Regulation And Mechanism Of MiR-200b In Corrneal Neovascularization In Rats

Objective To study the function of microRNA-200b(miR-200b)in the formation of corneal neovascularization(CNV)in rats and to explore the related mechanisms.Method 1.selecting 40 female SD rats,and chosing one eye as the CNV animal model(experimental group)which constructed by suture,and the other eye was used as the normal control group.the growth of corneal neovascularization on days 4,7,14,21 after modeling was observed under a slit lamp.RT-PCR(Real-time Quantitative Polymerase Chain Reaction)was used to detect the expressions of miR-200 b and VEGF mRNA in rat corneal tissue at different points in time after modeling.Hematoxylin-eosin staining was used to observe the corneal tissue structure and inflammatory infiltration.2.Transfecting the miR-200 b mimic?miR-200 b inhibitor and their respective negative controls(Scramble)in human umbilical vein endothelial cells(HUVECs)with lipidosome,RT-PCR was used to verify transfection efficiency.3.CCK-8method,scratch test method,transwell method and Matrigel tube-formation method were used to detect cell proliferation ? migration and tubule formation after transfection.4.Western blot was used to detect theexpression of VEGF? PIK3 CA and AKT protein in each group.Results 1.The rat CNV model was successfully constructed.RT-PCR results showed that miR-200 b expression in corneal tissues was significantly reduced on days 4,7,14 and 21 after sutures.It was lowest on day 7(P<0.05),and VEGF expression was significantly increased(P <0.05),the peak occurred on day 14,compared with the normal group,HE staining showed the tissue structure was disordered,the inflammatory cell infiltration increased on days 4,7,14 and 21 after suture(P <0.05),the peak occurred on day 14.2.24 hours after transfection,compared with the respective Scramble group,the relative expression of miR-200 b in the miR-200 b mimic group was significantly increased(P <0.01),while the relative expression of miR-200 b in the miR-200 b inhibitor group was significantly reduced.(P<0.05),which showed the transfection wassuccessful.3.Compared with the respective Scramble group,the miR-200 b mimic group showed a significant decrease in HUVECs proliferation ? migration and tubule formation ability(P <0.05),while the miR-200 b inhibitor group showed HUVECs proliferation ? migration and tubule formation ability increased significantly(P <0.05).4.48 hours after transfection,miR-200 b mimic can significantly reduce the expressions of VEGF ? PIK3 CA and p-AKT protein in HUVECs(P <0.05),while miR-200 b inhibitor can increase the expression of VEGF,PIK3 CA,p-AKT protein in HUVECs Significantly increased(P <0.05).Conclusion The expression ofmiRNA-200 b in rat corneal neovascularization induced by suture decreased,and miRNA-200 b could significantly inhibit the proliferation,migration and tube-formation of HUVECs.This may be related to inhibiting the signal transduction of PIK3 CA / AKT pathway.