| Section one:Characterization of miR-497 expression in corneal neovascularizationObjective: To predict and identify the correlation between micro RNA-497(micro RNA-497,miR-497)and vascular endothelial growth factor A(VEGFA)and fibroblast growth factor 2(FGF2)The expression changes of miR-497 after corneal alkali burn in mice.Methods: 1.Predict micro RNAs targeting VEGFA and its receptor KDR(VEGFR-2),FGF2 and its receptor FGFR1 that simultaneously target humans and mice by bioinformatics;2.Subsequently,20 female C57 BL / 6 wild-type mice were used to construct a mouse corneal alkali burn neovascularization model.Quantitative Real-time PCR(q PCR)was used to detect the corneal alkali burn at 0d,2d,The expression of miR-497 at 4d,7d,and 14 d and the m RNA expression of VEGFA and its receptor KDR(VEGFR-2),FGF2 and its receptor were observed,and their expression trends were observed.Results: 1.The star Base database based on miRanda,Pic Tar,and Target Scan predicts VEGFA and its receptor KDR(VEGFR-2),FGF2,and VEGFA from the miR-16 / 15/195/424/497 family and humans and mice.The receptor FGFR1 has a binding site;2.The q PCR results showed that after alkaline corneal burns in mice,compared with day 0,miR-497 expression increased on day 2 and peaked on day 4,and the expression gradually increased from day 7 to day 14 Compared with day 0,the relative expression of miR-497 m RNA on days 2,4,7,and 14 after corneal alkali burn was statistically significant(P <0.05).At the same time,the expressions of VEGFA m RNA,KDR m RNA and FGFR1 m RNA peaked on day 4,and gradually decreased from day 7 to day 14,and were statistically significant compared with day0.The trend was similar to the trend of miR-497 m RNA expression Consistently,while FGF2 m RNA peaked on Day 4,fell to near baseline levels on Day 7,and then increased again on Day 14.Conclusion: miR-497 can simultaneously target VEGFA and FGF2,and in the mouse corneal alkali burn neovascularization model,miR-497 m RNA expression increased,the highest on the fourth day,VEGFA m RNA,KDR m RNA and FGFR1 m RNA expression trend Consistent with miR-497 m RNA expression trend,FGF2 m RNA expression decreased on day 7 and then increased again.Section two:Effect of miR-497 expression on the biological characteristics of human corneal stromal cells and human umbilical vein endothelial cellsObjective: To verify that miR-497 can regulate the expression levels of VEGFA and FGF2,and can inhibit the proliferation and luminal formation of human umbilical vein endothelial cells(HUVECs)by targeting VEGFA and FGF2.Methods: 1.Construct 3’UTR wild-type and mutant luciferase reporter plasmids of VEGFA and FGF2,The dual luciferase test verified that miR-497 can target the 3’UTR that binds VEGFA and FGF2;2.Culture corneal stromal cells and transfect blank control,agomir NC,agomir497,antagomir NC,antagomir 497,ranibizumab(Ranibizumab),and then detect the expression of VEGFA and FGF2 in the cells by Western Blot(Western Blot)Protein;3.Cultivate HUVECs and transfect blank control,agomir NC,agomir 497,antagomir NC,antagomir 497,ranibizumab(Ranibizumab),and then measure the cell activity of each group by cell viability assay(CCK-8 method)The luminal forming ability of the cells of each group.Results: 1.Successfully constructed 3’-UTR wild-type and mutant luciferase reporter plasmids of VEGFA and FGF2.The dual luciferase reporter gene experiment proved that miR-497 can inhibit the 3’-UTR double of VEGFA and FGF2,respectively Luciferase activity,in which the inhibition of FGF2 activity is more obvious;2.Western Blot test results showed that,in corneal stromal cells,compared with the blank control group and agomir NC group,the relative expression of VEGFA and FGF2 protein in antagomir 497 group increased,and the relative expression of VEGFA and FGF2 protein in agomir 497 group decreased.3.The results of HUVECs cell activity measurement showed that compared with the blank control group,the proliferation ability of HUVECs in antagomir 497 group was enhanced(3.26 ± 0.61,P <0.01),and the proliferation ability of HUVECs in agomir 497 group was reduced(0.31 ± 0.08,P <0.01),better inhibition effect than ranibizumab group(0.86 ± 0.22).4.The results of HUVECs’ lumen formation showed that compared with the blank control group,the lumen formation of the antagomir 497 group was significantly increased,and the lumen formation of the agomir 497 group was reduced,compared with the ranibizumab group.less.Conclusion: 1.miR-497 can target the 3’UTR binding VEGFA and FGF2 to regulate its expression after transcription;2.Overexpression of miR-497 can inhibit the protein expression of VEGFA and FGF2 in corneal stromal cells,and the difference is statistically significant compared with the control group;3.Overexpression of miR-497 can inhibit HUVEC proliferation and lumen formation.Section three:miR-497 negative feedback regulates HIF-1α-induced VEGFA and FGF2 pathwaysObjective: To verify that hypoxia inducible factor-1α(HIF-1α)can regulate the expression of VEGFA and FGF2 through the transcription level and the post-transcription level mediated by miR-497,and verify whether VEGFA and FGF2 can synergistically regulate miR feedback-497 expression.Methods: 1.Use 16 female C57 BL / 6 wild-type mice to build a corneal alkali burn neovascularization model,and use quantitative real-time PCR(Quantitative Real-time PCR,q PCR)to detect mouse corneal alkali burn at 0d and 4d,7d,14 d HIF-1αm RNA expression changes,observe the expression trend;2.Construct the VEGFA and FGF2 promoter luciferase reporter genes,and then divide them into two groups of transfected blank plasmids and HIF-1α expression plasmids respectively,and verify the promoter fragments of HIF-1α and VEGFA and FGF2 through the luciferase reporter gene experiment Effect3.Incubate HUVECs under 1% hypoxia.After 24 hours,one group added HIF-1α inhibitor LW6(20u M),the other group added equal amount of dimethyl sulfoxide(DMSO),and continued incubation for 12 hours.q PCR to detect the expression of miR-497 m RNA,VEGFA m RNA and FGF2 m RNA;4.Transfect the HIF-1a overexpression plasmid in HUVECs for 36 hours,and then transfect the 3’-UTR reporter plasmid of VEGFA and FGF2(p MIR-FGF2-wt-3’-UTR / p MIR-VEGFA-wt-3 ’-UTR)12 hours,and measure luciferase activity.5.Divide HUVECs into 4 groups,add PBS,VEGFA,FGF2,VEGFA + FGF2(1:3)200ng and incubate for 48 hours,then q PCR detect miR-497 m RNA,VEGFA m RNA,KDR m RNA,FGF2 m RNA and FGFR1 m RNA expression.Results: 1.q PCR results showed that after alkali corneal burn in mice,compared with day 0,HIF-1α expression was lower on day 2 and gradually increased afterwards;2.Compared with the transfected blank plasmid,the HIF-1α expression plasmid significantly enhanced the luciferase activity of the VEGFA and FGF2 promoters by4.65 times and 2.13 times,respectively(p <0.01);3.qPCR detected the expression of miR-497,VEGFA and FGF2 m RNA.The results showed that compared with the DMSO control group,HIF-1α inhibited LW6-induced miR-497 expression to rise 8.62 times,LW6 inhibited VEGFA to 0.65 times,and LW6 had no obvious inhibitory effect on FGF2(P <0.01);4.The HIF-1α overexpression plasmid enhances the 3’UTR luciferase of VEGFA and FGF2 to 3.95 and 2.03 times of the blank plasmid(p <0.01).Conclusion: 1.HIF-1α can inhibit the expression of miR-497;2.HIF-1α can induce its m RNA expression at the transcription level through the promoters of VEGFA and FGF2;3.HIF-1α indirectly promotes the increased expression of VEGFA and FGF2,the targets of miR-497 after transcription,by inhibiting miR-497;4.VEGFA and FGF2 coordinate feedback to promote the expression of miR-497. |
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