G蛋白α亚基q类蛋白p.R183Q杂合突变对脑微血管内皮细胞增殖与氧化应激的影响

Effects of p.R183Q heterozygous mutation of GNAQ on proliferation and oxidative stress of brain microvascular endothelial cells

目的探讨脑面血管瘤病致病基因G蛋白α亚基q类蛋白(GNAQ)p.R183Q杂合突变对人脑微血管内皮细胞(HBMEC)增殖、氧化应激及内皮间充质转化(EndMT)的影响及机制。方法将HBMEC分为对照组、野生型组、突变型组及联合型组(n=3),分别转染空白载体、GNAQ过表达载体、GNAQ p.R183Q突变过表达载体及GNAQ过表达载体+GNAQ p.R183Q突变过表达载体。采用酶联免疫吸附试验检查丙二醛、超氧化物歧化酶(SOD)及谷胱甘肽过氧化物酶(GSH-Px)水平。实时荧光定量PCR检测平滑肌肌动蛋白α2(ACTA2)、波形蛋白(VIM)、E钙黏蛋白(CDH1)mRNA表达,蛋白质印迹检测GNAQ、磷酸化p38丝裂原活化蛋白激酶(p-p38 MAPK)、磷酸化NF-κB(p-NF-κB)p65及磷酸化核因子抑制蛋白(p-IκBa)蛋白表达。结果野生型组、突变型组及联合型组GNAQ蛋白表达较对照组明显升高(5.27±0.43,5.60±0.51,5.15±0.39 vs 1.00±0.08,P<0.05)。与对照组比较,野生型组细胞增殖能力、SOD、GSH-Px、CDH1 mRNA明显升高,而活性氧、超氧阴离子自由基及丙二醛水平明显降低,ACTA2 mRNA、VIM mRNA、p-p38 MAPK、p-NF-κB p65、p-IκBa蛋白表达明显下调(P<0.05)。与野生型组比较,突变型组和联合型组细胞增殖能力、SOD、GSH-Px、CDH1 mRNA表达明显降低,活性氧、超氧阴离子自由基、丙二醛水平明显升高,ACTA2 mRNA、VIM mRNA、p-p38 MAPK、p-NF-κB p65及p-IκBa蛋白表达明显上调(P<0.05)。结论GNAQ p.R183Q杂合突变发挥显性负效应抑制HBMEC细胞增殖、促进氧化应激与EndMT,机制可能与丝裂原活化蛋白激酶与NF-κB通路激活有关。

Objective To investigate the effect and underlying mechanism of p.R183Q heterozygous mutation of causative gene GNAQ of encephalofacial angiomatosis on the proliferation,oxidative stress and EndMT of HBMEC.Methods HBMEC were divided into control group,wild type group,mutant group and combined group(n=3),and were transfected with blank vector,GNAQ overexpression vector,GNAQ p.R183Q mutant overexpression vector,and GNAQ overexpression vector+GNAQ p.R183Q mutant overexpression vector,respectively.Thiazole blue colorimetry and 5-bromodeoxyuridine staining were used to detect cell proliferation,DCFH-DA fluorescent probe was employed to determine the levels of ROS and superoxide anion free radicals,and enzyme-linked immunosorbent assay was applied to measure the levels of MDA,SOD and GSH-Px.Real-time fluorescence quantitative PCR was carried out to detect the mRNA expression levels of smooth muscle actin alpha 2(ACTA2),VIM and E-cadherin(CDH1),and Western blotting was performed to measure the protein levels of phosphorylated p-p38 MAPK,p-NF-κB p65 and p-IκBa.Results The expression of GNAQ protein was significantly higher in the wild type group,mutant group and combined group than the control group(5.27±0.43,5.60±0.51 and 5.15±0.39 vs 1.00±0.08,P<0.05).The wild type group had stronger proliferation ability,increased SOD and GSH-Px contents and elevated CDH1 mRNA level,decreased productions of ROS and superoxide anion free radicals,and reduced MDA content,and down-regulated ACTA2 and VIM mRNAs and p-p38 MAPK,p-NF-κB p65 and p-IκBa proteins when compared with the control group(P<0.05).While,compared with the wild type group,the cell proliferation ability,SOD,GSH-Px,and CDH1 mRNA expression were significantly decreased,and the levels of ROS,superoxide anion free radicals,and MDA were significantly increased,and the expression levels of ACTA2 and VIM mRNAs and p-p38 MAPK,p-NF-κB p65 and p-IκBa proteins were significantly upregulated in the mutant group and the combined group(P<0.05).Conclusion GNAQ p.R183Q heterozygous mutation exerts a dominant-negative effect to inhibit cell proliferation and promote oxidative stress and EndMT in HBMEC,and the mechanism may be related to the activation of MAPK and NF-κB pathways.