| Background:Diabetic retinopathy(DR)is the most common complications of diabetes mellitus that affects three quarters of patients with diabetes more than 15 years,and 20% of these patients will progress to proliferative diabetic retinopathy(PDR)after 25 years of diabetes.It is predicted that 3.4 million diabetic patients over the age of 40 and 1.9 million patients over the age of 65 will afflicted with a sight-threatening by PDR at the year 2050.The pathological characteristics of PDR is hyperglycemia related abnormally epiretinal outgrowth and retinal neovascularization,and at its worst,resulted in resultant bleeding and retinal detachment,and leading to blindness in the end.Therefore,the control of high prolonged glucose induced neovascularization is essential in arresting the development of PDR.MicroRNAs(mi RNAs)are a group of 21-25 nucleotides lengths noncoding RNAs which could regulate the activity of m RNA by binding to the 3' untranslated region of its target transcript.Studies have found that many mi RNAs are abnormally expressed in DR,suggesting that mi RNAs might be pivotal factors associated with the pathogenesis of DR.mi R-21 is highly expressed in a variety of cancers and fibrosis related diseases.It is implicated in the regulation of cell proliferation,apoptosis,migration and angiogenesis.More importantly,There are studies that show that that mi R-21 was up-regulated in the vitreous humor of proliferative vitreoretinal disease patients,the proliferation and migration activity of retinal pigment epithelial cells was significantly changed by gain and loss function of mi R-21.However,the effect of mi R-21 on retinal angiogenesis are still not clear.In the present study,human retinal microvascular endothelial cells(HRMECs)were employed to investigate the effect of mi R-21-5p on high glucose induced angiogenesis and the underling mechanism was also studied.The results of this study provided a potential target for the diagnosis and treatment of diabetic retinopathy.Objective: To clarify the abnormal expression of mi R-21-5p in HRMECs induced by high glucose.The effects of mi R-21-5p silencing on the proliferation,migration and tube formation of retinal microvascular endothelial cells induced by high glucose were determined.To investigate the regulation of ERK and Akt signaling pathway in mi R-21-5p in HRMECs.It provides a theoretical basis for the diagnosis and treatment of mi R-21 for diabetic retinopathy.Methods: In order to study the role of mi R-21-5p in angiogenesis of PDR,HRMECs on high glucose induced were used to simulate the pathogenesis of PDR.The expression of mi R-21-5p,VEGF and VEGF-R2 was detected by real-time PCR and Western blot,and the effect of high sugar on the above indicators was determined.The mi R-21-5p inhibitor was synthesized and the negative control was set.HRMECs was transfected with high sugar induction 12 h,Cell proliferation was determined by MTT test,Ki6 was tested by mmunofluorescence,Scratch test is used to detect cell migration,The ability of the cell angiogenesis was detected by tube formation assay,Cell migration related protein mmp-2,mmp-9 was detected by Western blot,Protein VEGF,VEGF-R2,HIF-1? associated proteins found in angiogenesis was detected by Western blot,ERK,Akt signaling pathway related proteins and probability target gene maspin was detected by Western blot.HRMECs were respectively pre-incubated with LY294002 or U0126,the specific inhibitor of PI3K/AKT or ERK,to investigate the effect of these two pathways in high glucose treated HRMECs,then the ability of cell migration and cell angiogenesis was analyzed.The expression of VEGF?HIF-1??MMP-2?MMP-9 was detected by Western blot.Results:1.Cells were exposed to normal or high glucose for different time points to investigate its role on HRMECs viability.High glucose significantly increased cell viability at 24 h,48 h and 72 h,mannitol as a negative control didn't affect cell viability at all,p<0.05,p<0.01.2.The expression of mi R-21-5p under high glucose was up-regulated in 24 h 48h 72 h high glucose group,whereas,it was not changed in mannitol group,p<0.01.The level of VEGF m RNA and VEGFR2 m RNA was higher in glucose treated cells than mannitol group and control group,p<0.01.3.The protein level of VEGF and VEGFR2 was higher in glucose treated cells than mannitol group and control group,p<0.01.4.The proliferation of cells in high glucose group increased significantly compared with the control group,p<0.01.The proliferation of cells in mi R-21-5p inhibitor group decreased significantly compared with NC inhibitor group,p<0.01.5.Immunofluorescence results showed that the Ki67 positive cells were obviously increased after high glucose treatment,p<0.01.The number of Ki67 positive cells in mi R-21-5p inhibitor group decreased significantly compared with NC inhibitor group,p<0.05.6.Migration rate of high glucose group increased significantly compared with control group p<0.01.Migration rate of mi R-21-5p inhibitor group decreased significantly compared with NC inhibitor group,p<0.05.7.Tube formation assay showed that the branching points in high glucose treated cells were prominently higher than that in control group p<0.01,the branching points in mi R-21-5p inhibitor group cells were prominently less than that in NC inhibitor group and high glucose group.8.The protein level of MMP-2?MMP-9?p-AKT?AKT?p-ERK?ERK ?VEGF?HIF-1??maspin in mi R-21-5p inhibitor group cells were prominently less than that in NC inhibitor group and high glucose group.The protein level of MMP-2?MMP-9?p-Akt?Akt?p-ERK?ERK?VEGF?HIF-1??maspin in high glucose group was higher than that in control group,p<0.01.9.HRMECs were respectively pre-incubated with LY294002 or U0126.Pre-incubate with LY294002 or U0126 declined migration rate of high glucose treated HRMECs,that along with reduced MMP-2 and MMP-9 levels in HRMECs.10.High glucose induced increase of branching points were decreased by pre-incubation with LY294002 or U0126.11.The protein level of MMP-2?MMP-9?VEGF?HIF-1? in LY294002 group cells were prominently less than that in high glucose group cells,p<0.01.The protein level of MMP-2?MMP-9?VEGF?HIF-1? in U012 group cells were prominently less than that in high glucose group cells,p<0.01.Conclusions: The study found that high glucose promotes the proliferation activity of HRMECs,and increases the expression of mi R-21-5p,VEGF and VEGFR2 and protein in HRMECs.Inhibition of mi R-21-5p could suppresses high glucose induced proliferation and angiogenesis of HRMECs.mi R-21-5p may participate the proliferation and angiogenesis of HRMECs by the regulation of PI3K/AKT and ERK pathways via its target protein maspin. |
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