The Study On Mechanism Of Gga-miR-223 Targeting FOXO3 In Regulating Mycoplasma Gallisepticum Infection

Mycoplasma gallisepticum(Mycoplasmagallisepticum,MG)is the main pathogen causing chronic respiratory diseases in chickens and causes huge economic losses to the poultry industry.Many studies have shown that mi RNA plays a key role in the occurrence and development of many diseases.MG-HS is a virulent strain isolated from Hubei.Through high-throughput sequencing,it was found that the expression of gga-mi R-223 c was significantly inhibited in chicken embryos infected with MG-HS.In this study gga-mi R-223 was explored for its role and molecular mechanism in MG-HS infection.The main results were as follows:1.The relative expression of gga-mi R-223 in MG-HS-infected chicken embryos and DF-1 cells was detected by RT-q PCR.The results showed that the expression of gga-mi R-223 in lung tissue and DF-1 cells at 4,6 and 8 days after infection was significantly lower than that in normal control group(p<0.05),which was consistent with the results of high-throughput sequencing.2.Predicted by online software Target Scan and mi RDB,and combined with its function analysis,FOXO3 was preliminarily screened as the target gene of gga-mi R-223.Through RNAhybrid,it was found that gga-mi R-223 and FOXO3 3’UTR were strictly complementary to each other,and the binding sequences were highly conserved among different species.The analysis of double luciferase reporter system assay showed that overexpression of gga-mi R-223 in cells could significantly inhibit the luciferase activity of wild type vector FOXO3 3’UTR(p<0.05),but had no effect on the luciferase activity of mutant vector.Further verification by RT-q PCR and western blot showed that overexpression of gga-mi R-223 could significantly inhibit the relative m RNA and protein expression of FOXO3 in DF-1 cells(p<0.05).On the contrary,gga-mi R-223 inhibition could extremely significantly increase the relative m RNA and protein expression of FOXO3 in DF-1 cells(p<0.05).FOXO3 was identified as the direct target gene,and gga-mi R-223 negatively regulated the expression of FOXO3.3.The relative expression of FOXO3 m RNA in the lung tissue of chicken embryos infected with MG-HS was significantly higher than that in the normal group on the 4th,6th and 8th day after infection(p<0.05),and the relative expression of FOXO3 m RNA and protein in DF-1 cells infected by MG-HS was significantly higher than that in the normal group(p<0.05),which was opposite to the expression of gga-mi R-223.It wasfurther demonstrated that gga-mi R-223 played a key role by targeting FOXO3 in MG-HS infection.4.Cells proliferation,cycle and apoptosis were detected by CCK8 and flow cytometry after overexpression or inhibition of gga-mi R-223 in DF-1 cells infected by MG-HS.The results showed that MG-HS infection could significantly inhibit the proliferation of DF-1 cells,block the process of cell cycle and promote apoptosis(p<0.05).Overexpression of gga-mi R-223 could significantly promote the proliferation and cell cycle progression of MG-HS infected cells,make a large number of G1 phase cells enter S and G2 phases to significantly inhibit apoptosis,while inhibition of gga-mi R-223 could significantly inhibit the proliferation of infected cells and up-regulate the proportion of G1 phase cells to promote cell apoptosis(p<0.05).5.RT-q PCR detection showed that overexpression of gga-mi R-223 in DF-1 cells could significantly promote the expression of cell cycle marker genes CDK1,CDK6 and CCND1(p<0.05),while significantly inhibit the expression of cell apoptosis markers such as BIM,FASLG and TRAIL(p<0.05).However,the result of gga-mi R-223 inhibition was opposite to that of overexpression.When the target gene FOXO3 was knocked down in DF-1,the expression of the above cycle and apoptosis genes was consistent with the overexpression of gga-mi R-223 in DF-1.It was revealed that MG-HS down-regulateed the expression of gga-mi R-223 to inhibit the expression of cycle genes CDK1,CDK6 and CCND1 and promote the expression of apoptosis genes BIM,FASLG and TRAIL by targeting FOXO3,thus inhibiting the proliferation and promoting the apoptosis of infected cells.