CAFs促进ADH1B甲基化对卵巢癌细胞增殖及侵袭的影响

Effects of CAFs promoting ADH1B methylation on ovarian cancer cells proliferation and invasion

目的 探讨肿瘤相关成纤维细胞(CAFs)分泌的IL-6对卵巢癌细胞增殖及侵袭的影响及机制。方法 收取新鲜离体上皮性卵巢癌及正常卵巢上皮组织,分离纯化获得CAFs及正常卵巢成纤维细胞(NFs);蛋白质印迹和免疫荧光实验检测上皮细胞和成纤维细胞标志物α-平滑肌动蛋白(α-SMA)、上皮型钙黏附素(E-cadherin)表达;收集CAFs和NFs培养上清液与卵巢癌SKOV3细胞建立间接共培养体系,细胞分为SKOV3单独培养(SKOV3)组、SKOV3与NFs上清液(NFs)组及SKOV3与CAFs上清液(CAFs)组;细胞免疫组化检测SKOV3细胞共CAFs及NFs上清液培养后乙醇脱氢酶1B(ADH1B)表达;甲基化特异性PCR (MSP)、逆转录实时荧光定量PCR(RT-qPCR)、酶联免疫吸附试验(ELISA)及蛋白质印迹实验分别检测甲基化抑制剂5-氮杂-2′-脱氧胞苷(5-Aza-dC)干预前后各组细胞ADH1B mRNA表达及甲基化状态、信号转导和激活因子3(STAT3)蛋白磷酸化水平;细胞计数试剂盒8(CCK-8)法及Transwell实验分别检测IL-6抑制剂LMT-286及重组IL-6 (rhIL-6)对细胞增殖及侵袭能力的影响。结果 肿瘤成纤维细胞中高表达α-SMA,极低表达E-cadherin;相比较SKOV3组及NFs组,CAFs组ADH1B mRNA及蛋白表达明显下调,同时CAFs组细胞上清液中IL-6水平较SKOV3组及NFs组明显升高;5-Aza-dC作用后,ADH1B甲基化部分逆转;三组细胞ADH1B mRNA和蛋白表达均增加,CAFs组STAT3磷酸化水平下降;LMT-286及rhIL-6干预均仅抑制或促进CAFs组细胞增殖和侵袭,而SKOV3组和NFs组无明显改变。结论 CAFs通过IL-6/STAT3信号通路增强ADH1B甲基化促进卵巢癌细胞增殖和侵袭。

Objective To explore the influence of IL-6 secreted by cancer-associated fibroblasts(CAFs)on promoting the proliferation and invasion of ovarian cancer cells and the possible mechanisms.Methods CAFs and normal ovarian fibroblasts(NFs)were isolated and cultured respectively from epithelial ovarian cancer and normal ovarian epithelial tissues.Cell markers alpha-smooth muscle actin(α-SMA),E-cadherin were detected by Western blot and immunofluorescence.CAFs and normal ovarian fibroblasts(NFs)were collected and cultured,and their supernatants were used to establish an indirect co-culture system with ovarian cancer SKOV3 cells,including SKOV3 cells alone(SKOV3)group,SKOV3 combined with the supernatants of NFs(NFs)group and SKOV3 combined with the supernatants of CAFs(CAFs)group.Cell immunochemistry was used to detect the expression of alcohol dehydrogenase 1B(ADH1B)in SKOV3 cells co-cultured with the supernatant of CAFs or NFs.Before and after treatment with the methylation inhibitor 5-aza-2′-deoxycytidine(5-Aza-dC),methylation-specific PCR(MSP),Reverse transcription quantitative real-time PCR(RT-qPCR),enzyme-linked immunosorbent assay(ELISA),and Western blot were used to detect the mRNA level and methylation status of ADH1B,and the phosphorylation level of signal transducers and activators of transcription 3(p-STAT3).The cell counting kit-8(CCK-8)method and Transwell assay were used to investigate the effects of the IL-6 inhibitor LMT-286 and recombinant human interleukin-6(rhIL-6)on cell proliferation and invasion.Results The protein levels ofα-SMA was highly expressed,however,CAFs and NFs cells almost lacked the E-cadherin protein.Compared with the SKOV3 and NFs groups,CAFs group exhibited significantly downregulated mRNA and protein expression of ADH1B.After treatment with 5-Aza-dC,ADH1B methylation was partially reversed,and the mRNA and protein expression of ADH1B increased in all groups.The phosphorylation level of STAT3 proteins was significantly reduced in CAFs group,while there were no significant changes in SKOV3 and NFs groups.Intervention with LMT-286 and rhIL-6 only inhibited or promoted the proliferation and invasion of cells in CAFs group,while there were no significant changes in SKOV3 and NFs groups.Conclusion CAFs can enhance the methylation of ADH1B in ovarian cancer cells via IL-6/STAT3 pathway,and may promote the proliferation and invasion.