鼻咽癌细胞体外诱导肿瘤相关成纤维细胞形成并上调NNMT表达

Nasopharyngeal carcinoma cells induce cancer-associated fibroblast formation and upregu⁃late NNMT expression in vitro

目的:探讨烟酰胺-N-甲基转移酶(NNMT)在肿瘤相关成纤维细胞(CAFs)中的表达。方法:分别采用条件培养基(CM)和Transwell小室两种共培养模式建立CAFs体外诱导模型,将人胚肺成纤维细胞MRC-5与鼻咽癌细胞系5-8F共培养48 h,显微镜下观察细胞形态学变化。实时荧光定量PCR(RT-qPCR)法检测NNMT及CAFs相关标志物[α-平滑肌肌动蛋白(α-SMA)、成纤维细胞活化蛋白(FAP)、成纤维细胞特异性蛋白1(FSP-1)及波形蛋白(Vimentin)]基因表达,CCK-8法检测细胞增殖能力。分离纯化头颈肿瘤来源CAFs,RT-qPCR和Western blotting法检测CAFs相关标志物及NNMT的表达。结果:两种共培养模式均能成功诱导并上调MRC-5细胞中NNMT、α-SMA、FAP、FSP-1、Vimentin基因表达(P<0.05),其中Transwell小室共培养模式诱导效果较好。鼻咽癌细胞系5-8F-CM亦能促进MRC-5细胞生长;头颈肿瘤来源的CAFs中FAP、FSP-1、Vimentin、IL-6、NNMT表达量均升高(均P<0.05)。结论:Transwell小室共培养模式是体外诱导CAFs形成的良好建模方式,NNMT在CAFs中的表达上调。

Objective:To investigate the expression of nicotinamide-N-methyltransferase(NNMT)in cancer-associated fibroblasts(CAFs).Methods:Two co-culture modes,conditioned medium(CM)and Transwell,were used to establish an in vitro induction model of CAFs,and human embryonic lung fibroblasts MRC-5 were cocultured with nasopharyngeal carcinoma cell line 5-8F for 48 h.The morphological changes of the cells were observed under microscope.Gene expression of NNMT and CAFs-related markers includingα-smooth muscle actin(α-SMA),fibroblast activation protein(FAP),fibroblast-specific protein 1(FSP-1)and Vimentin were detected by real-time quantitative PCR(RT-qPCR),and cell proliferation ability was detected by CCK-8 assay.The head and neck cancer-derived CAFs were isolated and purified,and the expression of CAFs-related markers and NNMT were detected by RT-qPCR and western blotting.Results:Both co-culture modes successfully induced and up-regulated the expression of NNMT,α-SMA,FAP,FSP-1 and Vimentin genes in MRC-5 cells(P<0.05),with the Transwell co-culture mode showing better induction effect.The nasopharyngeal carcinoma cell line 5-8FCM also promoted the growth of MRC-5 cells.The expressions of FAP,FSP-1,Vimentin,IL-6,and NNMT were levated in head and neck cancer-derived CAFs(all P<0.05).Conclusion:Transwell co-culture mode is a good modeling approach to induce the forma-tion of CAFs in vitro,and the expression of NNMT was up-regulated in CAFs.