| Background and ObjectiveCancer stem cells,a special subgroup cells of tumor,with self-renewal potential and pluripotency,may be reprogrammed from the dedifferentiation of tumor cells,which contributing to the failure of clinical treatments.Deoxycholic acid(DCA),one of the components of gastroesophageal reflux contents,plays an important role in inducing the transformation of esophageal squamous epithelial cells into Barrett’s esophagus.Barrett’s esophagus is a precancerous lesion of esophageal adenocarcinoma(EAC).Considering esophageal adenocarcinoma grows in an inflammatory environment stimulated by deoxycholic acid,this experiment was conducted to investigate whether deoxycholic acid can affect the expression of reprogramming factors KLF4,OCT4 and Nanog,and have a malignant induction effect on the transformation of esophageal adenocarcinoma stem cells from esophageal adenocarcinoma cells,and whether the IL-6/STAT3 inflammatory signaling pathway is involved in the regulation of this process.Methods1.OE33 cell line of EAC was treated with 250uM DCA for Oh,3h,6h and 12h,before the mRNA expression of KLF4,OCT4,Nanog,IL-6 and Bcl-xL were detected by RT-PCR.The concentration of IL-6 protein in the supernatant of the media was detected by ELISA,while the expression of STAT3,pSTAT3,KLF4 and OCT4 protein were detected by Western Blot.2.The small interference RNA of STAT3 was used to treat OE33 cell line for 48 hours,before the detection of KLF4,OCT4 and Bcl-xL expression,which help to explore the upstream and downstream regulative relationship between STAT3 and KLF4,OCT4,Bcl-xL.3.Different concentrations of recombinant human IL-6(rhIL-6)were designed to treat OE33 cell line for 24 hours,in the purpose of detecting their effects on pSTAT3,KLF4,OCT4 and Bcl-xl expression,further confirming the the involvement of IL-6/STAT3 signaling pathway modulates the expression of reprogramming factors mentioned above.Results1.As DCA stimulated esophageal adenocarcinoma cells,it was shown that the fold change about mRNA expression of KLF4 were 1.87,6.34(P<0.01)and 4.57(P<0.05)after 3h,6h and 12h stimulation by DCA in the comparison of Oh stimulation.And the relative expression of KLF4 protein at Oh,3h,6h and 12h stimulation by DCA were 0.78,0.89,1.79(P<0.05)and 2.73(P<0.05),respectively.In addition to OCT4,the fold change of mRNA were 1.43,1.87(P<0.05)and 4.51(P<0.01)after 3h,6h and 12h DCA stimulation,respectively,while the relative protein expression of OCT4 were 0.53,0.70,1.87(P<0.05)and 2.36(P<0.05)at 0,3,6,12 hours of DCA stimulation.The relative mRNA expression of Nanog were 1.65,0.97 and 1.14 after 3,6 and 12 hours of stimulation with DCA,and there were no significant difference between them and Oh DCA stimulation.The fold change of mRNA expression in anti-apoptotic gene Bcl-xL were 2.16,2.31(P<0.05)and 5.38(P<0.05)after 3h,6h and 12h DCA stimulations,and the relative mRNA expression of IL-6 cytokine were 1.82,6.11(P<0.01)and 26.66(P<0.01)in the groups mentioned above,in comparision of DCA Oh treatment group.The concentration of secreted IL-6 protein in the supernatant of corresponding medium were 8.029 ng/L,17.004 ng/L and 20.532 ng/L in 3h,6h and 12h DCA stimulation groups,respectively,which were all significantly different from the DCA Oh group(P<0.01).Meanwhile,the Western Blot photogragh indicated that the downstream signal protein pSTAT3(Tyr705)of IL-6 increased with the prolongation of DCA stimulation.2.After STAT3 silencing,the relative mRNA expression of stem cell reprogramming factor KLF4,OCT4 and anti-apoptotic gene Bcl-xL were down-regulated to 13%(P<0.01),34%(P<0.05)and 37%(P<0.01),respectively.Meanwhile,the relative expression of KLF4 protein in negative control group and silencing group were 0.89 and 0.57(P<0.05),and that of OCT4 protein were 2.87 and 1.86(P<0.01),KLF4 and OCT4 protein were down-regulated with significance difference after STAT3 silencing,indicating that STAT3 is the upstream regulator of KLF4,OCT4 and Bcl-xL gene,and would play an important role in promotion of these genes expression.3.As recombinant human IL-6 stimulates esophageal adenocarcinoma cells,Western Blot photogragh showed that the expression of pSTAT3(Tyr705)is up-regulated in a concentration-dependent manner.Compared with the blank group,the fold change of KLF4 mRNA were 1.62 and 3.36(P<0.05),and the fold change of OCT4 mRNA were 2.65 and 3.87(P<0.05),after treated with rhIL-6 at 10 ng/ml and 100 ng/ml,respectively.It was showed the relative protein expression of KLF4 gene were 0.76,3.25(P<0.01)and 3.72(P<0.05)in blank group,10 ng/ml rhIL-6 group and 100 ng/ml rhIL-6 group,respectively.In addition,the relative protein expression of OCT4 gene were 2.74,5.08(P<0.01)and 7.86(P<0.01)in blank group,10 ng/ml rhIL-6 group and 100 ng/ml rhIL-6 group,respectively.The expression of KLF4 and OCT4 protein stimulated by recombinant human IL-6 were both with different significance.Meanwhile,in comparision to the blank group,the fold change of Bcl-xL mRNA expression at 10 ng/ml and 100 ng/ml rhIL-6 stimulation groups were 1.15 and 7.01(P<0.05),respectively.ConclusionsDeoxycholic acid has a malignant induction effect which activates the IL-6/STAT3 signaling pathway,and promotes the up-regulated expression of stem cell markers KLF4,OCT4 and anti-apoptotic gene Bcl-xL.This suggests that deoxycholic acid may induce the transformation of esophageal adenocarcinoma cells into cancer stem cells,improve the anti-apoptosis ability of tumors,and increase the malignancy of esophageal adenocarcinoma.Targeting on the IL-6/STAT3 signaling pathway and neutralizing deoxycholic acid may inhibit the formation of esophageal adenocarcinoma cancer stem cells and help to improve the clinical efficacy of esophageal adenocarcinoma. |
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