人脐带间充质干细胞外泌体靶向miR-126调节高糖诱导的人视网膜血管内皮细胞中血管内皮生长因子-A的表达

Human umbilical cord mesenchymal stem cell exosomes target miR-126 regulate the expression of vascular endothelial growth factor-A in high glucose-induced human retinal vascular endothelial cells

目的观察人脐带间充质干细胞(hUCMSC)外泌体(Exo)靶向miR-126对高糖诱导的人视网膜血管内皮细胞(hREC)中血管内皮生长因子(VEGF)-A水平的影响,初步探讨哺乳动物雷帕霉素靶蛋白(mTOR)/缺氧诱导因子1α(HIF-1α)通路在其中发挥的作用。方法将hREC置于30 mmol/L葡萄糖EGM-2-MV内皮细胞培养基中并于含1%O2的低氧细胞培养箱中培养,建立高糖低氧细胞模型。建模后分为Exo组、磷酸盐缓冲液(PBS)组、PBS+anti-miR126组、Exo+anti-miR126组、PBS+anti-mTOR组、PBS+anti-HIF-1α组。PBS组、Exo组高糖低氧诱导的hREC分别与PBS、100μg/ml的hUCMSC Exo共培养。PBS+anti-mTOR组、PBS+anti-HIF-1α组:浓度为500 nmol/L mTOR抑制剂ADZ2014、25μmol/L HIF-1α抑制剂YC-1分别预处理hREC 12 h后,再行高糖低氧诱导后与PBS共培养。PBS+anti-miR126组、Exo+anti-miR126组:miR-126 LNA Power Inhibitor探针转染高糖低氧诱导的hREC,转染6 h后分别与PBS、hUCMSC Exo共培养。实时荧光定量聚合酶链反应(qPCR)检测PBS组、Exo组共培养0、8、16、24 h细胞中miRNA-126表达水平。共培养24 h时,免疫荧光染色、蛋白质免疫印迹法(Western blot)、qPCR分别检测PBS组、Exo组细胞中mTOR、HIF-1α水平。Western blot、qPCR检测PBS+anti-mTOR组、PBS+anti-HIF-1α组细胞中VEGF-A表达水平。qPCR检测PBS+anti-miR126组、Exo+anti-miR126组细胞中VEGF-A、mTOR、HIF-1αmRNA表达。两组间比较采用t检验;多组间比较采用单因素方差分析。结果共培养0、8、16、24 h,Exo组细胞中miR-126 mRNA相对表达量逐渐增高,差异有统计学意义(F=95.900,P<0.05)。与PBS组比较,Exo组细胞中mTOR、HIF-1α蛋白表达(t=3.466、6.804)以及mTOR、HIF-1α、VEGF-A mRNA表达(t=8.642,7.897、6.099)均下调,差异有统计学意义(P<0.05);PBS+anti-mTOR组、PBS+anti-HIF-1α组细胞中VEGF-A蛋白(t=3.337、7.380)、mRNA(t=8.515、10.400)表达下降,差异均有统计学意义(P<0.05);Exo+anti-miR126组细胞中VEGF-A、mTOR、HIF-1αmRNA表达明显升高,差异均有统计学意义(t=4.664、6.136、6.247,P<0.05)。结论miR-126通过mTOR/HIF-1α通路参与调节hUCMSC Exo对高糖诱导的hREC中VEGF-A水平的影响。

Objective To explore the involvement of miR-126 and the role of mammalian target of rapamycin(mTOR)/hypoxia-induced factor 1α(HIF-1α)pathway in regulating human umbilical cord mesenchymal stem cells(hUCMSCs)exosomes(Exo)on vascular endothelial growth factor(VEGF)-A levels in high glucose-induced human retinal vascular endothelial cells(HRECs).Methods The hREC was cultured in EGM-2-MV endothelial cell culture medium with 30 mmol/L glucose and placed in hypoxic cell incubator by 1%oxygen concentration.The cell model of high glucose and low oxygen was established.After modeling,divided HRECs into Exo group,phosphate buffer saline(PBS)group,PBS+anti-miR126 group,Exo+antimiR126 group,PBS+anti-mTOR group,and PBS+anti-HIF-1αgroup.High-glucose and hypoxia-induced hREC in the PBS and Exo groups were respectively co-cultured with PBS and 100μg/ml hUCMSC Exo.PBS+antimTOR group,PBS+anti-HIF-1αgroup:500 nmol/L mTOR inhibitor ADZ2014,25μmol/L HIF-1αinhibitor YC-1 pretreatment for hREC 12 h,and then co-culture with PBS after High-glucose and hypoxia-induced.PBS+anti-miR126 group,Exo+anti-miR126 group:miR-126 LNA power inhibitor probe was transfected with high glucose,and co-cultured with PBS and hUCMSC Exo 6 h after transfection.Real-time polymerase chain reaction(qPCR)measured miRNA-126 expression levels in PBS,and Exo groups for 0,8,16 and 24 h.After 24 h of co-culture,the levels of mTOR and HIF-1αin the cells of PBS and Exo groups were detected by immunofluorescence,Western blot and qPCR,respectively.Western blot,qPCR detection of VEGF-A expression levels in cells of the PBS+anti-mTOR and PBS+anti-HIF-1αgroups.The expression of VE GF-A,mTOR,and HIF-1αmRNA was measured in cells of PBS+anti-miR126 group and Exo+anti-miR126 group by qPCR.Comparison between two groups was performed by t-test;one-way ANOVA was used for comparison between multiple groups.Results At 0,8,16 and 24 h,the relative mRNA expression of miR-126 gradually increased in the Exo group(F=95.900,P<0.05).Compared with the PBS group,The mTOR,HIF-1αprotein(t=3.466,6.804),mRNA in HRECs in the Exo group,VEGF-A mRNA expression(t=8.642,7.897,6.099)were all downregulated,the difference was statistically significant(P<0.05).The relative expression level of VEGF-A protein(t=3.337,7.380)and mRNA(t=8.515,10.400)was decreased in HRECs of the anti-mTOR+PBS group and anti-HIF-1α+PBS group,differences were statistically significant(P<0.05).The relative expression of VEGF-A,mTOR,and HIF-1αmRNA was significantly increased in the cells of the Exo+anti-miR126 group,the differences were all statistically significant(t=4.664,6.136,6.247;P<0.05).Conclusions miR-126 plays a role in regulating the effect of hUCMSCs exosomes on VEGF-A levels in high glucose-induced HRECs via mTOR-HIF-1αpathway.