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目的肿瘤相关成纤维细胞(cancer associated fibroblasts,CAFs)是肿瘤微环境中的重要细胞,它在肿瘤发生发展过程中扮演着重要角色。本研究探讨肿瘤相关成纤维细胞分泌CXC趋化因子配体1(chemokine C-X-C motif ligand 1,CXCL1)对胃癌细胞迁移侵袭能力的影响及分子机制。方法分离和培养胃癌相关成纤维细胞和胃正常黏膜成纤维细胞(normal fibroblasts,NFs);逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)、蛋白质印迹法和酶联免疫吸附剂试验(enzyme linked immunosorbent assay,ELISA)分别测定CAFs和NFs中CXCL1mRNA、蛋白水平表达量和条件培养基中CXCL1的含量;Transwell实验和细胞划痕实验观察外源性CXCL1和CXCL1抗体对胃癌细胞迁移侵袭能力的影响;蛋白质印迹法检测不同处理组MMP-3蛋白水平的表达变化。结果 CAFs中CXCL1mRNA、蛋白水平表达量和CAFs-CM分泌的CXCL1含量均显著高于NFs和NFs-CM组,P<0.01。外源性CXCL1组胃癌细胞穿透膜细胞数显著多于对照组,t=3.213,P=0.030;划痕伤口愈合率显著高于对照组,差异有统计学意义,t=3.006,P=0.04。而CXCL1抗体组胃癌细胞穿透膜细胞数显著少于对照组,t=3.811,P=0.020;划痕伤口愈合率显著低于对照组,差异有统计学意义,t=4.678,P=0.010。外源性CXCL1组胃癌细胞中基质金属蛋白酶3(matrix metalloproteinase-3,MMP-3)蛋白表达水平显著上调,t=4.153,P=0.009;而CXCL1抗体组胃癌细胞中MMP-3蛋白表达水平显著下调,差异有统计学意义,t=3.349,P=0.030。结论胃癌CAFs分泌的CXCL1可能是通过调节MMP-3蛋白表达促进胃癌细胞迁移和侵袭的。
Objective Cancer-associated fibroblasts (CAFs) are important cells in the tumor microenvironment. They play an important role in the development of tumors. This study was to investigate the effect of tumor-associated fibroblasts secreting chemokine C-X-C motif ligand 1 (CXCL1) on the migration and invasion of gastric cancer cells and its molecular mechanism. Methods Gastric cancer-associated fibroblasts and normal fibroblasts (NFs) were isolated and cultured; reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA) The enzyme-linked immunosorbent assay (ELISA) was used to determine the expression of CXCL1 mRNA and protein in CAFs and NFs, and the content of CXCL1 in conditioned media. Transwell and cell scratch experiments were used to observe the effects of exogenous CXCL1 and CXCL1 antibodies on gastric cancer cells. Effects of migration invasiveness; expression of MMP-3 protein in different treatment groups was detected by Western blotting. Results The expression of CXCL1 mRNA and protein in CAFs and CXCL1 in CAFs-CM were significantly higher than those in NFs and NFs-CM groups (P<0.01). The number of penetrating membrane cells in the exogenous CXCL1 group was significantly higher than that in the control group, t=3.213, P=0.030; the wound healing rate of the scratches was significantly higher than that of the control group, and the difference was statistically significant (t=3.006, P=0.04). . The number of penetrating membrane cells in the CXCL1 antibody group was significantly lower than that in the control group, t=3.811, P=0.020; the wound healing rate of the scratches was significantly lower than that of the control group, and the difference was statistically significant (t=4.678, P=0.010). The expression of matrix metalloproteinase-3 (MMP-3) protein in gastric cancer cells was significantly up-regulated in exogenous CXCL1 group, t=4.153, P=0.009, while the expression of MMP-3 protein was significantly increased in CXCL1 antibody group. Down-regulation, the difference was statistically significant, t = 3.349, P = 0.030. Conclusion CXCL1 secreted by gastric cancer CAFs may promote the migration and invasion of gastric cancer cells by regulating the expression of MMP-3 protein.