PGK1和ENO1介导的有氧糖酵解在脑胶质瘤血管生成中的作用

The role of PGK1 and ENO1 mediated aerobic glycolysis in angiogenesis of glioma

目的研究磷酸甘油酸激酶1(phosphoglycerate kinase 1,PGK1)和α-烯醇化酶(α-Enolase,ENO1)在脑胶质瘤微血管内皮细胞(Glioma microvascular endothelial cells,GECs)的表达,以及介导的有氧糖酵解在脑胶质瘤血管生成中的作用。方法培养正常人脑为血管内皮细胞(norma lmicrovascular endothelial cells,NECs)和GECs,应用Real-timePCR和WesternBlot方法检测PGK1和ENO1的表达水平;设计并构建PGK1和ENO1表达沉默的质粒以及相应的对照质粒,分别转染hCMEC/D3细胞,筛选出稳定表达细胞株后建立PGK1和ENO1表达沉默的GECs;应用葡萄糖检测试剂盒检测细胞的葡萄糖摄取量,无血清培养法检测乳酸产量,SeahorseXF24细胞能量代谢分析仪检测细胞的细胞外酸化率;应用CCK8实验检测细胞增殖能力变化,Transwell实验检测细胞迁移变化,Matrigel基质胶实验检测血管形成能力的变化。结果与NECs相比,PGK1和ENO1mRNA和蛋白表达水平在GECs中显著增高;PGK1和ENO1表达沉默均显著抑制了GECs的葡萄糖摄取量、乳酸产量和细胞外酸化率,显著降低了GECs的增殖、迁移和血管形成能力。结论PGK1和ENO1介导的有氧糖酵解促进脑胶质瘤微血管内皮细胞增殖、迁移和血管生成。

Objective To study the expression levels of phosphoglycerate kinase 1(PGK1) and α-Enolase(ENO1) in glioma microvascular endothelial cells(GECs), and the effects of PGK1 and ENO1-mediated aerobic glycolysis in angiogenesis of glioma. Method The expressions of PGK1 and ENO1 in normal microvascular endothelial cells(NECs) and GECs were were detected by Real-time PCR and Western blot. The silencing plasmids of PGK1 and ENO1 were designed and constructed, respectively, and the corresponding control plasmids. The above plasmids were transfected into hCMEC/D3 cells respectively, and the stable expression cell lines were selected to establish corresponding GECs. The glucose uptake of the cells was detected by glucose detection kit, and the lactate production was detected by serum-free culture method. Seahorse XF24 cell energy metabolism analyzer was used to detect the extracellular acidification rate(ECAR) of cells. CCK8 assay was used to detect the cell proliferation. Transwell chamber assay was applied to detect cell migration. Matrigel assay was used to detect the ability of angiogenesis. Results The expression levels of PGK1 and ENO1 were significantly higher in GECs than in NECs. Silencing of PGK1 and ENO1 significantly decreased the glucose uptake, lactate production and ECAR in GECs, and obviously inhibited the proliferation, migration and angiogenesis ability of GECs. Conclusion PGK1 and ENO1 promote the proliferation, migration and angiogenesis of GECs via mediating aerobic glycolysis.