Effect Of Regulation By MiR-34a To SIRT1 On The Hyperglycemic “Metabolic Memory”

Objective:This research through detecting the changes in the expression of miR-34 a,SIRT1,VEGF and the cell apoptosis in RPE-D407 cell lines and retina of SD rat from hygerglycemic "metabolic memory",and the changes of these indicators after down regulating miR-34 a level,to explore the role of miR-34 a in the development of DR, and provide a theoretical basis for the pathogenesis and the new treatment.Methods:293T cells were selected as tool cells,we synthesize the miR-34a?miR-34a-NC target gene plasmid,the SIRT1 3' UTR,SIRT1 3' UTR-NC,SIRT1 3' UTR-MU reporter plasmid,and the miR-146 b,miR-146b-NC,TRAF6 3' UTR positive control plasmid,then the miRNA plasmid and the corresponding 3 'UTR reporter plasmid were co-transfected into 293 T cells,detected by Luciferase after 48 h.Data induction and statistical analysis.RPE cells with good growth status were randomly divided into 7 groups.A(normal group):5mM normal glucose culture for 8 days(4 days as 1 generation);B(early treatment group):After 6h of 25 mM high glucose culture,5mM normal glucose culture for 8 days;C(high glucose group):25mM high glucose culture for 8 days;D(metabolic memory group):After 4 days of 25 m M high glucose culture,5m M normal glucose culture for 4 days;E(negative intervention group):After 4 days of 25 mM high glucose culture,transfect the transfection reagent without miR-34 a mimic/inhibitor and continue 5mM normal glucose for 4 days;F(miR-34 a mimic group):After 4 days of 25 m M high glucose culture,transfect the miR-34 a mimic and continue 5mM normal glucose culture for 4 days;G(miR-34 a inhibitor group):After 4 days of 25 mM high glucose culture,transfect the miR-34 a inhibitor and continue 5m M normalglucose culture for 4 days.In each group,RT-qPCR was used to detect the expression of miR-34 a,immunocytochemistry and Western blotting were used to detect the protein content of VEGF and SIRT1,cell apoptosis was detected by flow cytometry.Data induction and statistical analysis.6w old SD rats(clean grade,male,200±20g weight,60) were randomly divided into five groups:A.normal group;B.early treatment group;C.diabetes group;D.metabolic memory group;E.negative intervention group;F.miR-34 a inhibitor group.The diabetic model was made by intraperitoneal injection of STZ in each groups except the normal group.After successful modeling,B group immediately began intensive insulin therapy for 12 w,subcutaneous injection with insulin in the back or the neck for each rat 2 times a day, the total amount is 6-8 IU/D,the other groups were given subcutaneous injection of normal saline;6w after successful modeling,D group,E group,F group began intensive insulin therapy for 6w;Model 6w later,E group added intravitreal injection of Lentiviral packaging empty expression vector for one time,F group added intravitreal injection of Lentiviral packaging miR-34a-5p-inhibition expression vector for one time.Intravitreal injection 1w later,make ocular frozen sections,Observed transfection of Lentiviral miR-34a-5p-inhibition into rat's retina under a fluorescence microscope.After12 w,each group were taken eyeball and retina,RT-qPCR was used to detect the expression of miR-34 a,immunocytochemistry and Western blotting were used to detect the protein content of VEGF and SIRT1,cell apoptosis was detected by Tunel.Data induction and statistical analysis.Results:Luciferase reporter gene experiment results showed that,After the co-transfection of miR-34 a plasmid and SIRT13'UTR plasmid into 293 T cells,the relative expression of SIRT1 was significantly lower than other control groups, with statistical significance.The detection results of miR-34 a,SIRT1,VEGF and cell apoptosis in RPE cells showed that:After miR-34 a mimic transfected into the cell with metabolic memory,miR-34 a expression increased(t=65.035,P<0.01),SIRT1 expression decreased(t=12.054,P<0.01);Replace with miR-34 a inhibitor,miR-34 a expression decreased(t=6.211,P<0.01),SIRT1 expression increased(t=10.437,P<0.01).Compared to the normal group,mi R-34 a increased(t=12.717,P<0.01),SIRT1 decreased(t=32.797,P<0.01),VEGF and cell apoptosis increased(t=21.751,P<0.01) in the high glucose group.There was no difference between early treatment group and normal(t=5.233,t=5.905,t=7.483,P<0.01),the SIRT1 was lower than the normal group,(t=5.247,P<0.01),but higher than the metabolic memory group(t=5.427,P<0.01) and the negative intervention group(t=7.259,P<0.01).Conclusions:1. SIRT1 was one of the target genes of miR-34 a, and the expression of SIRT1 wasdownregulated by miR-34a;2. Early hypoglycemic therapy was effective,and the latter controls sugar levels tonormal,the expressions of miR-34 a,SIRT1,VEGF and the cell apoptosis in theRPE cells which cultured by high glucose and the retina of diabetic rats could notreturn to normal levels, there was a hygerglycemic "metabolic memory"phenomenon in DR;3. miR-34 a Inhibitor downregulated mi R-34 a, through the miR-34a/SIRT1 pathwayto reduce VEGF expression and cell apoptosis.By downregulating miR-34achanged epigeneticso which partially reversed the hygerglycemic "metabolicmemory",and possibly treated DR from the molecular level.