成纤维细胞生长因子21通过调控细胞脂代谢促进恶性黑色素瘤细胞生长

摘要

Objective To explore the role and underlying molecular mechanisms of fibroblast growth factor 21 ( FGF21) in the development of melanoma. Methods Quantitative RT-PCR was applied to investigate the mRNA levels of FGF21 in cells. FGF21 overexpression plasmid was used to transfect FGF21 in melanoma cells, and shRNA technology was used to knock down FGF21 expression in melanoma cells. CCK8 assay and BrdU staining were used to detect the abilities of cell growth and proliferation. RT-qPCR was used to evaluate the expression levels of fat acids-oxidation-associated genes PGC1a, CD36, CPT1a, CPT1b and CPT2. Melanoma cells with or without FGF21 overexpression were subcutaneously injected into nude mice to observe the tumorigenic ability of the cells.Results The mRNA expression level of FGF21 was about 10 times higher than that of melanocytes (P<0.05). After the specific knockdown of FGF21 or overexpression of FGF21 in melanoma cells, cell proliferation levels were significantly reduced or increased ( P<0.05) . In vitro experiments showed that the overexpression of FGF21 significantly promoted the fatty acid oxidation in melanoma cells in terms of increased PGC1a, CD36, CPT1a, CPT1b and CPT2 gene expression. Further studies found that after the inhibition of intracellular fatty acid oxidation by small molecule Etomoxir, the cell proliferation ability of Etomoxir group was significantly lower than that of the control group by CCK8 assay and BrdU staining ( P<0. 05) . Meanwhile, the tumor growth of the nude mice with FGF21-overexpressed melanoma cells was dramatically enhanced, and the tumor diameters were significantly increased. Conclusions FGF21 could promote the growth and proliferation of melanoma cells through regulating intracellular fatty acid oxidation, which accelerates the deterioration of melanoma.%目的 探讨成纤维细胞生长因子21(FGF21)在黑色素瘤发生发展中的作用和机制.方法 运用荧光定量PCR技术分析黑色素瘤细胞内基因的表达水平,运用FGF21过表达质粒转染,在黑色素瘤细胞中过表达FGF21,运用shRNA技术敲低黑色素瘤细胞内FGF21表达.运用CCK8染色以及BrdU染色检测黑色素瘤细胞的生长及增殖能力,运用荧光定量PCR技术检测肿瘤细胞脂肪酸氧化相关基因PGC1a、CD36、CPT1a、CPT1b、CPT2的表达变化.将过表达FGF21及非过表达FGF21的黑色素瘤细胞注射裸鼠皮下,观察细胞成瘤能力.结果 在肿瘤细胞中FGF21的mRNA表达水平平均高于黑色素细胞10倍左右,差异均具有统计学意义(P<0.05).在黑色素瘤细胞中过表达FGF21或者特异性敲低FGF21后,细胞的增殖能力出现显著的升高或降低,相同时间点实验组与对照组相比,差异均具有统计学意义(P<0.05).体外实验发现,FGF21过表达可明显促进黑色素瘤细胞中脂肪酸氧化水平,PGC1a、CD36、CPT1a、CPT1b、CPT2等与脂肪酸氧化相关基因的表达均高于对照组,差异具有统计学意义(P<0.05).进一步的研究发现在使用小分子Etomoxir抑制细胞内脂肪酸氧化后,经CCK8染色和BrdU染色检测,Etomoxir组细胞增殖能力明显低于对照组,二者比较差异均具有统计学意义(P<0.05).同时,过表达FGF21黑色素瘤细胞在裸鼠体内的成瘤能力明显升高,肿瘤直径显著增大,与非过表达黑色素瘤细胞比较,差异均具有统计学意义(P<0.05).结论 FGF21可以作用于黑色素瘤细胞,并通过提高细胞内线粒体脂肪酸氧化水平促进黑色素瘤的增殖,加速黑色素瘤恶化.