| Part 1:The expression of miR-126 and relative factors in rhesus macaque choroid-retinal endothelial cells under high glucose[Objectives]The aims of this study are to detect the expression of miR-126,VEGFA and PIK3R2 in rhesus macaque choroid-retinal endothelial cell line(RF/6A)cells cultured in normal glucose(NG),high glucose(HG)and osmosis(OSM),respectively.[Methods]Quantitative real time polymerase chain reaction(q-PCR)and western blot were used to detect the expression of miR-126,VEGFA and PIK3R2 in(RF/6A)cells cultured in nonnal glucose,high glucose and osmosis,respectively.[Results]1.Relative expressions of miR-126 in NQ HG and OSM groups were(1.00±0.00),(0.70±0.07)and(0.97±0.02),respectively.Compared to NG and OSM groups,relative expression of miR-126 were both decreased in RF/6A cells in HG group(P<0.05).Relative expression of miR-126 was reduced in OSM group compared with that in NG group(P>0.05).2.Relative expressions of VEGFA inRNA in NG,HG and OSM groups were(1.00±0.00),(3.92±0.43)and(0.97±0.13),respectively.Compared to NG and OSM groups,relative expression of VEGFA mRNA were both increased in RF/6A cells in HG group(P<0.05).The protein expression of VEGFA was similar with the mRNA expression.3.Relative expressions of PIKK3R2 mRNA in NQ HG and OSM groups were(1.00±0.00),(1.52±0.09)and(0.99±0.09),respectively.Compared to NG and OSM groups,relative expression of PIKK3R2 mRNA were both increased in RF/6A cells in HG group(P<0.05).The protein expression of PIKK3R2 was similar with the mRNA expression.[Conclusions]In RF/6A cells,high glucose could lead to the decreasing expression of miR-126,the increasing expression of VEGFA and PIK3R2.Part 2:miR-126 overexpression inhibits high glucose-induced migration and tube formation of rhesus macaque choroid-retinal endothelial cells[Objectives]To investigate the effect of miR-126 on the migration and tube formation capacity of RF/6A cells.[Methods]After transfection of miR-126 mimics and miR-126 inhibitor,we investigated the function of miR-126 in RF/6A cells by scratch wound,transwell migration and tube formation assays.[Results]1.Results of scratch wound assay showed that relative wound area of RF/6A cells in Control group and HG group were(1.00 ±0.05)and(0.92 ±0.09),respectively.Compared to Control group,relative wound area of RF/6A cells in HG group was smaller(P<0.05).Relative wound area of RF/6A cells in HG+miR-126 mimics group was(1.02 ±0.13),compared to HG group,relative wound area of RF/6A cells in HG+miR-126 mimics group was bigger(P<0.05).Relative wound area of RF/6A cells in HG+miR-126 inhibitor group was(0.88 ±0.09),compared to HG group,relative wound area of RF/6A cells in HG+miR-126 inhibitor group was smaller(P<0.05).2.Results of transwell assay showed that average number of migrated RF/6A cells in Control and HG group were(33.40±3.05)and(55.20±1.79),respectively.Compared to Control group,average number of migrated RF/6A cells in HG group was more(P<0.05).Average number of migrated RF/6A cells in HG+miR-126 mimics group were(38.00±2.35),compared to HG group,average number of migrated RF/6A cells in HG+miR-126 mimics group was less(P<0.05).Average number of migrated RF/6A cells in miR-126 inhibitor group were(64.20±3.83),compared to HG group,average number of migrated RF/6A cells in HG+miR-126 inhibitor group was increased(P<0.05).These results indicated that high glucose induced the high migration of RF/6A cells.Compared to HG group,RF/6A cells in HG+miR-126 mimics group revealed lower migration.Compared to HG group,RF/6A cells in HG+miR-126 mimics group showed high migration.3.Results of tube formation assay showed that average number of tube branch points of RF/6A cells in Control and HG group were(16.00±0.71)and(27.8±2.86),respectively.Compared to Control group,average number of tube branch points of RF/6A cells in HG group was more(P<0.05).Average number of tube branch points of RF/6A cells in HG+miR-126 mimics group were(8.00±1.22),compared to HG group,average number of tube branch points of RF/6A cells in HG+miR-126 mimics group was less(P<0.05).average number of tube branch points of RF/6A cells in HG+miR-126 inhibitor group were(36.2±3.49),compared to HG group,average number of tube branch points of RF/6A cells in HG+miR-126 inhibitor group was more(P<0.05).These results demonstrated that overexpression of miR-126 could effectively suppress high glucose-induced capillaries formation,and interference of miR-126 led to opposite results.[Conclusions]By scratch wound,transwell migration and tube formation assays,we found that miR-126 overexpression could inhibit the migration and sprouting of RF/6A cells induced by high glucose.Part 3:Study about the molecular mechanisms of miR-126 inhibits the angiogenesis of RF/6A cells[Objectives]The aims of this study are to investigate the relative regulation between miR-126 and VEGF/PI3K/AKT signaling pathway in retinal vascular endothelial cells.[Methods]After addition of miR-126 mimics and miR-126 inhibitor,we investigated the effect of miR-126 on the expression of VEGFA,PIK3R2 and AKT.Moreover,bioinformatics analysis and luciferase array were used to confirm the direct or specific regulation of miR-126 to VEGFA or PIK3R2.[Results]1.Through bioinformatics analysis,we found that miR-126 could regulated VEGFA/PIK3R2.Then,by dual luciferase experiment,we found that relative luciferase activity was decreased in cells co-transfected with miR-126 and 3'UTR of VEGFA or PIK3R2,and co-transfection of "seed sequence" based mutation of miR-126 binding site of VEGFA-3'UTR(mut)and PIK3R2-3'UTR(mut)both restored the luciferase activity.2.VEGFA:Relative expression of VEGFA mRNA in HG and HG+miR-126 mimics groups were(3.92±0.23)and(2.42±0.17),respectively.Compared to HG group,relative expression of VEGFA mRNA in HG+miR-126 mimics group was lower.Relative expression of VEGFA mRNA in HG+miR-126 inhibitor group was(4.94±0.21),compared to HG group,relative expression of VEGFA mRNA in HG+miR-126 mimics group was increased.The results of western blot further verified results of q-PCR.PIK3R2:Relative expression of PIK3R2 mRNA in HG and HG+miR-126 mimics groups were(1.52±0.09)and(0.85±0.077),respectively.Compared to HG group,relative expression of PIK3R2 mRNA in HG+miR-126 mimics group was decreased.Relative expression of PIK3R2 mRNA in HG+miR-126 inhibitor group was(2.25±0.016),compared to HG group,relative expression of PIK3R2 mRNA in HG+miR-126 mimics group was increased.The results of western blot further verified results of q-PCR.AKT1:Relative expression of AKT1 mRNA in HG and HG+miR-126 mimics groups were(1.22±0.09)and(0.92±0.075),respectively.Compared to HG group,relative expression of AKT1 mRNA in HG+miR-126 mimics group was decreased.Relative expression of AKT1 mRNA in HG+miR-126 inhibitor group was(1.54±0.01),compared to HG group,relative expression of AKT1 mRNA in HG+miR-126 mimics group was increased.Results of western blot showed that total AKT1 in each groups revealed little change.While,compared to HG group,phosphorylation-AKT1(p-AKT1)in HG+miR-126 mimics group was reduced.Compared to HG group,p-AKT1 in miR-126 inhibitor group was increased.SDF-1?:Relative expression of SDF-1? mRNA in HG and HG+miR-126 mimics groups were(1.62±0.07)and(0.91±0.11),respectively.Compared to HG group,relative expression of SDF-1? mRNA in HG+miR-126 mimics group was reduced.Relative expression of SDF-1? mRNA in HG+miR-126 inhibitor group was(2.35±0.21),compared to HG group,relative expression of SDF-1? mRNA in HG+miR-126 mimics group was enhanced.VCAM-1:Relative expression of VCAM-1 mRNA in HG and HG+miR-126 mimics groups were(1.85±0.11)and(1.26±0.07),respectively.Compared to HG group,relative expression of VCAM-1 mRNA in HG+miR-126 mimics group was decreased.Relative expression of VCAM-1 mRNA in HG+miR-126 inhibitor group was(2.18±0.21),compared to HG group,relative expression of VCAM-1 mRNA in HG+miR-126 mimics group was increased.SPRED1:Relative expression of SPRED1 mRNA in HG and HG+miR-126 mimics groups were(1.45±0.16)and(0.79±0.07),respectively.Compared to HG group,relative expression of SPRED1 mRNA in HG+miR-126 mimics group was less.Relative expression of SPRED1 mRNA in HG+miR-126 inhibitor group was(2.08±0.21),compared to HG group,relative expression of SPRED1 mRNA in HG+miR-126 mimics group was enhanced.[Conclusions]Overexpression miR-126 inhibited the increased expression of VEGFA,PIK3R2,SDF-1?,VCAM-1,SPRED1,and the activation of AKT1 induced by high glucose and HG+miR-126 inhibitor caused the opposite results which were determined by q-PCR and western blot.In addition,by luciferase assay,we found that miR-126 could directly negative regulate VEGFA and PIK3R2.Our results suggest that miR-126 overexpression inhibits the migration and sprouting of RF/6A cells induced by high glucose which might possibly by blocking VEGFA and PIK3R2 in the VEGF/PI3K/AKT signaling pathway. |
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