南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (6): 943-951.doi: 10.12122/j.issn.1673-4254.2023.06.09

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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

邓 婷,杜伯雨,郗雪艳   

  1. 湖北医药学院基础医学院免疫教研室,湖北 十堰 442000
  • 出版日期:2023-06-20 发布日期:2023-07-06

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

DENG Ting, DU Boyu, XI Xueyan   

  1. Department of Immunology, School of Basic Medical Sciences, Hubei University of Medicine, Shiyan 442000, China
  • Online:2023-06-20 Published:2023-07-06

摘要: 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。

关键词: 肿瘤微环境;结直肠癌;癌症相关成纤维细胞;ERK

Abstract: 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 up-regulated mRNA expression levels of CAFs markers (including α-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 of α-SMA protein and ERK phosphorylation in the cells (P<0.05). The ERK inhibitor SCH772984 obviously inhibited the expression of α-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.

Key words: tumor microenvironment; colorectal cancer; cancer-associated fibroblasts; ERK singaling