Gpr124对高糖环境下视网膜微血管内皮细胞增殖、迁移和血管形成的调控作用

Regulatory effect of Gpr124 on proliferation,migration and angiogenesis of retinal microvascular endothelial cells under high-glucose condition

目的研究高糖环境下人视网膜微血管内皮细胞(human retinal microvascular endothelial cell,HRMEC)中G蛋白偶联受体124(Gpr124)表达的变化,以及干扰Gpr124对HRMEC的调控作用。方法免疫荧光观察Gpr124在HRMEC中的表达;高糖刺激HRMEC,将细胞分为空白组和高糖组,CCK-8、EdU检测细胞活力与增殖情况;病毒抑制Gpr124表达后,将细胞分为空白对照组、高糖对照组、高糖+shRNA组以及高糖+Gpr124 shRNA组,划痕实验评估细胞迁移,基质凝胶评估细胞小管形成能力。Western blot检测Gpr124、VEGFA、MMP9和β-catenin相对蛋白表达水平。结果相较于空白组,HRMEC在高糖组中显示出显著提升的细胞活力和增殖细胞数(P<0.01)。高糖环境下,Gpr124、VEGFA以及MMP9的表达水平也显著增加(P<0.01)。同时,在各个时间点,高糖对照组的迁移面积以及体外血管形成面积和管径长度均显著高于空白对照组(P<0.01)。然而,在敲低Gpr124的表达后,高糖+Gpr124 shRNA组的HRMEC在迁移和体外血管形成能力上相较于高糖+shRNA组有显著的降低(P<0.01),并且高糖+Gpr124 shRNA组相比于高糖+shRNA组VEGFA、MMP9以及β-catenin蛋白表达减少(P<0.01)。结论Gpr124能够调控高糖环境下HRMEC的增殖、迁移以及管形成能力,并且还可以抑制VEGFA和MMP9的过度表达,这一影响可能是通过调节Wnt/β-catenin经典通路来实现的。

ObjectiveTo investigate the changes in G protein-coupled receptor 124(Gpr124)expression in human retinal microvascular endothelial cells(HRMEC)under high-glucose condition and the regulatory effect of Gpr124 interference on HRMEC.MethodsImmunofluorescence assay was used to observe the expression of Gpr124 in HRMEC.The cells were divided into a blank group and a high glucose group.CCK-8 assay and EdU cell proliferation assay were used to detect cell viability and proliferation,respectively.The Gpr124 expression was knocked down by transducting lentiviral virus carrying shRNA targeting Gpr124.Thus,the cells were divided into a blank control group,a high glucose control group,a high glucose+shRNA group,and a high glucose+Gpr124 shRNA group.Cell scratch test was performed to evaluate cell migration,and Matrigel plug assay was employed to assess the cell tubule formation.Western blotting was applied to detect the protein levels of Gpr124,VEGFA,MMP9,andβ-catenin.ResultsThe HRMEC from the high glucose group showed enhanced cell viability and increased number of proliferating cells compared to the cells of the blank group(P<0.01).High glucose treatment also induced increased expression levels of Gpr124,VEGFA and MMP9(P<0.01),and larger migratory area and in vitro angiogenic area as well as longer tube diameter at all time points when compared with the conditions in the blank control group(P<0.01).However,knockdown of Gpr124 resulted in a significant decrease in HRMEC migration and in vitro angiogenic capacity,accompanied by decreases in protein expression of VEGFA,MMP9 andβ-catenin(P<0.01).ConclusionGpr124 can regulate the proliferation,migration,and tube formation ability of HRMEC under high-glucose condition,and also inhibit the overexpression of VEGFA and MMP9,which may be through regulating the classical Wnt/β-catenin pathway.