The Research Of The Effect Of MiR-145 Modulates TGF-β/Smad Pathway In MO Infected Lung Fibroblasts

Mycoplasma ovipneumoniae (MO) is a prokaryotic pathogenic microorganisms. If the sheep infected by MO will be panting, cough, progressive emaciation and interstitial pneumonia and other symptoms, and even lead to death of sheep. It brings great harm to sheep breeding industry. At the same time, it causes serious economic losses. Pathological changes of interstitial pneumonia including pulmonary interstitial fibrosis. This is a kind of extracellular matrix deposition caused by progressive disease. TGF-β/Smad profibrotic pathway plays an important role in pulmonary fibrosis. MicroRNA is a kind of endogenous non coding small RNA, which can be targeted to the target mRNA and the translation of the target mRNA is inhibited. It is about 21-25 nucleotides and is highly conserved in evolution. MiR-145 is a highly conserved miRNA, which is a length of 22nt. At present, there is little research about the regulation of miR-145 in pulmonary interstitial fibrosis. In particular, the regulation of TGF-β/Smad signaling pathway by miR-145 in lung fibroblasts (MRC-5) infected with MO has not been reported yet. Therefore, it is of great value and significance to study the role of miR-145 in the TGF-β/Smad pathway of MO infected MRC-5 cells.In this study, we first verify the existence of miR-145 in sheep cells. The target genes of in the miR-145 TGF-β/Smad signaling pathway were predicted by the target gene bioinformatics software. Then the target genes were verified by the luciferase reporter system. The MO infected MRC-5 cell model was successfully constructed, by inhibiting and over expressing miR-145. It is important to explore the process of MO anti infection process. MiR-145 regulates the expression of target gene Smad3 in the regulation of TGF-β/Smad pathway. To provide the experimental basis for exploring the mechanism of miR-145 in MO anti infection process. The main results are as follows:(1) By gene amplification and sequencing technology to determine the sheep genome contains miR-145, which is consistent with the sequence of miRNA expression in the previous experiment.(2) We predicted the target genes of miR-145 were Smad3 and Smad4 in TGF-β/Smad pathway by using bioinformatics software, and the results of the dual luciferase reporter gene system experiment proved that miR-145 directly targeted to Smad3 is the target gene of miR-145 in TGF-β/Smad pathway.(3) We successfully established the MO infected MRC-5 cell model.The expression of miR-145 and fibrosis related factors in the cells was significantly increased after MO infected MRC-5 cells.(4) Overexpression and inhibition the expression of miR-145 in MO infected the MRC-5 cells. We determined that miR-145 target to Smad3 and affect the pathway downstream factor expression in mRNA and protein levels. To achieve the negative regulation role of miR-145 to the TGF-β/Smad pathway.The results of the study show that in the process of MO infected host cells, the expression of miR-145 and pro fibrosis related factors can be stimulated, while miR-145 has a negative regulatory effect on the TGF-β/Smad pathway. The experimental results provide experimental basis for the study of the regulation of MO infection induced fibrosis process.