结直肠成纤维细胞通过激活ERK信号通路促进结直肠癌细胞的恶性生物学行为

doi:10.12122/j.issn.1673-4254.2024.10.04

摘要/Abstract

摘要：

目的探究人结直肠成纤维细胞（CCD18-Co）条件培养基对结直肠癌（CRC）细胞恶性进展的影响，为CRC治疗提供思路。方法利用RTCA、克隆形成和创伤愈合实验测定CRC细胞增殖、克隆形成和迁移能力；Western blotting检测CCD18-Co-CM激活的CRC细胞ATK、ERK和STAT3信号通路，同时检测相应信号通路阻断后CRC细胞增殖、克隆形成和迁移能力；肿瘤球形成实验检测CCD18-Co-CM对CRC细胞成球能力的影响；RT-PCR方法检测CRC细胞干性标志物的表达情况。结果 CCD-18Co-CM能够促进CRC细胞的增殖、克隆形成和迁移能力（P<0.05）。CCD-18Co-CM能够增强CRC细胞的成球能力及干性标志物的表达（P<0.05）。CCD-18Co-CM 能够激活CRC细胞ERK信号通路（P<0.05），ERK信号通路抑制剂SCH772984能够降低CRC细胞增殖、克隆形成和迁移能力及成球能力和干性标志物的表达（P<0.05）。结论人正常结直肠成纤维细胞可通过激活ERK通路促进CRC细胞的恶性进展。

关键词: 结直肠成纤维细胞, 结直肠癌, 干性, ERK

Abstract:

Objective To investigate the effect of human colorectal fibroblast (CCD-18Co)‑conditioned medium (CCD18-Co-CM) on biological behaviors of colorectal cancer (CRC) cells and explore the possible molecular mechanisms. Methods Real-time cellular analysis (RTCA), clone formation assay and wound healing assay were used to analyze the changes in proliferation, clone formation, and migration abilities of CRC cell lines HCT116 and Caco-2 treated with CCD18-Co-CM. Western blotting was used to detect the changes in ATK, ERK and STAT3 signaling pathways in the CRC cells activated by CCD18-Co-CM. The effect of CCD18-Co-CM on spheroidization ability of the cells was assessed with sphere-formation assay, and the changes in expressions of CRC stemness markers were detected using RT-PCR. Results CCD-18Co-CM significantly promoted proliferation, colony formation, and migration of HCT116 and Caco-2 cells, enhanced sphere-forming ability and expressions of CRC stemness markers, and increased ERK phosphorylation in the cells. Treatment with SCH772984 effectively inhibited CCD-18Co-CM-induced ERK signaling pathway activation, suppressed the malignant phenotype, and lowered the sphere-forming ability and expression of stemness markers of the two CRC cells. Conclusion Colorectal fibroblasts promote malignant phenotype of CRC cells by activating the ERK signaling pathway.

Key words: fibroblasts, colorectal cancer, stemness, ERK

郗雪艳, 邓婷, 杜伯雨. 结直肠成纤维细胞通过激活ERK信号通路促进结直肠癌细胞的恶性生物学行为[J]. 南方医科大学学报, 2024, 44(10): 1866-1873.

Xueyan XI, Ting DENG, Boyu DU. Colorectal fibroblasts promote malignant phenotype of colorectal cancer cells by activating the ERK signaling pathway[J]. Journal of Southern Medical University, 2024, 44(10): 1866-1873.

图/表 6

表1 RT-PCR的引物序列

Tab.1 Primer sequences for RT-PCR

Gene name	Primer sequences
CD29	Up: 5'-TCCAACCTGATCCTGTGTCC-3'
CD29	Down: 5'-ACAATTCCAGCAACCACACC-3'
CD44	Up: 5'-GGCACCCGCTATGTCCAGAA-3'
CD44	Down: 5'-CCTCCTGAAGTGCTGCTCCT-3'
CD166	Up: 5'-GTCTGCTCTTCTGCCTCTTG-3'
CD166	Down: 5'-CGTCAAGTCGGCAAGGTATG-3'
MYC	Up: 5'- CGTCTCCACACATCAGCACAA-3'
MYC	Down: 5'-TCTTGGCAGCAGGATAGTCCTT -3'
EPCAM	Up: 5'-GGCTCTTTAAGGCCAAGCAG-3'
EPCAM	Down: 5'-CCAGTAGGTTCTCACTCGCT-3'
GAPDH	Up:5'-AGCTCACTGGCATGGCCTTC-3'
GAPDH	Down: 5'-CGCCTGCTTCACCACCTTCT-3'

图1 CCD-18Co-CM可增强CRC细胞的增殖、克隆形成及迁能力

Fig.1 CCD-18Co-CM promotes proliferation (A, B), clone formation (C, D) and migration (E, F; original magnification: ×100) of CRC cells analyzed using real-time cellular analysis, colony forming assay and Transwell assay (Mean±SD, n=3). *P<0.05, **P<0. 01 vs control.

图2 CCD-18Co-CM促进CRC细胞的干性特征

Fig.2 CCD-18Co-CM promotes stemness characteristics of CRC cells. A, B: Relative mRNA expression levels of CD29, CD44, CD166, MYC and EPCAM in HCT116 and Caco-2 cells after CCD-18Co-CM treatment detected by RT-qPCR (Mean±SD, n=3). C, D: Morphology of the tumor spheres formed by HCT116 and Caco-2 cells after CCD-18Co-CM treatment (Scale bar = 50 μm). *P<0.05, **P<0.01 vs control.

图3 CCD-18Co-CM激活CRC细胞中ERK信号通路

Fig.3 CCD-18Co-CM activates the ERK signaling pathway in CRC cells. A: Western blotting for detecting expression levels of STAT3, p-STAT3, AKT, p-AKT, ERK and p-ERK proteins in HCT116 and Caco-2 cells treated with CCD-18Co-CM. B: Western blotting for detecting expression levels of ERK and p-ERK in HCT116 and Caco-2 cells treated with different concentrations of SCH772984. C: Western blotting for detecting expression levels of ERK and p-ERK in HCT116 and Caco-2 cells after combined treatment with CCD-18Co-CM and 100 nmol/L SCH772984. Data are presented as Mean±SD (n=3). *P<0.05 vs control; #P<0.05, ##P<0.01 vs CCD-18Co-CM.

图4 iERK可抑制CCD-18Co-CM诱导的CRC细胞增殖、克隆形成和迁移能力的增强

Fig.4 SCH772984 (iERK) inhibits CCD-18Co-CM-induced proliferation, clone formation, and migration of CRC cells. A, B: RTCA for assessing proliferation of HCT116 and Caco-2 cells treated with CCD-18Co-CM and iERK (100 nmol/L). C, D: Colony-forming assay of HCT116 and Caco-2 cells treated with CCD-18Co-CM and 100 nmol/L iERK (×100). E, F:Transwell assay of HCT116 and Caco-2 cells treated with CCD-18Co-CM and 100 nmol/L iERK (×100). Data are presented as Mean±SD (n=3). *P<0.05 vs control, #P<0.05 vs CCD-18Co-CM.

图5 iERK抑制CCD-18Co-CM诱导的CRC细胞干性特征的增强

Fig.5 iERK decreases cancer stemness of CRC cells induced by CCD-18Co-CM. A, B: RT-qPCR for detecting mRNA expression levels of stemness-related genes in HCT116 and Caco-2 cells treated with CCD-18Co-CM and SCH772984 (Mean±SD, n=3). C, D: Morphology of tumor spheres formed by HCT116 and Caco-2 cells after CCD-18Co-CM and SCH772984 treatment (Scale bar=1 mm). *P<0.05, **P<0.01 vs control; #P<0.05 vs CCD-18Co-CM.

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