Expression And Identification Of Differencial MicroRNA In Hopoxia Human Retinal Pigment Epithelial Cell Line ARPE-19

Part 1 Expression character of Hopoxia Human Retinal Pigment Epithelial Cell Line ARPE-19Objective To study the influence of hypoxia on appearance and proliferation of human retinal pigment epithelial(RPE)cells and expression of VEGF and PEDF.Methods The ARPE-19 cells were put into normal and hypoxic chamber respectively.After 0hr,8hr,24hr,48hr,the proliferation of ARPE-19 cells was evaluated by[3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromid, MTT]-test.Apoptosis rate of ARPE-19 were detected by hochest staining and flow cytometry.After 24 hours,the VEGF mRNA and PEDF mRNA were detected by RT-PCR.Image processing and quantitative analysis were applied with computer.Results The value detected by MTT in the hypoxia groups were lower than that in the normal group,especially at 48h point(P＜0.001).Apoptosis rate of ARPE-19 that determined by hochest staining and flow cytometry were higher in the hypoxia groups,especially at 24h point(P＜0.001).The expression of VEGF mRNA in ARPE-19 cells increased obviously under hypoxia,and the expression of PEDF mRNA in ARPE-19 cells decreased obviously under hypoxia.Conclusion Hypoxia can stimulate the apoptosis of ARPE-19 cells,destroy the balance of VEGF and PEDF expression,The results indicate that controlling the activity of RPE during hypoxia is essential to inhibit the formation of choroidal neovascularization. Part 2 Expression of Differencial MicroRNA in Hopoxia Human Retinal Pigment Epithelial Cell Line ARPE-19Objective MicroRNA(miRNA)play a global role in regulating gene expression and have important tissue-specific functions.But little is known about their role in the emerge of CNV in region of ophthalmology.The purpose of this study was to find those different miRNAs that predicted to target genes involved in emerge of CNV.Methods Retinal pigment epithelium cell line ARPE-19 were put into hypoxic chamber for 24 hours and RNA were extracted for detection further.Different miRNAs were predicted using Microarray assay(LC Sciences)with RNA that extracted from ARPE-19 cells.Different miRNAs that signal numerical value above 1000 were verificated by quantitative PCR(q-PCR).Results 42 miRNAs were predicted to target emerge of CNV,within these miRNAs 18 miRNAs expressed as up-regulation,and 24 miRNAs expressed as down-regulation,that ten highest signal numerical value include Mir-1,Mir-1469,Mir-663,Mir-1915,Mir-29c,Mir-574-5p,Mir-638,Mir-29b-1,Mir- 1308,Mir-10a,within these miRNAs Mir-1,Mir- 29c,Mir- 1308 expressed as down-regulation,the others expressed as up-regulation.Within 28 miRNAs that signal numerical value above 1000 included Mir-638,Mir-1308,Mir-125b,Mir-155,Mir-30c,Mir-125a-5p which expressed coincidence with the results of Microarray assay.Through verification Mir-155 expressed as the most significant. Conclusion Many miRNAs likely to regulate genes important for emerge of CNV were present in the ARPE-19 in hypoxic chamber. ARPE-19 after Mir-155 transfectionObjective The purpose of this study was to find whether Mir-155 has some activity of anti-hypoxia and play very important role in emerge of CNV to human retinal pigment epithelium cell line ARPE- 19.Methods The ARPE-19 cells were transfected with Mir-155,and then put into hypoxic chamber for 0hr,8hr,24hr,48hr,the proliferation of ARPE-19 cells was evaluated by[3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromid, MTT]-test.Apoptosis rate of ARPE-19 were detected by flow cytometry. Results The value detected by MTT in the transfected groups were higher, especially at 48h point.Apoptosis rate of ARPE-19 that determined by flow cytometry were lower in the transfected groups(P＜0.001),especially at 48h point.Conclusion Mir-155 enduce some activity of anti-hypoxia after transfection within ARPE- 19 and play very important role in emerge of CNV.