miR-148/152家族调控内皮细胞糖酵解相关基因的表达分析

Role of miR-148/152 family in regulating the expression of glycolysis-related genes in endothelial cells

目的探究miR-148/152家族在多能干细胞衍生内皮细胞(ECs)中对糖酵解相关基因的调控作用。方法本研究以人胚胎干细胞(hESCs)株H1(WT)和采用CRISPR/Cas9基因编辑技术构建的miR-148/152家族全敲除多能干细胞系(TKO)为基础;利用免疫荧光技术检测干细胞标志物NANOG的表达,评估WT和TKO干细胞的多能性;通过添加化学小分子和细胞因子如Wnt信号通路激活剂、碱性成纤维细胞生长因子、血管内皮生长因子和骨形态发生蛋白4等,定向诱导hESCs向ECs分化;采用RT-qPCR检测miR-148/152家族在ECs中的敲除效率,并探究糖酵解关键酶和代谢转换关键基因在WT和TKO干细胞分化ECs中的表达差异。两组间比较采用独立样本t检验。结果与WT比较,TKO多能干细胞中miR-148a(1.00±0.03比0.00±0.00)、miR-148b(1.00±0.07比0.13±0.06)、miR-152(1.01±0.15比0.05±0.03)丰度降低,差异具有统计学意义(P均<0.001)。WT和TKO hESCs均表达核定位的多能性标志分子NANOG,且均可定向分化为CD31阳性的ECs。与WT比较,TKO ECs中miR-148a(1.00±0.05比0.00±0.00)、miR-148b(1.00±0.08比0.12±0.05)、miR-152(1.00±0.08比0.13±0.07)检测丰度降低,差异具有统计学意义(P均<0.001)。与WT比较,TKO ECs中糖酵解关键酶如磷酸甘油酸激酶(1.00±0.09比0.20±0.02)、己糖激酶(1.02±0.20比0.55±0.12)、磷酸果糖激酶(1.00±0.05比0.67±0.14)、乳酸脱氢酶(1.00±0.04比0.53±0.05)、丙酮酸激酶(1.00±0.03比0.83±0.09)、3-磷酸甘油醛脱氢酶(1.00±0.03比0.59±0.09)的mRNA表达水平下调,而糖代谢向氧化磷酸化代谢转换过程中的重要基因丙酮酸脱氢酶激酶1(1.00±0.08比2.90±0.23)在敲除系中表达量升高,差异有统计学意义(P均<0.05)。与WT比较,TKO ECs中糖酵解抑制因子磷脂酶和张力蛋白同源物的表达量升高(1.01±0.11比3.83±0.81,P<0.001)。结论miR-148/152家族是调控ECs糖代谢的重要因子,可能在维持ECs糖酵解平衡,抑制其向氧化磷酸化代谢转换中发挥重要作用。

Objective To explore the role of miR-148/152 family in the regulation of glycolysis-related genes in pluripotent stem cell-derived endothelial cells(ECs).Methods In this study,we used the wild-type human embryonic stem cells(hESCs)strain H1(WT)and the miR-148/152 family knockout hESCs line(TKO),which was generated from H1 hESCs using CRISPR/Cas9 gene editing technology.The expression of pluripotent marker NANOG was detected by immunofluorescence technology.ECs were differentiated from both WT and TKO hESCs by subsequent treatment with chemical small molecules and cytokines such as Wnt signaling pathway activator,basic fibroblast growth factor,vascular endothelial growth factor and bone morphogenetic protein 4.RT-qPCR was performed to detect the knockout efficiency of miR-148/152 in the TKO ECs,and explore the effects of miR-148/152 family on the glycolytic metabolic enzymes and the metabolic transformation-related genes.Data between the two groups were compared by t test.Results Compared with the WT,the TKO hESCs showed significantly decreased detection abundance of miR-148a(1.00±0.03 vs 0.00±0.00),miR-148b(1.00±0.07 vs 0.13±0.06),and miR-152(1.01±0.15 vs 0.05±0.03),(all P<0.001).A comparable expression of the pluripotency marker NANOG was detected in the nuclei of WT and TKO hESCs.Both WT and TKO hESCs can differentiate into CD31-positive ECs.Compared with the WT,the ECs derived from TKO hESCs showed significantly decreased detection abundance of miR-148a(1.00±0.05 vs 0.00±0.00),miR-148b(1.00±0.08 vs 0.12±0.05),and miR-152(1.00±0.08 vs 0.13±0.07),(all P<0.001).The mRNA expression levels of key glycolytic enzymes,including phosphoglycerate kinase 1(1.00±0.09 vs 0.20±0.02),hexokinase 2(1.02±0.20 vs 0.55±0.12),6-phosphofructo-2-kinase(1.00±0.05 vs 0.67±0.14),lactate dehydrogenase A(1.00±0.04 vs 0.53±0.05),pyruvate kinase M(1.00±0.03 vs 0.83±0.09),glyceraldehyde-3-phosphate dehydrogenase(1.00±0.03 vs 0.59±0.09),were significantly reduced in TKO hESC-derived ECs,when compared to that in the WT hESC-derived ECs(P<0.05).Moreover,the mRNA expression of pyruvate dehydrogenase kinase 1(1.00±0.08 vs 2.90±0.23,P<0.001),a key gene in the process of metabolic transformation from glucose metabolism to oxidative phosphorylation metabolism,was significantly upregulated in TKO hESC-derived ECs.Consistently,expression level of the glycolysis suppressor phosphate and tension homology in the TKO ECs increased by about 4 folds when compared to that in the WT ECs(1.01±0.11 vs 3.83±0.81,P<0.001).Conclusion The miR-148/152 family is an important factor in regulating the glucose metabolism of ECs,and may contribute to maintaining the balance of glycolysis and inhibiting the glycolysis-to-oxidative phosphorylation transition.