| ObjectiveSorafenib is the standard first-line treatment for advanced hepatocellular carcinoma(HCC),even though acquired resistance to sorafenib has been found in many HCC patients,resulting in poor prognosis.Accumulating evidence demonstrates that liver cancer stem cells(LCSCs)contribute to sorafenib resistance in HCC.The inflammatory factor interleukin 6(IL-6)plays an important role in sorafenib resistance in HCC.However,the mechanism by which IL-6 in LCSCs is involved in the process of HCC sorafenib resistance remains elusive.We aimed to establish stable sorafenib drug-resistant HCC cell lines,to isolate liver cancer stem cells from them,blocking their IL-6/STAT3 signaling pathway,and to explore the regulation mechanism of IL-6/STAT3 signaling pathway targeting liver cancer stem cells for sorafenib drug-resistant HCC through in vitro and in vivo experiments.Methods(1)Human HCC cell line PLC/PRF/5 was cultured in vitro with standard methods.Sorafenib was added into cell culture medium,stable drug-resistant HCC cell line PLC/PRF/5-R was established by concentration gradient method,and PLC/PRF/5-R cell activity was detected by CCK-8 method,and IC50 was calculated.The expression levels of multidrug resistance genes(MDR),E-cadherin(E-cadherin)and vimentin(Vimentin)in PLC/PRF/5-R were tested by the real-time fluorescent quantitative PCR and Western blotting,To clarify the stability of PLC/PRF/5-R to sorafenib resistance,and the epithelial-mesenchymal transition(EMT)characteristics of resistant strains.(2)According to the currently recognized surface marker molecules(Ep CAM and CD44)of liver cancer stem cell,flow cytometry was used to sorting the positive(Ep CAM+/CD44+)and negative(Ep CAM-/CD44-)cells in PLC/PRF/5 and PLC/PRF/5-R which have been cell proliferation and apoptosis test.The tumorigenicity of the positive and negative cells after sorting was detected by the tumorigenic ability of subcutaneous transplanted tumors in nude mice to determine the success of the sorting,isolation and culture of the drug-resistant sorafenib liver cancer cell line LCSCs-R for subsequent experiments.(3)The expression levels of IL-6,IL-6R,STAT3 and GP130 in LCSCs and LCSCS-R were detected by real-time fluorescence quantitative PCR and Western blotting to determine the activation status of IL-6/STAT3 signaling pathway in LCSCs-R.IL-6R-sh RNA was transfected into LCSCs-R by plasmid-mediated cell transient transfection,and the expression level of IL-6 receptor(IL-6R),a key factor in the IL-6/STAT3 signaling pathway,was detected by real-time fluorescence quantitative PCR and Western blotting to confirm that the IL-6/STAT3 signaling pathway was blocked.The expression levels of LCSCs marker(Ep CAM,CD44),stemness marker(Oct3/4,β-catenin)and hepatocyte differentiation marker(G-6-P,AFP)were determined and compared with the control group.(4)Animal experiments in vivo: Cell transfected IL-6R-sh RNA into LCSCs-R through lentivirus-mediated to construct a stable LCSCs-R cell line with blocks IL-6,and establish a nude mouse subcutaneous xenograft model to monitor the general condition and size of transplanted tumor in nude mice with blocking IL-6 and/or sorafenib.The expression levels of LCSCs markers(Ep CAM and CD44),stemness markers(Oct3/4 and β-catenin)and angiogenic factors(VEGF and VEGFR)in tumor tissues were detected at the end of the experimental period to evaluate the role of IL-6in sorafenib resistance in HCC.ResultsWe established a stable sorafenib-resistant HCC cell line PLC/PRF/5-R successfully with an IC50 of 12.18 μM,the expression level of MDR was increased,E-cadherin was decreased or absent,and Vimentin was increased,presenting obvious characteristics of EMT.There was no significant statistical difference in the detection of cell proliferation and apoptosis of positive and negative cells after the flow cytometry separation,but the results of tumor formation experiment in nude mice suggested that the LCSCs-R was isolated from PLC/PRF/5-R were more tumorigenic than those in the control group.The expression levels of IL-6,IL-6R,STAT3 and GP130 in LCSCs-R were significantly increased,indicating that the IL-6/STAT3 signaling pathway was activated in LCSCs-R.Blocking IL-6 expression restored the sensitivity of LCSCs-R to sorafenib,suggesting that the IL-6/STAT3 signaling pathway plays a key role in inducing sorafenib resistance.Furthermore,a xenograft tumor model showed that IL-6 downregulation improved the antitumor effect of sorafenib.ConclusionsLCSCs play an important role in sorafenib-resistant HCC,and inhibition of the IL-6/STAT3 signaling pathway improves the antitumor effects of sorafenib against HCC in vitro and in vivo.These findings demonstrate that IL-6 in LCSCs may function as a novel target for combating sorafenib resistance in HCC. |
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