| Mycoplasma gallisepticum(MG)is the main pathogen causing chronic respiratory disease(CRD)in chickens.Chickens of different breeds and ages can be infected all the year round.So far,there are no effective prevention and control measures.The pathogenic mechanism of MG Needs further study.Studies have shown that micro RNAs(miRNAs)play an important role in the occurrence and development of poultry diseases.Early miRNAs sequencing of the research team found that the expression of Let-7d in the lung tissue of chicken embryos infected with MG was significantly down-regulated.This experiment uses Let-7d as the research object to explore its role in the process of MG infection and its molecular mechanism.The main results are as follows:1.In the MG infection cell test,compared with the normal group,the expression of Let-7d in DF-1 cells and CP-Ⅱ cells was significantly reduced after MG infection(p<0.05),which was consistent with the sequencing results,while the MKP1 The m RNA and protein expression levels were significantly higher than those in the normal group(p<0.05),which was contrary to the expression results of Let-7d.Overexpression of Let-7d can significantly up-regulate the m RNA and protein expression of p MGA1.2 in CP-Ⅱ cells infected with MG.It is predicted that Let-7d may regulate the infection of MG.2.The use of miRDB,Target Scan software and dual luciferase reporter gene system detection showed that let-7d was overexpressed in CP-Ⅱ cells,and the dual luciferase activity of the wild-type MKP1 3’UTR plasmid was significantly inhibited.There was no significant change in the dual luciferase activity of transfected mutant plasmids.It is proved that Let-7d can bind to the seed sequence of MKP1.Overexpression of Let-7d in CP-Ⅱ cells can significantly down-regulate the protein expression of MKP1(p<0.01);while the inhibition of Let-7d results in the opposite.It shows that MKP1 is the direct target gene of Let-7d,and Let-7d negatively regulates the MKP1 gene.3.Inhibition of Let-7d can significantly down-regulate the expression of P38,ERK,JNK m RNA and phosphorylated protein in MG-infected CP-Ⅱ cells(p<0.05),while the overexpression of Let-7d results in the opposite.Overexpression of MKP1 can significantly inhibit the MAPK signaling pathway and inhibit the m RNA expression of P38,ERK,and JNK genes,while inhibiting MKP1 has the opposite result.It shows that the down-regulated Let-7d regulates the infection of MG by up-regulating MKP1.4.Inhibition of Let-7d can significantly down-regulate the m RNA expression of AP-1,immunochemokines RANTES and MIP-1β and the protein expression of pro-inflammatory factors TNF-α and IL-6 in CP-Ⅱ cells infected with MG(p<0.05),while the results after overexpression of Let-7d are just the opposite.Overexpression of MKP1 can significantly inhibit the protein expression of pro-inflammatory factors IL-6and TNF-α in MG-infected CP-Ⅱ cells(p<0.05),while inhibiting MKP1 has the opposite result.It shows that Let-7d,which is down-regulated in infected cells,resists the inflammation caused by MG infection by inhibiting the MAPK signal pathway.5.MG infection caused a significant increase in the apoptotic rate of CP-Ⅱ cells,while a significant decrease in the cell proliferation rate.Inhibition of Let-7d significantly promotes the proliferation of CP-Ⅱ cells after MG infection,inhibits cell apoptosis,inhibits Let-7d and interferes with MKP1,but inhibits the proliferation of CP-Ⅱ cells and promotes apoptosis;while overexpression of Let-7d The result is the opposite.It is proved that after MG infection,let-7d with low expression promotes proliferation and inhibits apoptosis by up-regulating MKP1,thereby resisting MG infection.The results of this study reveal that Let-7d,which is significantly down-regulated after MG infection,inhibits the MAPK signaling pathway by up-regulating the target gene MKP1,effectively alleviating inflammation,promoting cell proliferation,and inhibiting cell apoptosis to resist MG infection. |
