miR-373靶向VEGFA对糖尿病视网膜病变大鼠的作用

Effect of miR-373 on rats with diabetic retinopathy by targeting VEGFA

目的:探讨miR-373靶向血管内皮生长因子A(VEGFA)对糖尿病视网膜病变(DR)大鼠的作用。方法:将40只大鼠随机分为对照组(n=10)和DR组(n=30),造模成功后的DR组大鼠分为模型组(n=10)、miR-373 agomir组(n=10)、agomir-NC组(n=10),右眼玻璃体腔分别注射200μL生理盐水、miR-373 agomir(200nmol)、agomir-NC(200nmol),每周1次,共处理12wk。通过RT-qPCR检测各组大鼠视网膜组织中miR-373和VEGFA mRNA表达水平,双荧光素酶实验验证miR-373与VEGFA靶向关系,Western-blot检测各组大鼠视网膜组织中VEGFA、Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2基因(Bcl-2)、磷脂酰肌醇3-激酶(PI3K)、磷酸化-磷脂酰肌醇3-激酶(p-PI3K)、丝苏氨酸蛋白激酶(AKT)、磷酸化-丝苏氨酸蛋白激酶(p-AKT)蛋白表达水平。结果:与对照组比,模型组和agomir-NC组大鼠视网膜组织miR-373及Bcl-2蛋白表达水平显著下降,VEGFA mRNA、Bax、p-PI3K和p-AKT蛋白表达量显著升高(均P<0.05)。与agomir-NC组比,miR-373 agomir组大鼠视网膜组织miR-373及Bcl-2蛋白表达水平显著升高,VEGFA mRNA及蛋白、Bax、p-PI3K和p-AKT蛋白表达量显著下降(均P<0.05)。双荧光素酶实验证实VEGFA是miR-373的靶基因。结论:miR-373通过靶向VEGFA抑制糖尿病视网膜病变,其机制可能与miR-373抑制PI3K/AKT信号通路有关。

AIM:To investigate the effect of microRNA-373(miR-373)by targeting vascular endothelial growth factor A(VEGFA)on diabetic retinopathy(DR)rats.METHODS:Totally 40 rats were randomly divided into control group(n=10)and DR group(n=30).The rats after successful modeling in DR group were divided into model group(n=10),miR-373 agomir group(n=10),and agomir-NC group(n=10).The right eyes vitreous cavity separately were injected with 200μL normal saline,miR-373 agomir(200nmol)and agomir-NC(200nmol)were treated once a week for 12wk.The expression levels of miR-373 and VEGFA mRNA in each group were detected by RT-qPCR.Dual luciferase experiment was used to verify the targeting relationship between miR-373 and VEGFA.Western-blot was used to detect VEGFA,Bcl-2 related X protein(Bax),b-cell lymphoma-2(Bcl-2),phosphatidylinositol 3-kinase(PI3K)and phospho phosphatidylinositol 3-kinase(p-PI3K),serine threonine protein kinase(AKT),phospho serine protein kinase(p-AKT)protein expression levels.RESULTS:Compared with the control group,the expression levels of miR-373 and Bcl-2 protein in the retina tissue of model group and agomir-NC group were significantly decreased,and the expression levels of VEGFA mRNA,Bax,p-PI3K and p-AKT protein were significantly increased.Compared with the agomir-NC group,the expression levels of miR-373 and Bcl-2 protein in the retina tissue of miR-373 agomir group were significantly increased(all P<0.05),while the expression levels of VEGFA mRNA and protein,the Bax,p-PI3K,p-AKT protein in the retina tissue of miR-373 agomir group were significantly decreased(all P<0.05).Dual luciferase assay confirmed that VEGFA is the target gene of miR-373.CONCLUSION:miR-373 can inhibit diabetic retinopathy by targeting VEGFA,which may be related to the inhibition of PI3K/AKT signaling pathway by miR-373.