The Role And Mechanism Of MiRNA-424(322)-5p In Rat Corneal Neovascularization

Objective To explore the role and mechanism of microRNA-424(322)-5p(miR-424(322)-5p)in the formation of corneal neovascularization(CorNV)in rats.Method 1.Thirty SD female rats were randomly grouped according to different time periods.The left eye was the experimental group and the contralateral eye was the control group.The rat CorNV model was induced by suture method.On the 0th,4th,7th,14 th after model,it was used to observe the corneal structure,blood vessel formation and protein expression that the corneal tissues were tested by HE staining and immunohistochemistry(IHC).On the 14 th days after models,miR-424(322)-5p and vascular endothelial growth factor(VEGF)mRNA expression in rat corneal tissue were detected by RT-PCR assay.2.Human umbilical vein endothelial cells(HUVECs)were transfected by Liposome and divided into miR-424(322)-5p mimic group and negative control(miR-424(322)-5p NC)group,blank control(CON)group,respectively;MiR-424(322)-5p inhibitor group,negative control(miR-424(322)-5p NC)group,blank control(control,CON)group,respectively.3.The transfection efficiency of miR-424(322)-5p was tested by RT-PCR assay.It was detected by CCK-8 assay,scratch assay,Transwell assay,Matrigel Tube assay that the proliferation,migration,and tube forming ability of HUVECs in each group,respectively.4.The relative expression of VEGF and Raf/MEK/ERK pathway proteins was detected by Western blot.Results 1.The rat CorNV model was successfully constructed.The HE staining of the corneal tissue showed that the corneal structure of the experimental group,compared with the control group,was disordered and the corneal edema gradually increased,the new blood vessels were gradually formed,and the inflammatory cells were gradually increased on the 4th,7th,and 14 th days after models.2.Immunohistochemistry(IHC)results showed that the experimental group,compared with the control group,had expression of VEGF protein,which was brownish yellow.3.RT-PCR showed the expression of miR-424(322)-5p and VEGF mRNA in rat corneal tissue was significantly increased after the 14 th day of modeling(P<0.01).4.The expression level of miR-424(322)-5p mimic group transfected with HUVECs by Liposome was significantly higher than the NC group and the CON group(P<0.01).The results of CCK-8,scratch test,Transwell test and Matrigel gel tube test showed that the cell proliferation,migration and tube formation ability of miR-424(322)-5p mimic group was significantly higher than the NC group and the CON group(P<0.01);the expression of miR-424(322)-5p inhibitor group transfected with HUVECs by liposomes was significantly lower than the NC group and the CON group(P<0.01).The results of CCK-8,scratch test,Transwell test,and Matrigel gel tube test showed that the cell proliferation,migration and tube-forming ability of the miR-424(322)-5p inhibitor group was lower than the NC group and the CON group(P<0.01).5.Western blot showed that the The miR-424(322)-5p mimic group,s relative expression of VEGF and Raf/MEK/ERK pathway protein,compared with the NC group and the CON group,was significantly increased(P<0.01);the miR-424(322)-5p inhibitor group,s relative expression of VEGF and Raf/MEK/ERK pathway protein,compared with the NC group and the CON group,was significantly reduced(P<0.01).Conclusion The relative expression of miR-424(322)-5p and VEGF mRNA in rats CorNV is up-regulated;miR-424(322)-5p may activate VEGF and Raf/MEK/ERK pathways to promote the cell migration,proliferation and proliferation of HUVECs,thereby participating in the formation of CorNV.