结直肠癌细胞通过激活成纤维细胞的ERK通路诱导癌症相关成纤维细胞的形成

Colorectal cancer cells induce the formation of cancer-associated fibroblasts by activating the ERK signaling pathway in fibroblasts

目的探究结直肠癌(CRC)细胞(HCT116、Caco-2)条件培养基促进癌症相关成纤维细胞(CAFs)形成的机制,为CRC治疗提供新的思路。方法将对数生长期的人正常结直肠成纤维细胞(CCD-18Co)分为对照组、HCT116细胞条件培养基处理组(HCT116-CM组)、Caco-2细胞条件培养基处理组(Caco-2-CM组)、300 nmol/L ERK抑制剂SCH772984组(iERK组)、HCT116-CM联合ERK抑制剂组(HCT116-CM+iERK组)、Caco-2-CM联合ERK抑制剂组(Caco-2-CM+iERK组)。采用RT-PCR和细胞免疫荧光检测CAFs相关分子标志物表达水平;利用RTCA、克隆形成和创伤愈合实验测定细胞增殖、克隆形成和迁移能力;Western blot检测HCT116-CM和Caco-2-CM激活的成纤维细胞信号通路,同时检测相应信号通路阻断后CAFs形成情况。结果HCT116-CM和Caco-2-CM可上调CCD-18Co中CAFs标志物α-平滑肌肌动蛋白(α-SMA)、成纤维细胞活化蛋白(FAP)、纤连蛋白(FN)和转化生长因子-β(TGF-β)的mRNA表达水平并促进成纤维细胞向CAFs转化(P<0.05)。HCT116-CM和Caco-2-CM促进CCD-18Co细胞的增殖、克隆形成和迁移(P<0.05)。HCT116-CM和Caco-2-CM增强CCD-18Co细胞中α-SMA蛋白的表达和ERK磷酸化修饰水平(P<0.05),ERK抑制剂SCH772984可抑制α-SMA的表达并抑制CRC细胞条件培养基的促CAFs形成作用(P<0.05)。结论CRC细胞可通过激活成纤维细胞的ERK通路诱导CRC相关CAFs的形成。

Objective To investigate the mechanism by which conditioned medium of colorectal cancer cells promotes the formation of cancer-associated fibroblasts(CAFs).Methods Normal human colorectal fibroblasts(CCD-18Co cells)in logarithmic growth phase were treated with the conditioned media of colorectal cancer HCT116 cells(HCT116-CM)or Caco-2 cells(Caco-2-CM)alone or in combination with 300 nmol/L ERK inhibitor SCH772984.The expression levels of CAFs-related molecular markers were detected in the treated cells with real-time quantitative PCR(RT-qPCR)and immunofluorescence assay,and the changes in cell proliferation,colony formation and migration were assessed with RTCA,colony formation and wound healing assays;Western blotting was performed to detect the activated signaling pathways in the fibroblasts and the changes in CAFs formation after blocking of the signaling pathway.Results HCT116-CM and Caco-2-CM significantly upregulated mRNA expression levels of CAFs markers(includingα-SMA,FAP,FN and TGF-β)in CCD-18Co cells,and strongly promoted fibroblast transformation into CAFs(P<0.05).The two conditioned media also promoted the proliferation,colony formation and migration of CCD-18Co cells(P<0.05)and significantly increased the levels ofα-SMA protein and ERK phosphorylation in the cells(P<0.05).The ERK inhibitor SCH772984 obviously inhibited the expression ofα-SMA and the transformation of CCD-18Co cells into CAFs induced by the conditioned medium of colorectal cancer cells(P<0.05).Conclusion Colorectal cancer cells may induce the formation of colorectal CAFs by activating the ERK pathway in the fibroblasts.