Effect And Significance Of Ranibizumab On MicroRNAs Levels In Corneal Neovascularization And The Expression And Correlation Analysis Of Mmu_circ₀001052 In Corneal Neovascularization In Mice

Part 1 Effect and significance of Ranibizumab on microRNAs levels in corneal neovascularizationObjective To investigate the therapeutic effect of Ranibizumab on corneal neovascularization in rats and to screen the differentially expressed miRNAs,after treatment.To provide a new direction for corneal neovascularization therapy.Methods Twenty-four SPF SD rats were randomly divided into three groups:blank group?n = 8??group A?.Taking the left eye as the experimental eye,the rat corneal neovascularization model was established by suture and divided into model group?group B?and subconjunctival injection of Ranibizumab treatment group?group C?.On the 8th day after operation,6 rats in each group were randomly selected to measure the length and area of corneal neovascularization.The expression of CD31 was detected by histopathological examination and immunofluorescence staining.Combined with gene chip and bioinformatics database,miRNAs,real-time fluorescence quantitative PCR related to neovascularization was selected to verifythe difference of miRNAs and VEGF-A expression in each group.Bioinformatics methods were used to analyze the target genes of differential miRNA.Results On the 8th day after operation,the length and area of corneal neovascularization in group C were lower than those in group B?P<0.01?.Histopathological examination showed that neovascularization and infiltration of inflammatory cells were found in group B,but only a small number of neovascul-arization and inflammatory cells were found in group C.Tissue immunofluorescence showed that the number of CD31 positive microvessels in group B was?9.83±1.85?per 400 × visual field.The number of microvessels positive for CD31 staining in group C was?4.58±1.38?per 400 × visual field.The number of blood vessels in the three groups was statistically different?F=163.654,P<0.01?.The expression of VEGF-AmRNA in group C was lower than that in group B,while the expression of miR-15b,miR-16 and miR-29c in group C was higher than that in group B?P<0.01?.The target genes of differential miRNA are mainly enriched in angiogenesis,protein binding and other items.Conclusion Subconjunctival injection of Ranibizumab can reduce corneal neovascularization in rats and decrease the levels of VEGF-A,changed the levels of related miRNAs.The mechanism of angiogenesis may though regulating the path-ways of miRNAs.Part 2 The expression and correlation analysis of mmu_circ₀001052 in mouse corneal neovascularizationObjective To investigate the expression of mmu circ 0001052 and MMP16 in corneal neovascularization induced by alkali burn in mice and their relationship,so as to provide new ideas for the diagnosis and treatment of corneal neovascularization.Methods Mouse corneal neovascularization model was established by Alkali burn,and the expressions of mmu circ 0001052,miR-184 and MMP16 were detected by real-time quantitative PCR?qRT-PCR?.Search the circBase database formiRNAs in which the mmu_circ₀001052 sequences are highly complementary and paired,and search the Targetscan database for target genes downstream of miRNAs.The expression of MMP16 was detected by immunohistochemistry,and the correla-tion between mmu_circ₀001052,target miRNA and downstream mma was analyzed by spearman method.Results On the 2nd to 4th day after the establishment of the model,the blood vessels around the corneal limbus of the mice became thicker and partly sprouted to the center.On the 7th to 14th day,the neovascularization was thick and dense,and most of the neovascularization intersected into a network.After 21 days,the neovascularization of the cornea gradually disappeared.Compared with the blank group,mmu_circ₀001052 in corneal tissue was higher on the 7th?14th and 21st days after alkali burn than that in the blank group,and reached its peak on the 14th day.The difference is statistically significant.MMP16mRNA expression was up-regulated at all time points after alkali burn and reached a peak oin the 14th day.miR-184 was down-regulated at all time points,and its expression level was the lowest on the 7th day.The difference in the relative expression level of miR-184mRNA at different time points was statistically significant?P<0.01?.Under HE staining of corneal tissue,normal mouse corneal tissue epithelium and inner cortex were observed to be intact,each layer of structure was removed,and collagen f-ibers in matrix were arranged regularly.On the 4th,7th,14th and 21st days after alkali burn,corneal epithelial layer was edematous,and many new blood vessels with different diameters and a large number of red blood cells began to appear in the stroma.The inflammatory cells and blood vessels were the most on the 14th day,and the layer decreased on the 21st day.MMP16 was not expressed in cornea of blank group mice.The protein expression of MMP16 in conreal tissue increased obviously at various time points after alkali burn,showing brown color,and the expression of MMP16 protein was up-regulated.Correlation analysis of mnmu_circ₀001052 is positively correlated with MMP16 and negatively correlated with miR-184.Conclusion mmu_circ₀001052 and MMP16 may play a role in the occurrence and development of corneal neovascularization in mice.The increase of mmu_circ₀001052 may enhances the inhibition of miR-184 expression and thus promotes the expression of MMP16.