MiRNA-424(322)-5p promotes corneal neovascularization in rats via activating Raf/MEK/ERK pathways

Abstract

Objective To explore the role and possible mechanism of microRNA-424(322)-5p (miR-424(322)-5p) in the formation of corneal neovascularization (Cor NV) in rats. Methods Corneal neovascularization model was established in 20 female rats by suturing. Their left eye was used as the experimental group and the right eye was used as the normal control group. On the 0th, 4th, 7th, 14th days after modeling, corneal structure, blood vessel formation and protein expression in the corneal tissues were observed by HE staining and immunohistochemistry (IHC). On the 4th day after modeling, the mRNA expression of miR-424(322)-5p and vascular endothelial growth factor (VEGF) in the corneal tissue was detected by RT-PCR. Human umbilical vein endothelial cells (HUVECs) were transfected with liposomes and divided into miR-424(322)-5p mimic group, negative control (miR-424(322)-5p NC) group and blank control (CON) group, respectively. The transfection efficiency of miR-424(322)-5p was tested by RT-PCR. CCK-8 assay, scratch assay, Transwell assay and Matrigel tube assay were performed to measure the proliferation, migration, and tube forming ability of HUVECs in each group, respectively. The levels of VEGF and Raf/MEK/ERK pathway proteins were detected by Western blotting. Results The rat Cor NV model was successfully established. HE staining showed that the rat corneal tissues of the experimental group were in disordered arrangement, corneal edema and thickening, neovascularization, and inflammatory cell infiltration on the 4th, 7th and 14th day after modeling. The results of IHC indicated that the expression of VEGF was enhanced significantly after modeling when compared with the normal control group. RT-PCR displayed that the expression of miR-424(322)-5p and VEGF mRNA in rat cornea tissues was significantly enhanced on the 4th day after modeling (P < 0.01). The expression level of miR-424(322)-5p in the mimic group after HUVECs transfected with liposomes was significantly higher than that in the NC group and the CON group (P < 0.01). The results of CCK-8 assay, scratch assay, migration assay and Matrigel tube assay displayed that miR-424(322)-5p promoted the migration, proliferation and tube forming ability of HUVECs (P < 0.01). Western blotting showed that the protein levels of VEGF and Raf/MEK/ERK pathway proteins were significantly up-regulated (P < 0.01). Conclusion Overexpression of miR-424(322)-5p promotes the migration, proliferation and tube formation of HUVECs by activating VEGF and Raf/MEK/ERK pathways, and thereby participates in the formation of Cor NV.

Keywords

• mir-424(322)-5p
• corneal neovascularization
• human umbilical vein endothelial cells
• raf/mek/erk pathway