantly increased in the G2+S phase after transfection with gga-mi R-130b-3p mimics.In contrast,the transfection of gga-mi R-130b-3p inhibitor resulted in a significant increase in the proportion of cells in the G1 phase and a significant decrease in the proportion of cells in the G2+S phase.This study shows that gga-mi R-130b-3p upregulated in chicken infected with MG inhibits the expression of target gene PTEN,activates PI3 K / AKT / NF-κB signaling pathway,accelerates cell cycle progression,and promotes the proliferation of tissue cells to resist the body’s MG infection. |
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