二甲双胍对缺氧环境中RPMI 8226细胞新生血管生成的影响

The Effect of Metformin on Angiogenesis in Hypoxic RPMI 8226 Cells

目的 探讨缺氧条件下二甲双胍对多发性骨髓瘤(MM)细胞株RPMI 8226新生血管生成的影响。方法 选取常氧和缺氧条件下RPMI 8226细胞的基因表达综合数据库(GEO)数据集GSE110113进行差异表达基因(DEGs)和相关信号通路富集分析。采用100μmol/L氯化钴(CoCl_(2))构建化学缺氧模型;采用CCK-8试剂盒检测不同浓度的二甲双胍作用于缺氧条件下MM细胞株RPMI 8226后的细胞增殖活力;采用Western-blot检测缺氧诱导因子-1α(HIF-1α)和血管内皮生长因子(VEGF)蛋白的表达情况;采用ELISA检测培养基上清液VEGF蛋白的表达含量;采用体外新生血管生成实验检测新生血管生成。结果 基因本体论(GO)、京都基因和基因组百科全书(KEGG)信号富集分析显示,DEGs富集在“缺氧反应”和“HIF-1信号通路”;RPMI 8226细胞中HIF-1α和VEGF蛋白的表达随CoCl_(2)作用时间增加而增加;二甲双胍以剂量依赖性方式降低缺氧诱导RPMI 8226细胞中HIF-1α和VEGF蛋白的表达及培养上清液中VEGF蛋白的表达水平;二甲双胍可抑制CoCl_(2)构建的缺氧环境下RPMI 8226细胞的增殖,并促进细胞株凋亡,抑制其诱导的新生血管生成。结论 二甲双胍可抑制缺氧条件下MM细胞株RPMI 8226中HIF-1α和VEGF蛋白的表达及新生血管生成。

Objective This study aims to investigate the effects of metformin on angiogenesis in multiple myeloma(MM)RPMI 8226 cell line under hypoxic conditions.Methods Differentially expressed genes(DEGs)analysis and enrichment analysis of related signaling pathways were performed on gene expression omnibus(GEO)dataset GSE110113 of RPMI 8226 cells under normoxic and hypoxic conditions.A chemical hypoxia model was created using 100μmol/L cobalt chloride(CoCl_(2)).The proliferation activity of RPMI 8226 cells under hypoxic conditions after treatment with different concentrations of metformin was evaluated using the CCK\|8 assay.The expression of hypoxia inducible factor-lα(HIF-1α)and vascular endothelial cell growth factor(VEGF)proteins was detected by Western-blot,while the expression level of VEGF protein in the culture supernatant was measured by ELISA.In addition,an in vitro angiogenesis experiment was conducted to assess neovascular formation.Results GO and KEGG pathway enrichment analysis showed significant enrichment of DEGs in"hypoxia response"and"HIF-1 signaling pathway".The expression of HIF-1αand VEGF proteins in RPMI 8226 cells significantly increased with the duration of CoCl_(2) treatment.Metformin dose-dependently reduced the expression of HIF-1αand VEGF proteins induced by hypoxia in RPMI 8226 cells and decreased the expression level of VEGF protein in the culture supernatant.Moreover,metformin significantly inhibited cell proliferation,promoted apoptosis and suppressed angiogenesis in RPMI 8226 cells under the hypoxic environment induced by CoCl_(2).Conclusion Metformin can significantly inhibit the expression of HIF-1αand VEGF proteins as well as angiogenesis in RPMI 8226 cells under hypoxic conditions.