他莫昔芬通过GPR30介导CAF外分泌CXCL16促进乳腺癌MCF-7细胞迁移及侵袭

Tamoxifen enhances the abilities of migration and invasion of breast cancer MCF-7 cells via external secretion of CXCL16 from CAF cells mediated by GPR30

目的 :探讨他莫昔芬(tamoxifen,TAM)通过雌激素G蛋白偶联受体30(estrogen G-protein-coupled receptor 30,GPR30)介导肿瘤相关成纤维细胞(cancer-associated i broblast,CAF)外分泌CXC型趋化因子配体16(CXCchemokine ligand 16,CXCL16)对乳腺癌MCF-7细胞迁移和侵袭的影响。方法 :TAM和TAM联合GPR30特异性抑制剂G15分别处理CAF后,通过基因芯片检测2组细胞差异表达的基因,并从中挑选出CXCL16,应用实时荧光定量PCR和蛋白质印迹法进行验证,应用ELISA法检测细胞上清液中CXCL16的浓度。用TAM和TAM联合G15处理后的CAF上清液、不做任何处理的CAF上清液、添加CXCL16或CXCL16中和抗体的培养液分别培养MCF-7细胞,采用MTT法、划痕愈合实验和Transwell小室法检测细胞的增殖、迁移和侵袭能力。结果 :与TAM联合G15处理组相比,TAM处理组CAF中2 358个基因表达上调,2 178个基因表达下调;TAM处理组CAF中CXCL16 m RNA和蛋白的表达水平及上清液中CXCL16的浓度均显著上调(P值均<0.01),且该效应可被G15阻断(P值均<0.01)。TAM处理组CAF上清液和添加CXCL16培养液培养的MCF-7细胞,其迁移和侵袭能力均增强(P值均<0.05),而增殖能力则无显著变化(P值均>0.05)。结论 :CAF经TAM处理后CXCL16分泌增多,该效应由GPR30介导,而外分泌的CXCL16可促进乳腺癌MCF-7细胞的迁移和侵袭能力。

Objective：To explore the effects of external secretion of CXCchemokine ligand 16（CXCL16） from cancer-associated fibroblast（CAF） mediated by estrogen C-protein-coupled receptor 30（CPR30） via tamoxifen（TAM） on proliferation,migration and invasion of breast cancer MCF-7 cells.Methods：After CAFs were treated with TAM or combination of TAM and CPR30 specific inhibitor G15,the differentially expressed genes were detected by gene chip and the CXCL16 was sorted out.The expression levels of CXCL16 mRNA and protein were verified by realtime fluorescent quantitative PCR and Western blotting,respectively.The concentration of externally secreted CXCL16 was detected by ELISA test.The breast cancer MCF-7 cells were cultured with cell culture supernatant of CAF after treatment with TAM,combination of TAM and G15,medium containing CXCL16 or CXCL16 neutralizing antibody or without any treatment,the abilities of proliferation,migration and invasion of MCF-7 cells were detected by MTT method,wound-healing assay and Transwell chamber assay,respectively.Results：As compared with the combination of TAM and G15 treatment group,there were2 358 up-regulated genes and 2 178 down-regulated genes in TAM treatment group.The expression levels of CXCL16 mRNA and protein as well as the concentration of externally secreted CXCL16 in cell supernatant of CAF after treatment with TAM were up-regulated（all P 〈 0.01）,and these effects could be blocked by G15（all P 〈 0.01）.The abilities of migration and invasion of MCF-7 cells cultured with cell supernatant of CAF treated by TAM or medium containing CXCL16 were improved（all P 〈 0.05）,but the proliferative ability had no obvious change（all P 〉 0.05）.Conclusion：After treatment with TAM,secretion of CXCL16 from CAF is increased,and this effect is mediated by CPR30.The external secretion of CXCL16 enhances the abilities of migration and invasion of breast cancer MCF-7 cells.