MiR-200b通过调控PIK3CA/AKT通路抑制大鼠角膜血管新生

MiR-200b inhibits corneal angiogenesis in rats by regulating PIK3CA/AKT pathway

目的探讨microRNA-200b(miR-200b)在大鼠角膜新生血管(corneal neovascularization,CNV)形成中的作用及其调节机制。方法24只雌性SD大鼠采用一眼经缝线法构建CNV模型(实验组),另一眼作为正常对照组,于裂隙灯下观察建模后第4、7、14天角膜新生血管情况,采用HE染色观察角膜组织结构改变及炎性细胞浸润情况,RT-PCR检测造模后第14天各组大鼠角膜组织中miR-200b以及血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达。利用脂质体转染人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),分为miR-200b模拟物(miR-200b mimic)组、阴性对照(miR-NC)组、空白对照(CON)组,利用RT-PCR验证转染效果,分别采用CCK-8检测HUVECs增殖能力,划痕实验检测HUVECs迁移能力,Matrigel胶成管实验检测HUVECs管腔形成能力。并通过Western blot检测VEGF及PIK3CA、AKT蛋白的表达。结果成功构建大鼠CNV模型,HE染色显示与正常组相比,缝线后第4、7、14天,大鼠角膜组织排列紊乱,炎性细胞浸润逐渐加重(P<0.05),RT-PCR结果显示缝线后第14天角膜组织中miR-200b表达明显降低,而VEGF表达明显升高(P<0.01)。与CON组和miR-NC组相比,miR-200b mimic组中miR-200b相对表达量显著升高(P<0.01),而增殖、迁移及管腔形成能力显著下降(P<0.05),转染miR-200b mimic后,HUVECs中VEGF、PIK3CA、p-AKT蛋白表达均明显下降(P<0.05)。结论miRNA-200b可显著抑制HUVECs的增殖、迁移及成管能力,可能通过抑制PIK3CA/AKT通路参与CNV的发生和发展。

ObjectiveTo investigate the role of microRNA-200b(miR-200b)in corneal neovascularization(CNV)in rats and explore the possible mechanisms.MethodsTwenty-four female SD rats were subjected to corneal suture to induce CNV in one eye,with the other eye as the normal control.On the 4th,7th and 14th days after the suture,CNV was observed under a slit lamp,and the structure and inflammatory cell infiltration in the corneal tissue were observed using HE staining.On day 14 after the suture,the expression levels of miR-200b and vascular endothelial growth factor(VEGF)mRNA in the cornea were detected using qRT-PCR.Human umbilical vein endothelial cells(HUVECs)were transfected via liposome with a miR-200b mimic or a negative control sequence(miR-NC),and the cell proliferation,migration and tube formation abilities were evaluated using CCK-8 assay,Transwell chamber assay and Matrigel assay,respectively.The expression levels of VEGF and PIK3CA/AKT proteins in the cells were detected by Western blotting.ResultsCNV models were successfully established by corneal suture in the rats.HE staining showed that corneal suture resulted in disordered alignment of the corneal tissue and progressively increased inflammatory cell infiltration on days 4,7 and 14 after the suture(P<0.05).RT-PCR showed that miR-200b expression decreased and VEGF expression increased significantly in the corneal tissue on day 14 after the suture(P<0.01).Compared with the cells without transfection or transfected with miR-NC,the HUVECs transfected with the miR-200b mimic exhibited significantly attenuated proliferation,migration and lumen formation abilities(P<0.05)with also significantly lowered expression of VEGF,PIK3CA and p-AKT proteins(P<0.05).ConclusionmiRNA-200b can significantly inhibit the proliferation,migration and tube formation of HUVECs,and may participate in the occurrence and development of CNV by inhibiting the PIK3CA/AKT pathway.