| Background: Most hepatocellular carcinoma(HCC) cases are associated with chronic hepatitis and fibrosis. Hepatic stellate cells(HSCs) contribute to fibrosis under the influence of chronic hepatitis and constitute the microenvironment of HCC. Aim: To evaluate the sensitivity change of HCC cells to Sorafenib and the phenotype change of HCC cells using HSCs-HCC cell lines coculture system. To investigate the underlying mechanisms through which HSCs enhance the malignant behavior of HCC cells.Methods: The change of sensitivity of HCC cells to Sorafenib in coculture system was evaluated by MTT assay. Potential genes, cytokines and proteins mediating Sorafenib resistance were determined by ELISA, western blotting and q PCR assay. Specific inhibitors were used to interuput the effects of coculture and to explore potential significant signialing pathways. On the other hand, EMT associated proteins and genes were detected by Western Blotting and q PCR, respectively. Cell migration and invasion were determined by wound-healing and transwell assay. Specific si RNA was used to interfere key factors that may induce EMT during coculture. At the same time, migration and invasion were also evaluated after si RNA transfection. HCC cells with or without HSCs were injected into the liver of nude mouse. 4 weeks later, pulmonary metastasis of primary tumor was determined.Results: Firstly, Sorafenib inhibited the proliferation of HCC cells through induction of apoptosis. Decreased sensitivity to Sorafenib was observed after when HCC cells were cocultured with HSCs. The levels of apoptosis related proteins including cleaved Caspase-3, Caspase-9 and PARP were reduced. HGF in the conditional culture medium from HSCs was higher than in the HCC cells. Addition of HGF as well as coculturing with HSCs minimized the effect of Sorafenib on HCC cells. Inhibition of AKT or c-Met attenuated Sorafenib resistance induced by HSCscoculture while p-STAT3 was not influenced. Increased sensitivity to Sorafenib was also observed after the usage of JAK and STAT3 inhibitor. It should be noticed that the STAT3 inhibitor S3I-201 not only induced apoptosis in HCC cells independently but also increased the efficiency of Sorafenib.Secondly, stronger migration and invasion abilities of HCC cells were observed after coculturing with HSCs, which was accompanied with the morphology change of HCC cells. This morphology change was similar with EMT induced by TGF-β. Reduced E-Cadherin and increased Vimentin indicated that HCC cells underwent EMT in the coculture system. These results were further confirmed by q PCR and immunofluorescence. The activities of pathways involved in EMT including AKT, and STAT3 were upregulated in HCC cells when cocultured with HSCs. Through si RNA interference, we found that AKT pathways may not be required in HSCs induced HCC cells EMT. On the contrary, si RNAs against STAT3 significantly inhibited EMT induced by coculture and reduced the migration and invasion abilities of HCC cells. The TGF-β levels in the culture medium of HSCs and HCC cells were similar. Addition of IL-6 also did not directly induce EMT while S1 P induced persistent STAT3 activation and EMT in HCC cells. Migration and invasion abilities of highly metastatic HCC cells were reduced by S1PR1 si RNA. HSCs mediated EMT was also reversed by interfering S1PR1 expression. Inoculation of both HCC cells and HSCs resulted in higher pulmonary metastasis rate compared with HCC cells alone.Conclusions: HSCs secreted HGF to activated c-Met/AKT in HCC cells which further induced Sorafenib resistance.Activation of JAK/STAT3 pathway by HSCs may also contribute to Sorafenib resistance. Specific inhibitor of c-Met, AKT, JAK and STAT3 increased the sensitivity of HCC cells to sorafenib.HSCs coculture induced EMT and facilitated the migration and invasion of HCC cells.S1P may be a significant mediator of HSCs induced EMT by activating S1PR1/STAT3/Slug pathway in HCC cells.
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