miR-519d-3p靶向HIF-1α抑制高糖诱导的人视网膜微血管内皮细胞功能障碍及血管生成

miR-519d-3p alleviates high glucose-induced human retinal microvascular endothelial cells dysfunction and inhibits angiogenesis by targeting hypoxia inducible factor 1 subunit alpha

目的:明确miR-519d-3p对高糖诱导的人视网膜微血管内皮细胞(HRMEC)功能障碍与血管生成的影响,并阐明其对低氧诱导因子-1α(HIF-1α)的调控机制。方法:通过5、30mmol/L葡萄糖分别诱导HRMEC建立正常(NG)和高糖(HG)细胞模型。将HRMEC分为对照组(HG细胞模型转染阴性对照模拟物)、甘露醇组(对照组加入25mmol/L甘露醇)、miR-519d-3p过表达组(HG细胞模型转染miR-519d-3p模拟物)、miR-519d-3p联合HIF-1α过表达组(HG细胞模型共转染miR-519d-3p模拟物和HIF-1α过表达载体)。实时荧光定量PCR法检测各组miR-519d-3p的表达情况。Western blotting法检测各组HIF-1α蛋白的表达情况。荧光素酶报告基因实验检测miR-519d-3p和HIF-1α的结合位点情况。CCK-8法检测各组细胞增殖情况。Hoechst 33342染色法检测各组细胞凋亡情况。ELISA法检测各组细胞外液炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6蛋白的表达情况。小管形成实验检测各组新生毛细血管管腔样结构形成情况。结果:与NG相比,HG细胞模型中miR-519d-3p表达显著减少,而HIF-1α蛋白表达显著增加(均P<0.01)。与对照组比较,miR-519d-3p过表达组中HIF-1α蛋白表达显著降低(P<0.01)。miR-519d-3p中“CGUGAAA”序列可以与HIF-1α3'-非编码区(3'-UTR)中“GCACUUU”序列特异性结合。与对照组比较,miR-519d-3p过表达组细胞24、48、72h吸光度值均显著增加,细胞凋亡率显著减少,细胞外液TNF-α、IL-1β、IL-6浓度均显著减少,新生毛细血管管腔样结构数量显著减少(均P<0.01)。与miR-519d-3p过表达组比较,miR-519d-3p联合HIF-1α过表达组细胞24、48、72h吸光度值均显著减少,细胞凋亡率显著增加,细胞外液TNF-α、IL-1β、IL-6浓度均显著增加,新生毛细血管管腔样结构数量显著增加(均P<0.01)。对照组和甘露醇组中上述各指标比较无差异(均P>0.05)。结论:高糖诱导HRMEC模型中miR-519d-3p表达下调,而HIF-1α蛋白表达上调。HIF-1α是miR-519d-3p的靶基因,miR-519d-3p靶向HIF-1α增加细胞增殖并降低细胞凋亡和炎症反应,从而减轻高糖诱导的HRMEC功能障碍并抑制血管生成。

AIM:To clarify the effect of miR-519d-3p on high glucose-induced human retinal microvascular endothelial cells(HRMEC)dysfunction and angiogenesis,and to elucidate the regulatory mechanism of miR-519d-3p on hypoxia inducible factor 1 subunit alpha(HIF-1α).METHODS:The normal glucose(NG)and high glucose(HG)cell models were established by inducing HRMEC with 5 and 30 mmol/L glucose,respectively.Control group:HG cell model was transfected with negative control mimics;mannitol group:the control group was added with 25 mmol/L mannitol;miR-519d-3p overexpression group:HG cell model was transfected with miR-519d-3p mimics;miR-519d-3p combined with HIF-1αoverexpression group:HG cell model was co-transfected with miR-519d-3p mimics and HIF-1αoverexpression vector.The expression of miR-519d-3p in each group was tested by real-time fluorescence quantitative PCR.The expression of HIF-1αprotein in each group was tested by Western blotting.The binding sites between miR-519d-3p and HIF-1αwere detected by luciferase reporter gene assay.The cell proliferation of each group was detected by CCK-8.The cell apoptosis of each group was tested by Hoechst 33342 staining.The protein expression of extracellular fluid inflammatory factors tumor necrosis factor-α(TNF-α),interleukin(IL)-1βand IL-6 in each group was tested by ELISA.The formation of new capillary lumen-like structures was detected by tubule formation assay.RESULTS:Compared with the NG,miR-519d-3p expression was significantly reduced in the HG cell model,while HIF-1αprotein expression was significantly increased in the HG(all P<0.01).Compared with the control group,HIF-1αprotein expression was significantly reduced in the miR-519d-3p overexpression group(P<0.01).The“CGUGAAA”sequence of miR-519d-3p could specifically bind to the“GCACUUU”sequence of HIF-1α3-untranslated region(3’-UTR).Compared with the control group,the miR-519d-3p overexpression group showed a significant increase in 24,48 and 72h absorbance values,a significant decrease in cell apoptotic rate,a significant decrease in the concentrations of TNF-α,IL-1βand IL-6,and a significant decrease in the number of new capillary lumen-like structures(all P<0.01).Compared with the miR-519d-3p overexpression group,the miR-519d-3p combined with HIF-1αoverexpression group showed a significant decrease in 24,48 and 72h absorbance values,a significant increase in cell apoptotic rate,a significant increase in the concentrations of TNF-α,IL-1βand IL-6,and a significant increase in the number of new capillary lumen-like structures(all P<0.01).There was no difference between the control group and mannitol group in the comparison of the above indicators(all P>0.05).CONCLUSION:miR-519d-3p expression is down-regulated while HIF-1αprotein expression is up-regulated in high glucose induced HRMEC model.HIF-1αis a target gene of miR-519d-3p.The miR-519d-3p targets HIF-1αto increase cell proliferation and reduce cell apoptosis and inflammation,thereby alleviating high glucose-induced HRMEC dysfunction and inhibiting angiogenesis.