| Objective This study was to investigate the role of micro RNA-21(mi R-21)in mouse alkali-induced corneal neovascularization(Cor NV)and treatment of Cor NV by antagomi R-21 via inhibiting mi R-21,which could provide experimental basis for studying pathological mechanism of Cor NV and exploring a new method in treating Cor NV.Methods(1)Analysis of mi R-21 expression in mouse Cor NV induced by alkali burn.The right eyes of eighty-five healthy BALB/c mice were received 1mol/L Na OH solution on the central corneas for 30 s,which were divided into experimental group,and the left eyes were untreated as normal control group.Frontal section photographs were taken on days 4,7,14,21,28 post alkali injury to observe Cor NV morphological changes and calculate area of Cor NV.On days 4,7,14 and 28 after alkali burn,corneal pathological structures and pathological changes of Cor NV were observed by hematoxylin-eosin(HE)staining;CD31 staining and protein concentratins of HIF-1α and VEGF-A expression in the corneas were detected by immunohistochemical(IHC)staining.Illumina mi RNA sequencing technology was conducted to detect mi RNAs expression profiles in the corneas on day 4 and 7 post injury.Mi R-21,HIF-1α m RNA and VEGF-A m RNA expression were detected by quantitative real-time polymerase chain reaction(Real-time PCR).(2)Inhibition of mouse Cor NV induced by alkali burn via antagomi R-21.Ninety healthy BALB/c mice were used to establish the models of Cor NV induced by alkali burn: right central corneas were received 1mol/L Na OH solution for 30 s and left eyes kept untreated.All the mice were randomly divided into five group and each group contained eighteen mice.Right eyes in each group received subconjuctival injections of 0.9% normal saline(negative control group),5nmol scrambled sequence(antagomi R-negative control treatment group),1nmol antagomi R-21(low dose ofantagomi R-21 treatment group),5nmol antagomi R-21(high dose of antagomi R-21 treatment group),2nmol Ranibizumab(positive control group)on days 2 and 8 post injury,respectively.Frontal section photographs were taken on days 4,7,14 post injury to observe Cor NV morphological changes and calculate area of Cor NV.On days 7 and 14 after alkali burn,corneal pathological structures and pathological changes of Cor NV were observed by HE staining;CD31 staining and protein concentratins of HIF-1α and VEGF-A expression in the corneas were detected by IHC staining;mi R-21,HIF-1α m RNA and VEGF-A m RNA expression were detected by Real-time PCR.Results(1)The area of Cor NV reached peak on day 14 after alkali-induced injury,there were statistical significance between two groups(P<0.05);thick and big new vessels could be seen in the stroma of cornea by HE staining;then Cor NV regressed.Illumina mi RNA sequencing results revealed that nineteen mi RNAs expression in the corneas altered significantly(two folds and above)compared to normal corneas on both days 4 and 7.Meantime,mi R-21 at the two investigative time points were upregulated approximately 6.84 and 6.27 fold changes.Real-time PCR and IHC staining showed that mi R-21,HIF-1α(m RNA and protein),VEGF-A(m RNA and protein)signfiantly higher than that in normal group(P<0.05),and all of their expression reached maximum on day 7 after alkali burn.The changes of mi R-21 had positive correlations with HIF-1α(m RNA and protein)or with VEGF-A(m RNA and protein)expression.(2)Frontal eye section photograph,corneal HE staining and CD31 staining revealed that areas of Cor NV and neovessesls in 5nmol antagomi R-21 group and Ranibizumab group were all signfiantly lower than saline group(P<0.05)on days 7 and 14 after alkali burn.Real-time PCR showed compared with saline group,mi R-21,HIF-1α m RNA and VEGF-A m RNA relative expression in 5nmol antagomi R-21 group were all significantly lower on days 7,14 post injury(P<0.05);mi R-21 and HIF-1α m RNA relative expression in 1nmol antagomi R-21 group were lower on day 7 post injury,while VEGF-A m RNA relative expression was lower just on day 14(P<0.05);mi R-21 and VEGF-A m RNA relative expression in Ranibizumab group weresignificantly lower on days 7,14 post injury,and HIF-1α m RNA relative expression was lower on day 7(P<0.05).IHC staining revealed that on days 7 and 14 after alkali burn,HIF-1α and VEGF-A protein expression in 5nmol antagomi R-21 group decreased compared to saline group(P<0.05);VEGF-A protein expression in 1nmol antagomi R-21 group decreased only on day 7 and decreased in Ranibizumab group on days 7,14 post injury(P<0.05),while there was no signifiant change of HIF-1α protein expression at the two investigative time points.Conclusions(1)Mi R-21,HIF-1α and VEGF-A were upregulated significantly in the process of Cor NV induced by alkali burn,and mi R-21 showed positive correlation with HIF-1α and VEGF-A expression,mi R-21 could play a role in stimulating Cor NV.(2)Proper amount of antagomi R-21 suppressing mi R-21 via subconjuctival injection might inhibit formation of Cor NV induced by alkali burn through downregulating HIF-1α and VEGF-A expression.(3)Mi R-21 could stimulate mouse Cor NV induced by alkali burn through HIF-1α/VEGF-A signal pathway.Antagomi R-21 could be expected to become a new potential method for treating Cor NV. |
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