The Role Of TLR4-Snail Signal Pathway In The EMT Of Human Biliary Epithelial Cells Induced By LPS

Objective: To study the role of TLR4 and the function of Nuclear transcription factor--snail in the EMT of human biliary epithelial cells induced by LPS, the si RNA of TLR4 be used to silence the expressing of TLR4 in human biliary epithelial cell lines. This study will provide new therapy for hepatic duct fibrosis and improve the curative effects of hepatic duct fibrosis. Methods: the normal biliary epithelial cell lines of human(HIBEpi C)was cultured, after training to the logarithmic growth phase, the cell were divided into four groups: ①The control group(normal biliary epithelial cell lines); ②The LPS-induced group(the cell was induced by LPS but the m RNA of TLR4 were not silenced); ③NC+LPS group(the cell were transfected by blank plasmid and induced by LPS); ④KD+LPS group(after the m RNA of TLR4 were silenced and the cell were induced by LPS). After all of the cell had been induced by LPS except control group at0h、14h、24h、48h and 72 h, the m RNA of TLR4 and snail and the epithelial markers--E-cadherin were identified by fluorescent quantitative PCR. All of the results were analyzed by statistical software of SPSS17.0. Results:1. With the extension of LPS inducing, the form of normal biliary epithelial cell gradually elongated, the number of protuberances increased and the intercellular contact becomes looser, finally the morphology of the HIBEpi C was similar to fibroblast cell. While the cell which had silenced the m RNA of TLR4 had no change and keep the normal epithelial phenotype. The result suggested that TLR4 play an important role in the EMT of HIBEpi C induced by LPS.2. The expression of TLR4 m RNA in LPS-induced group were significant increased compared with control group(P<0.01),and maintained at a higher level with the extension of LPS inducing. And the expression of TLR4 m RNA in KD+LPS group were decreased at the beginning, the transfection rate of TLR4 m RNA at 0h、14h、24h、48h、72h were 73.5%、81.5%、89.9%、93.9%、82.6% respectively. This result suggested that TLR4-si RNA can specifically silence the expression of TLR4 m RNA.3.The increase of snail can be detected after LPS inducing HIBEpi C for 14 h, and maintained at a higher level with the extension of LPS inducing(P<0.01).While the expression of snail in KD+LPS group were decreased with the extension of culture, especially at the time of 14 h,24h and 48h( P < 0.01).This result suggested that TLR4-si RNA can down regulate the expression of snail in HIBEpi C.4.The expression of E-cadherin was decreased after the HIBEpi C was induced by LPS in the 14 h to 72 h. but the expression of E-cadherin in KD+LPS group were increased with the extension of culture,14 h,24h and 48 h can detect the expression of E-cadherin were up regulationed(P<0.01), this result suggest that TLR4-si RNA can up regulate the expression of E-cadherin.5. LPS inducing the HIBEpi C which were transfection by negative control virus, the EMT related gene expression is consistent with CON+LPS group every time, there was no statistically significant difference(P > 0.05). This result suggested that virus vector itself to this had no effect on the result of the experiment. Conclusion: 1. The using of TLR4-si RNA which can silence the expression of TLR4 m RNA chould block the EMT of HIBEpi C induced by LPS. 2. After the TLR4 m RNA was silenced by TLR4-si RNA, the expression of epithelial markers--E-cadherin in HIBEpi C was increased and the EMT induced by LPS was inhibited significantly.