| Aims:Hepatocellular carcinoma(HCC)is one of the most common malignant tumors.Liver cancer stem cells(LCSCs),which have the capacity of self-renewal,metastatic,chemotherapy resistance and tumor initiation,contribute to treatment resistance and tumor relapse in hepatocellular carcinoma(HCC).Recent studies have shown that cancer-associated fibroblasts(CAFs)are involved in regulating the properties of CSCs.Therefore,the purpose of this study is to explore the role and mechanism of CAFs in promoting the stemness characteristics of CD24~+HCC cells.Methods:We firstly evaluated the expression of CD24 by Immunohistochemistry(IHC)in tumor specimens and matched adjacent normal specimens from 63 HCC patients.And then we investigated the correlation between CD24 expression and clinical-pathologic characteristics.CD24~+cells were isolated from HCC cell lines(HLE and MHCC-97L cell lines),and their stemness characteristics were demonstrated in vitro and in vivo by using sphere formation assay,Transwell invasion and migration assays,chemotherapy resistance assay and tumor formation assay.The expressions of stemness-related genes were evaluated by qRT-PCR and Western blot analysis.Second,we identified the fibroblasts in HCC tissues by IHC with the activated fibroblasts marker alpha-smooth muscle actin(?-SMA)in tumor tissues.And then we evaluated the correlation between the expression of?-SMA in CAFs and that of CD24 in HCC cells.CAFs isolated from fresh HCC samples and the conditional medium(CM)of CAFs were collected.CAF-CM were co-cultured with CD24~+HCC cells,and CSC functions were measured.ELISA was performed to confirm the cytokines secreted by CAFs.To further confirmed that the cytokines secreted by CAFs,we added the recombinant protein or neutralizing antibodies of the cytokines.And then the stemness characteristics of CD24~+HCC cells were detected.Finally,to understand the molecular mechanism by which CAFs secreted cytokines to promote CD24~+HCC cells,we used inhibitors,siRNA and lentiviruses to inhibit related pathways in CD24~+HCC cells.And then co-cultured with CAF-CM,the stemness properties of CD24~+HCC cells were measured.Results:(1)We found that the expression of CD24 was high in HCC tissues,and positively correlated with the poor prognosis;(2)CD24~+cells isolated from HCC cell lines(HLE and MHCC-97L cell lines)exhibited higher self-renewal,chemotherapy resistance,invasion and migration abilities in culture,and formed more xenograft tumors in mice than CD24~-cells;(3)We found that high expression of?-SMA was significantly correlated with worse clinical-pathological.And there are a positive correlation between the expression of?-SMA in CAFs and that of CD24 in HCC cells;(4)CAFs originated from HCC tissues promoted the stem properties of CD24~+HCC cells;(5)ELISA assay showed that CAFs secreted a significant amount of HGF and IL6,and recombinant human HGF and IL6 promoted the stemness properties of CD24~+HCC cells.Moreover,the neutralizing antibodies of HGF and IL6 blocked the CAF-enhanced stemness characteristics of CD24~+HCC cells;(6)Western blot analysis showed that HGF/c-Met,IL6/IL6R and STAT3 pathways were activated in CD24~+HCC cells co-cultured with CAF-CM,and CAF-induced STAT3phosphorylation completely blocked by simultaneous MET knockdown and treatment with Tocilizumab(an anti-IL6 receptor antibody);(7)We evaluated that high expression of p-STAT3 was significantly correlated with worse clinical-pathological and shorter survival;(8)When inhibited STAT3 signaling by lentivirus-based shRNA or the inhibition of STAT3phosporylation in CD24~+HCC cells,the effect of CAF-enhanced stemness characteristics was completely abolished.Conclusion:These findings suggested that HGF and IL6 secreted by CAFs promoted the stemness properties of CD24~+HCC cells through the activation of STAT3 signaling.Our findings provide a more understanding of the crosstalk between the tumor microenvironment and HCC CSCs.Moreover,our study revealed a potential therapeutic target in HCC. |
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