The Role Of TLR4/miR-181b Axis In The Epithelial-mesenchymal Transition(EMT) Induced By TGF-β And Its Molecular Mechanism

Transforming growth factor β1(TGF-β1) was considered as one of the most important signaling pathways that induced epithelial-mesenchymal transition(EMT),widely involved in various pathological or physiological process such as embryonic development,tumor metastasis and fibrosis.Toll-like receptor 4(TLR4) was the first discovery of TLRs family members,which played an important role in the process of tumor metastasis and fibrosis,in addition to the leading regulatory effect of body’s natural and acquired immune.However,it was not fully clear to the role of TLR4 signaling pathway in the epithelial-mesenchymal transition(EMT) induced by TGF-β and its mechanism.In this study,we found that TGF-β1 could reduce the expression of epithelial marker E-cadherin in A549 cells,increase the expression of interstitial marker N-cadherin and Vimentin,and induce epithelial-mesenchymal transition.After stimulating A549 cells with TLR4 agonist LPS,the role of TGF-β1 inducing EMT decreased significantly,suggesting that TLR4 activation could inhibit TGF-β1 inducing EMT.Through Deep Sequencing and q RT-PCR,we found that LPS stimulation rised significantly mi R-181 b expression in A549 cells.The results of Ch IP experiments and luciferase assay showed that transcription factor AP1 could bind to mi R-181 b promoter.And AP1 knock-down could block the upregulation of mi R-181 b induced by LPS stimulation in A549 cells.Further study showed that overexpressing mi R-181 b could inhibit obviously the downregulation of E-cadherin and the upregulation of N-cadherin and Vimentin induced by TGF-β1.Through a database query,we found that two 8mer sequence of TGFBR1 gene 3’UTR side matched the mi R-181 b.Luciferase assay verification showed that two sequence of TGFBR1 gene 3’UTR side could bind to mi R-181 b.And western blot results showed that mi R-181 b mimic could reduce TGFBR1 protein expression in A549 cells,whereas mi R-181 b inhibitor could upregulate TGFBR1 protein levels.The above results showed that TLR4 activation could raise mi R-181 b expression by AP1,and mi R-181 b could inhibit TGF-β1 inducing EMT through downregulation of the target gene TGFBR1 expression.This study not only contributed to understanding the TLR4 complex network mechanisms in the EMT process,but also prompted by intervening in the TLR4/mi R-181 b signal axis to explore new methods and strategies of tumor metastasis and fibrosis treatment.