The Effect of miR-200a-3p on Corneal Neovascularization in Alkali Burned Mice by Targeting and Regulating ALKBH5
ZHANG Jiasheng1, LIN Shan2*, LIU Shiliang3
This study aimed to explore the effect of miR (microRNA)-200a-3p on corneal neovascularization in alkali burned mice by targeting and regulating ALKBH5 (RNA demethylase AlkB homologue 5). Totally 108 mice were assigned into the control group, the alkali burn group, the AAV-NC group, the AAV-pre-miR-200a-3p group, the AAV-pre-miR-200a-3p+pc-NC group, and the AAV-pre-miR-200a-3p+pc-ALKBH5 group, with 18 per group. Except for the control group, the mice in the other groups were all used to construct corneal alkali burn models with filter pa per soaked in 1 mol/L NaOH. Corneal opacity was blind assessed by ophthalmologist. The histopathological changes of corneal tissue was evaluated by HE staining. Collagen deposition was evaluated by Masson tricolor staining. Apop tosis was measured by TUNEL staining. The expression of CD31 (platelet-endothelial cell adhesion molecule) was measured by immunofluorescence staining. The level of miR-200a-3p was detected by RT-qPCR (real-time reverse transcription PCR). The expression of ALKBH5 protein was detected by Western blot. In addition, the targeting rela tionship between miR-200a-3p and ALKBH5 was determinethed by dual-luciferase reporter gene and RIP experiment. The results showed that compared with the control group, the corneal opacity score of mice in the alkali burn group raised, the inflammatory cells infiltrated the corneal stroma, the collagen structure of the stroma relaxed, and the cor neal thickness raised. The collagen volume fraction, corneal apoptosis rate, relative fluorescence intensity of CD31 and protein expression of ALKBH5 all increased, while the expression of miR-200a-3p declined (P<0.05). Compared with the AAV-NC group, the corneal opacity score of mice in the AAV-pre-miR-200a-3p group declined, the corneal thick ness declined, the collagen volume fraction, corneal apoptosis rate, relative fluorescence intensity of CD31 and protein expression of ALKBH5 declined, while the expression of miR-200a-3p raised (P<0.05). Compared with the AAV-pre miR-200a-3p+pc-NC group, the corneal opacity score and corneal thickness of mice in the AAV-pre-miR-200a-3p+pc ALKBH5 group increased, the collagen volume fraction, corneal cell apoptosis rate, relative fluorescence intensity of CD31 and protein expression of ALKBH5 raised (P<0.05). The results of dual-luciferase activity and RIP experiment showed that miR-200a-3p had a targeting relationship with ALKBH5 (P<0.05). In conclusion, miR-200a-3p inhibits corneal neovascularization in alkali burned mice by suppressing the expression of ALKBH5.
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