LPS Induceshuman Intrahepatic Biliary Epithelial Cell Epithelial-mesenchymal Transition ViaTLR4

Objective:To investigate the role of TLR4 in LPS induces epithelial-mesenchymalt ransition(EMT)of human intrahepatic biliary epithelial cells(HIBECs) and to exploret he possible mechanisms involved. Methods:HIBECs are cultured in vitro. CON+LPS group is treated with 2ug/ml L PS, CON group without LPS. Cellsare collected after 72 hours.Morphologyof cells are observed with inversion phase contrast microscope. Real-time fluorescentPCR de tects mRNA level expression ofTLR4, nucleartranscription factor Snail, ep-ithelial m aker E-cadherin, mesenchymal makers N-cadherin and Vimentin. Protein l-evel expre ssion of all indicators mentioned obove are detected with western blot. Transwell ce ll migration experiment evaluates the cells mobility. To construct TLR4 siRNAlentiv iral vector in KD+LPSgroup, NC+LPS group as negative control. To observe effect of cilencing TLR4 gene on LPS induces EMT.Results:1.LPS induces HIBECs 72 h, CON+LPS group cell morphology changes from e pithelial to mesenchymal. Compared with CON group, CON+LPS group epithelial maker E-cadherin mRNA expression down-reglates(P<0.05), on the contrary, mesenchymal makers N-cadherin and Vimentin mRNA expression up-regulates(P<0.05,P<0.05). Protein level expression of all indicators mentioned obove are consistent with mRNA expression. Transfer rate have no difference between CON+LPS and CON groups. These results suggest LPS can induce EMT of HIBECs.2.CON+LPSgroup TLR4 mRNA level expresses higher than CON group(P<0.05).The protein expression keeps in step with mRNA. This show that LPS lead to TLR4 increase in HIBECs.3.CON+LPS group Snail mRNA level expresses higher than CON group(P<0.05).The protein expression keeps in step with mRNA level. This show that LPS cause Snail of HIBECs increase.4. To compare with NC+LPS group, KD+LPS group TLR4 mRNA expresses lower(P<0.05). The protein expression keeps in step with mRNA. This suggests TLR4 SiRNA can attenuated LPS lead to TLR4 increase in HIBECs.5.To compare with NC+LPS group, KD+LPS group Snail mRNA expresses lower(P<0.05).The protein expression keeps in step with mRNA. This suggests cilencing TLR4 genecan weaken LPS cause Snail increase in HIBECs.6.To compare with NC+LPS group, KD+LPS group cell morphology change f-ro m epithelial to mesenchymal isn't obvious. Epithelial maker E-cadherin mRNA e-xp ression up-reglates(P<0.05)and mesenchymal makers N-cadherin and Vimentin m RNA expression down-regulates(P<0.05,P<0.05). Protein level expression of allin dicators mentioned obove are consistent with mRNA expression.These results indi-ca te that silencing TLR4 gene can inhibit LPS induce EMT of HIBECs. Conclusion:1. This study demonstrates LPS induces EMT of HIBECs via TLR4.2. LPS inducesEMT of HIBECs may regulated by nuclear transcription factor Snail.