Effect Of MiRNA-1 On Regulating Human Trabecular Meshwork Cells’ Function And Fibronectin

PURPOSE: Glaucoma is a kind of eye disease characterized by optic disk cupping and visual field loss that caused by pathological elevated intraocular pressure.The increase of trabecular outflow resistance is the main cause of glaucomatous optic nerve damage.The excessive accumulation of extracellular matrix(ECM)in primary open-angle glaucoma(POAG)is the major factors resulting in obstruction of aqueous humor outflow and higher intraocular pressure.In this experiment,we hope to study the role of micro RNA-1(mi RNA-1)on fibronectin(FN)in human trabecular meshwork cells(HTMCs)under oxidative stress and reveal the pathway of roles of mi RNA-1METHODS: HTMCs were treated with 300 μM H2O2 for 2 hours to establish oxidative stress.The expression levels of mi RNA-1 and the m RNA level of fibronectin in HTMCs under oxidative stress were evaluated by quantitative polymerase chain reaction(q PCR).The cells proliferation and migration were measured by Cell Counting Kit-8(CCK-8)and transwell assay when the cells were transfected with over-expressed mi RNA-1 plasmid by Lipofectamine 2000.The effects of mi RNA-1 on the fibronectin were detected by q PCR,western blot and immunofluorescence technique,Luciferase reporter assay and bioinformatics were used to test the target of mi R-1.。RESULTS:1.HTMCs were incubated with 300 μM H2O2 for 2 hours to establish oxidative stress,The expression level of mi RNA-1 was downregulated and the m RNA level of fibronectin was upregulated in HTMCs under oxidative stress2.The q PCR showed that the expression level of mi RNA-1was increased compared with the control group when transfected with mi RNA-1,The changes of proliferation activity of HTMCs was detected by CCK-8 when transfected plasmid,the relative expression of pc DNA3 control group was 0.84+0.01 and the relative expression of pc DNA3/pri-1 was 0.93+ 0.03;which showed that there was significant difference compared with the two groups(p<0.05).The changes of migration ability of HTMCs was detected by Transwell when transfected plasmid,the relative expression of control group that transfected pc DNA3 was 111+1.86 and the relative expression of experimental group that transfected pc DNA3/pri-1 was 210+4.9,which was significant difference compared with the two groups(p<0.05).The proliferation and migration ability of HTMCs were enhanced when transfection with pc DNA3/pri-1plasmid;3.q PCR showed overexpression of mi RNA-1 might inhibit the m RNA level of fibronectin,the relative expression of experimental group that transfected pc DNA3/pri-1 was 0.66 + 0.12,Western blot showed overexpression of mi RNA-1could inhibit the protein level of FN,the relative expression of experimental group that transfected pc DNA3/pri-1 was 0.62 + 0.11;the results of immunofluorescence further verified the role of mi RNA-1 on fibronectin;4.Three mi RNA target gene prediction software and luciferase reporter assayshowed that mi RNA-1 could target fibronectin through 3’-untranslated region(3’-UTR).CONCLUSIONS:The expression level of mi RNA-1 was downregulated and the m RNA level of fibronectin was upregulated in HTMCs under oxidative stress.The overexpression of mi RNA-1 increased the viability of cells proliferation and the ability of cells migration.Mi RNA-1 could directly regulate the expression of fibronectin in HTMCs under oxidative stress,which maybe provide a novel therapeutic strategy in glaucoma.