CXCL-1促进胃癌细胞迁移和侵袭机制探讨

Effect of CXCL1 on gastric cancer cells migration and invasion

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, which play vital roles in the initiation and progression of tumor. The study aimed to investigate the effects and molecular mechanism of CXCL1 secreting from cancer-associated fihroblast on gastric cancer cells migration and invasion. METH- ODS Isolated and primary cultured CAFs and normal fibroblasts （NFs）. To determine the CXCL1 mRNA and protein expression level using RT-PCR and Western blot assay, and determined CXCL1 concentration in CAFs-CM （conditioned media） and NFs-CM using ELISA assay. We further investigated the effects of exogenous CXCL1 and CXCL1 antibody on the migration and invasion of gastric cancer cells using Transwell and healing assay. We determined the effects of exog- enous CXCL1 and CXCL1 antibody on the expression level of MMP-3 protein via Western blot assays. RESULTS The relative expression levels of CXCL1 mRNA and protein in CAFs and secretion concentration of CXCL1 in CAFs-CM were significantly higher than that of NFs and NFs-CM （P〈0.01）. The number of cells penetrated membrane and wound clo- sure rate of CXCL1 group were significantly more and lower than that of the control group （t=3. 213,P=0. 030; t= 3. 006,P=0. 040）, respectively. The number of cells penetrated membrane and wound closure rate of CXCL1 antibodygroup were sign Kieantly higher than that of control group （t= 3. 811, P = 0. 020 ; t = 4. 678, P =0. 010）. The expression level of MMP-3 protein is up-regulated in gastric cancer in exogenous CXCL1 group and down regulated in CXCL1 anti- body group （t = 4.153, P= 0. 009 ; t= 3. 349, P= 0. 030）. CONCLUSION CXCL1 secreting from CAFs promotes the mi- gration and invasion of gastric cancer cells probably by regulating MMP-3 protein.