Study On MiR-19a Targeting TBK1 To Inhibit NF-?B Signaling Pathway

Endometritis in cows is a common inflammatory injury of uterine tissue,which is often caused by childbirth injury and bacterial infection.It can cause implantation failure,abortion and infertility of animals,and bring huge economic losses to the breeding industry.Because the pathogenesis of endometritis is not very clear,so far there is no good treatment for endometritis.Currently,it is mainly treated with antimicrobial drugs in clinical practice,but the problems of antibiotic residues and drug-resistant bacteria formed in the treatment process will cause serious food safety problems.Therefore,it has been banned or strictly restricted in many countries.The occurrence of endometritis is not only related to pathogenic factors,but also closely related to the autoimmune status of the body.Recent studies have shown that miRNAs can regulate the occurrence,development and prognosis of a variety of diseases.Their mechanism is mainly through bounding to the 3'-UTR of the target gene to silent its expression.MiR-19 a belongs to the polycistron miR-17-92 cluster and plays an important role in the pathogenesis of colitis,pneumonia,atherosclerosis and a variety of cancers.Bacterial endotoxins such as LPS bind to the pattern recognition receptor 4(TLR4)on the cell surface to activate the NF-?B pathway,induce an inflammatory response,and promote the expression of cytokines such as TNF-?,IL-1? and IL-6.In order to further elucidate the mechanism of endometritis,and provide new methods and ideas for clinical treatment of endometritis,bovine endometrial epithelial cells(bEECs)inflammation model and mouse endometritis models were established to explore the regulatory effect of miRNAs on LPS(Lipopolysaccharide)-induced endometritis.In this study,LPS was used to stimulate mouse uterine tissue and bEECs respectively,miR-19 a mimics or inhibitor was used to interfere with the expression level of miR-19 a to test the function of miR-19 a,and the function of TBK1 was verified by siRNA technology.The results showed that the mouse endometritis model was successfully established by stimulating the mouse uterus with LPS,and the expression level of miR-19 a was about 3 times lower than that of the control group(p<0.05).In order to further determine whether miR-19 a is involved in this inflammatory process,LPS was used to stimulate bEECs.The results showed that with the increase of the stimulation time and concentration,the expression level of miR-19 a decreased by 2 to 3 times compared with the control group,which was manifested as a significant down-regulation in time and dose-dependent manner(p<0.05).In order to verify the function of miR-19 a,we detected the changes in the expression of related inflammatory factors and proteins after interfering with the expression of miR-19 a.The results showed that miR-19 a significantly alleviated the phosphorylation of related proteins p65 and I?B? in the NF-?B signaling pathway and reduced the expression level of inflammatory factors by about 2~4 times(p<0.05).In order to study the mechanism of miR-19 a,we predicted and verified its target genes.Experiments confirmed that TBK1 was a target gene of miR-19 a in the NF-?B pathway.To test whether miR-19 a plays a role by silencing the expression of target genes,we examined the function of TBK1.The results showed that the silencing of TBK1 reduced the levels of inflammatory factors by about 2~3 times and decreased the phosphorylation of related proteins p65 and I?B?(p<0.05).Conclusion: this study mainly studied the effect and mechanism of miR-19 a on LPS-induced endometritis,and the results showed that miR-19 a can mediate the negative regulation of TLR4/NF-?B signaling pathway by targeting TBK1,down-regulate the phosphorylation level of p65 and I?B?,reduce the release of inflammatory cytokines,and then alleviate the inflammatory injury.