| Background and objective:Diabetic retinopathy(DR) is the main cause of blindness in people over 40 years of age. According to a population-based epidemiological survey held in 2010 complications of diabetes appeared in about 1/3 diabetic patients and 1/10 diabetic patients had vision-threatening complications, such as diabetic macular edema(DME) and proliferative diabetic retinopathy(PDR). The proliferation of neovascular fiberous membrane in DR is one of the most serious complications in patients with advanced diabetic retinopathy and operation is the only possible treatment for this stage of disease. But how to identify the proliferative activity of the membrane, is a clinical problem to solve. The purpose of this study is to analyse the proliferative membrane activity, to screen the mi RNA related to proliferation activity of membrane,and to further explore the role and possible signal transduction pathway of the relative mi RNA.Subjects and methods1. to verify the activity of proliferative activity of fibrovascular epiretinal membrande by anti-Ki-67 antigen immunohistochemical staining.2. mi RNA q PCR chip array. Vitreous specimens from Ki-67(+) and Ki-67(-) patients were running mi RNA q PCR microarray to detected mi RNA associated with cell proliferation, migration and angiogenesis.3. mi R-451 expression of RPE stimulated by high glucose was deteced by Real-time PCR4. Effect of mi R-451 a on RPE in vivo biological characteristics was examined. ARPE-19 cell line were transfected with mi R-451 a mimics, mimics control, mi R-451 a antagomir, antagomir NC, then cell proliferation and migration ability were detect by MMT, cell counting kit8, and cell scratch test.5. mi R-451 a target genes were predicted. ATF-2, one of the target gene of mi R-451 a, m RNA was deteced by RT-PCR, protein was detected by Western Blot. Luciferase reporter system was done to determine whether mi R-451 a targeted ATF-2 directly.6. The relational signal pathway was detected.7. Statistical analysisSpss 13.0 software was used for statistical analysis. Data were presented as Mean SEm of at least 3 independent experiments. Differences were analyzed using th nonparametric test Mann-Whitney-Wilcoxon. Relative quantification value() of QPCR were analyzed through One-way ANOVA, with the SNK, LSD or Dunnedtt’s T3 tests for multiple comparisons.Result1. The proliferative index of epiretinal membrane was 4.18% 4.58%(0%-16.67%). Ki-67(+) cases accounted for 78.6% of all cases.2. According to mi RNA q PCR array of all the 83 mi RNAs in vitreous sample, only 16 mi RNAs were down-regulated in Ki-67(+) sample compared with Ki-67(-) sample < 2 fold. Of the other 67 up-regulated mi RNAs, the expression of mi R-451 a were 32.89728 fold.3. In vitro, high glucose up-regulated mi R-451 a expression of ARPE-19 cells at 24 hours and 48 hours(P=0.02,0.022). The expression of mi R-451 a at 72 hours was same with control group(P=0.531).4. According to MTT assay, overexpression of mi R-451 a inhibited RPE proliferation at 24 hour and 48 hour after transfection(P<0.05). While downexpression of mi R-451 a improved RPE proliferation at 48 hours. According to CCK-8 assay, overexpression of mi R-451 a inhibited RPE proliferation at 24 hours and 48 hours after transfection(P=0.036 0.018). While down-expression of mi R-451 a improved RPE proliferation at 24 hours and 48 hours(P=0.0410.015). Mi R-451 a antagomir promote cell proliferation and migration5. Luciferase reporter assay system. After construct psi CHECK-ATF-2 3’UTR vector, psi CHECK-ATF-2 3’UTR and mi R-451 a mimics, psi CHECK and mi R-451 a mimics, psi CHECK-ATF-2 3’UTR and mimics-control were cotransfected respectively, luciferase activity were changed significantly(F=63.041 P=0.000). And mi R-451 a and target to 3’UTR of ATF-2 directly can be confirmed by dual luciferase reporter assay system. After transfection with mi R-451 a mimics and inhibitor 24 hour and 48 hour, m RNA expression of ATF-2 changed with levels of mi R-451a(F=50.645 F=59.898 P=0.000 P=0.000).Western Blot results showed that protein expression of ATF-2 also changed with levels of mi R-451a(F=16.626,p=0.000).6. PCR results showed that m RNA expression of matrix metalloproteinase-2, Cyclin A1, Cyclin D1 were changed with mi R-451 a expression. Cylcin D1, Cyclin A1 and MMP2 expression had statistically difference at 48 hour and 72 hour seperately(F=14.113,10.223,7.143,46.382,18.549,13.865; P=0.000,0.000, 0.000,0.000, 0.000,0.000).Conclusion:1. mi R-451 a related to cell proliferation and migration2. miR-451 a mimics can inhibit the proliferation of RPE3. mi R-451a/ATF-2/ MMP2,Cyclin A1,Cyclin D1 may be a promising singal passway of RPE proliferation and migration. |
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