| Diabetic retinopathy(DR)is a common ocular disease characterized by the serious microvascular complications of DM.The worldwide prevalence of DR from a pooled metaanalysis of population studies conducted in USA,Australia,Europe and Asia reported an overall prevalence of 35%for any type of DR.In the coming years the prevalence of DR will rapidly increase with the increasing number and lifespan of people with diabetes.It is caused by abnormal retinal blood vessels that are either proliferative(proliferative diabetic retinopathy,PDR)or functionally incompetent,leaking fluid and lipid into the retina.Visual impairment occurs when edema affects the central retina or macula(diabetic macular edema,or DME).Lead angiogenesis inhibitors present an attractive treatment option for DR.microRNAs(miRNAs)are short(about 22 nucleotides long)and highly conserved sequences of endogenous RNA that do not encode for any protein,and represent a new powerful class of gene modulators.They are expressed in all human cell-types and are involved in the main biological processes including cell growth,differentiation,and apoptosis,but studies refered to their roles in physiology and pathology is still an ongoing process.Recently,miRNAs have been suggested to par-ticipate in the regulation of diverse biological processes,and their deregulation or dysfunction plays important roles in kinds of physiopathological progresses.Even several studies reported the role of miRNA in the development of DR,the detailed mechanism remain largely illusive.Recently,emerging evidence has suggested that deregulated miR-128 was involved in the pathogenesis of kinds of diseases;however the role of miR-218 in the DR was still unknown.HOXB gene is involved in the migration,proliferation and differentiation of vascular endothelial cells.It is believed that HOXB is involved in angiogenesis via regulating TGF-?,VEGF,CXCL1,IL-8,Ang-2 and MMP-9.Considering the comprehensive effect of Hox gene,it could be conjectured that there was potential relationship between HOX gene group and DR incidence.The Hoxb3 gene,which is a member of HOXB family,is a pro-angiogenic homeobox gene that regulates the morphology of newly forming vessels.However,the exact role of HoxB3 in DR development and the pathological mechanism was still unclear by now.Bioinformatics has shown that the 3'-UTR of HoxB3 contains a putative binding site for miR-128.Therefore in the present study,we determined the expression levels of miR-128 in RPE cells under high glucose condition.Then we assessed the effect of miR-128 on proliferation,apoptosis and the expression of HOXB3 of RPE cells in high glucose condition.In addition,we conducted several experiments to detect the detailed signaling pathways for the protective effect of miR-128.Purpose1.To investigate miR-128 expression in retinal tissue of DM models;2.To investigate miR-128,HoxB3 and PI3K/AKT/m-TOR expression in cultured human retinal pigment epithelial cells(ARPE-19)under high glucose conditions;3.to clarify effects of miR-128 on biological properties of ARPE-19 cells,and indentify the regulation of HoxB3 and PI3K/AKT/m-TOR by miR-128.Methods1.Male Sprague-Dawley rats were randomly assigned to either the control or DM group.DM was induced by a single intraperitoneal injection of streptozotocin at a dose of 60 mg/kg body weight,and was defined as a blood glucose level above 16.7 mmol/l determined at 3 days after injection.Control rats received a single intraperitoneal injection of citrate buffer.miR-128 expression were analysis by qRT-PCR.2.ARPE-19 cells were exposed to 5.5 mM and 25 nm glucose for 6 h,12 h,24 h and 48 h.Cell viability assays of cells from different group were determined by MTT.The apoptotic rates were analyzed with flow cytometry.miR-128 expression were tested by qRT-PCR.VEGF content in culture supernatant were evaluated by ELISA method.HoxB3 expression and PI3K/AKT/m-TOR pathway were discussed by Western-blot.3.ARPE-19 in normal and high glucose condition were treated with miR-128 mimic(miR-126-mimic)or miR-128 inhibitor(miR-126-inhibitor).ARPE-19 cells were transfected with 100 nM miR-128 mimic and 200 nM miR-126 inhibitor using lipofectamine 2000 reagent.Cells were collected for the further determination.Cell viability assays of cells from different group were determined by MTT.The apoptotic rates were analyzed with flow cytometry.miR-128 expression were tested by qRT-PCR.VEGF content in culture supernatant were evaluated by ELISA method.HoxB3 expression and PI3K/AKT/m-TOR pathway were discussed by Western-blot.Results1.There was a significant weight loss in DM rats compared with age-matched controls.STZ-treated rats lost weight during the first 2 weeks after injection.At 10 weeks after injection,the rats had lost a significant amount of weight,such that their weights were similar to those at the time of injection.STZ-treated rats showed a notable increase in blood glucose levels in the first week after injection.At 10 weeks after injection,blood glucose levels were more than 5-fold higher than those of the age-matched control rats.2.MiR-128 displayed increased expression in the retinas of DM rats.3.In order to determine the expression of miR-128 in the high glucose condition,the quantitative RT-PCR detection was conducted between the RNA extracted from culture ARPE-19 in high glucose compared with the normal condition.Our data showed that miR-128 was significantly decreased in ARPE-19 cells in high glucose compared with the control group.4.First,the cell viabilities in different time points were presented in Figure 2.Compared with the control group,cell group with high glucose treated for 6 h showed no significant difference.However,with the increase of high glucose treatment duration,the cell viability reduced significantly.In the 12 h group,the cell viability was significantly decreased after high glucose treated(P<0.01).In the longer treatment group,the cell viability was depressed more significantly(P<0.001).Considering that the 48 h demonstrated a significantly and stably decreased cell viability level,this time point was used in the following experiment.Besides,we conducted advanced study to detect the effect of miR-128 on the ARPE-19 cells.The cell viability in the miR-128 treated in the normal condition ARPE-19 cells was not significant different from control group.While it was detected that miR-128 application in the high glucose group improved the ARPE-19 cells viability significantly.5.Apoptosis plays key roles in kinds of abnormal pathologic processes,including the decreased cell viability induced by high glucose.To investigate whether miR-128 protect the ARPE-19 cells from apoptosis induced by high glucose,flow cytometry was used for the detection of apoptotic rate of cultured cells.The data showed that ARPE-19 cells incubated with high glucose for 48 h showed significant higher apoptosis rate compared with the control group.However,ARPE-19 cells in high glucose treated with miR-128 for 6 h showed significantly decreased apoptosis rate.Besides,the application of miR-128 inhibitor would increase the apoptosis rate compared with the RPE cells in the high glucose condition.All the detailed data was presented in the summary histogram.6.We used TargetScan 6.2 software to search the potential target gene of miR-128.HOXB3 was predicted to be a target of miR-128.In the western blot analysis,it was found that HOXB3 was up-regulated in the high glucose group.In the miR-128 treatment group,miR-128 could down-regulate the expression HOXB3 in the ARPE-19 cells.Besides the miR-128 inhibitor would lead to the up-regulation of HOXB3.To confirm this correlation between miR-128 and HOXB3,the miR-128 binding sequences present at the 3'-UTR of HOXB3 mRNA(WT-3'-UTR),its mutant site(HOXB3-3'UTR-mut)were subcloned downstream of the luciferase reporter gene and then co-transfected into ARPE-19 cells.The relative luciferase activity of the reporter that contained wild-type 3'-UTR was decreased to 54.6%when miR-128 was co-transfected.However then luciferase activity assay showed that miR-128 significantly suppressed the WT 3'-UTR but not that of Mut 3'-UTR of PTEN luciferase activity in ARPE-19 cells.7.To further investigate the mechanism of miR-128 protecting RPE cells in high glucose condition,we evaluated the protein levels of PI3K/AKT-mTOR pathway.The PI3K/AKT-mTOR pathway was associated with HOXB3 gene function and played key roles in different pathologic processes.The total protein of PI3K and AKT was not significantly different in each group.Ho-wever,the phosphorylation of PI3K and AKT were significantly increased in the high glucose group.Besides miR-128 treatment prevented the increase of p-PI3K and p-AKT.Besides the miR-128 inhibitor treatment would increase the expression of p-PI3K and p-AKT.For the m-TOR expression level,it would be up-regulated in the high glucose group;however,miR-128 would significantly reduce the m-TOR expression.Conclusion1.miR-128 were abnormally expressed in DR retinas.miR-128 expression levels in retinas could be affected by diabetes,and are closely associated.with DR development.2.High glucose significantly alter viability of ARPE-19 cells and inhibit VEGF expression.3.High glucose inhibit miR-128 expression and promote HoxB3 expression.4.High glucose stimulate the activation of PI3K/AKT/m-TOR pathway.5.HoxB3 were identified as the functional downstream target of miR-128 by directly targeting the 3'-UTR.miR-128 exert its protective effects in DR at least in part by suppression of HoxB3.6.miR-128 produced the protective effect through down-regulating of HOXB3 and inhibiting PI3K/AKT-mTOR pathway. |
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