氯化钴低氧对黑色素瘤细胞系迁移能力及FSTL1表达分泌的影响

Effects of cobalt chloride-induced hypoxia on cell migration and expression and secretion of FSTL1 in melanoma cell line

目的探讨氯化钴(CoCl_2)化学模拟低氧对黑色素瘤细胞系B16F10迁移的影响,以及Follistatin-like 1(FSTL1)蛋白在此过程中的转录、表达和分泌情况。方法 CoCl_2模拟低氧作用于小鼠B16F10细胞,实验分为3组:0μmol/L CoCl_2对照组、50μmol/L和100μmol/L CoCl_2处理组。用MTT法测定细胞活力;用Transwell法测定细胞迁移能力;qRT-PCR检测Fstl1 m RNA表达;Western blot检测细胞内外FSTL1蛋白表达。结果 CoCl_2模拟低氧可以导致B16F10细胞存活率显著下降,并呈浓度和时间依赖性;50μmol/L CoCl_2处理组(0.158±0.006)、100μmol/L CoCl_2处理组(0.203±0.002)B16F10细胞迁移能力均明显高于对照组(0.107±0.001,均P<0.05);50μmol/L CoCl_2处理组(1.573±0.114)、100μmol/L CoCl_2处理组(2.219±0.085)Fstl1 m RNA表达均显著高于对照组(0.962±0.054,均P<0.05),而胞内FSTL1蛋白表达与Fstl1 m RNA表达趋势一致。同时也发现,CoCl_2处理组细胞外FSTL1蛋白表达均低于对照组,且100μmol/L CoCl_2处理组几乎检测不到FSTL1表达。结论 CoCl_2模拟低氧促进黑色素瘤细胞迁移,可能与FSTL1的表达和分泌有关,但其功能和作用机制还需进一步探讨。

Objective To explore the effects of cobalt chloride（CoCl_2）-induced hypoxia on migration of melanoma cells, and to detect the transcription, expression and secretion of Follistatin-like 1（FSTL1） in this process. Methods B16F10 melanoma cell line was treated with CoCl_2 in order to mimic hypoxia. Experimental cells were divided into three groups： 0μmol/L, 50 μmol/L and 100 μmol/L CoCl_2 treatment groups. MTT assay was used to assure cell viability, and to determine the treatment concentration of CoCl_2. Transwell assay was used to determine the migration ability of B16F10 melanoma cell line. Real-time PCR was used to measure the m RNA expression of Fstl1. Western blot assay was used to detect the intracellular and extracellular protein expression of FSTL1. Results The cell viability of B16F10 melanoma cell line was significantly reduced by CoCl_2 treatment, with a time and concentration-dependent manner. The migration ability of B16F10 cell line was significantly increased in CoCl_2 treated group compared with that of control group（P 0.05）. The m RNA level of Fstl1 was obviously higher in CoCl_2 treated group than that of control group（P 0.05）. The intracellular expression of FSTL1 protein was consistent with the expression trend of Fstl1 m RNA. Simultaneously, the extracellular protein level of FSTL1 was significantly decreased compared with that of control group. There was no expression of FSTL1 in 100 μmol/L CoCl_2 treatment group. Conclusion The migration ability of melanoma cell line is enhanced by CoCl_2 treatment, which may be associated with expression and secretion of FSTL1, however, the relevant mechanism still needs further investigation.