| ObjectiveTo investigate the role and the mechanism of LPS on the induction of EMT in humangastric carcinoma cells in vitro.MethodsHuman gastric carcinoma cells were treated with LPS for 48 hours. Cell morphology changes were then observed under phase-contrast microscopy. EMT-related molecular markers were detected by real-time PCR and Western blot. The migration and invasion ability were evaluated by wound-healing and transwell invasion assay. The activation of Shh signal pathway was evaluted by real-time PCR and Western blot. To investigate the role of Shh signal pathway in LPS induced EMT, cyclopamine, a selective Shh signal pathway inhibitor, was added together with LPS to study the effects of cyclopamine on EMT, migration and invasion in gastric carcinoma cells. To further investigate whether LPS-induced Shh signal related to NF-κB activation, BAY11-7082, a selective NF-κB signal pathway inhibitor, was added together with LPS to study the effects of BAY11-7082 on Shh activation in gastric carcinoma cells and to determine if the activation of Shh was regulated by LPS through NF-κB signaling pathway.ResultsThe AGS cells underwent apparent changes in cell scattering compatible with EMT after 48 h of 10 μg/m L LPS treatment, while no phenotypic changes were observed in HGC-27 and SGC-7901 cells. Real time PCR showed that the expression of epithelial phenotype marker E-cadherin m RNA in cells treated with 10 μg/m L LPS for 48 h was no obviously less than that in control group, and mesenchymal phenotype marker N-cadherin m RNA in cells treated with 10 μg/m L LPS for 48 h was slightly higher than that in control group and the difference was no statistically significant(P<0.05). However, Western blot showed that the protein expression of E-cadherin was obviously less than that in control group and the protein expression of N-cadherin, Slug and Vimentin was obviously higher than that in control group, and the difference was statistically significant(P<0.05). When cyclopamine was administered, the LPS-induced expression change of E-cadherin, N-cadherin, Slug and Vimentin was abrogated, and the differences was statistically significant(P < 0.05). wound-healing and transwell invasion assay showed that LPS promoted migration and invasion mobility dramatically. We also showed that the expression of Shh, and Gli2 and Ptch1 was significantly increased in LPS-treated cells compared with control cells, and the differences was statistically significant(P<0.05). However, cyclopamine decreased LPS-induced the expression of Shh, Gli2 and Ptch1 and LPS-promoted migration and invasion, and the difference between the combined group and control group was statistically significant(P < 0.05). Added LPS to gastric cancer SGC-7901 cells induced the increase of the NF-κB p65 protein expression and the decrease of the IκBα protein expression and the differences was statistically significant(P<0.05). When BAY11-7028 was administered, the LPS-induced expression change of Shh, Gli2 and Ptch1 was abrogated, and the differences was statistically significant(P<0.05).ConclusionsThis study demonstrates that LPS could induce EMT in human gastric carcinoma cells and contribute to migration and invasion. LPS induced EMT through activation of the Shh signal pathway in a NF-κB-dependent manner. |
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