Micrornas The Targeted Pten/pi3k Signaling Pathways Regulation Of Early Diabetic Nephropathy

Objective To explore the microRNA (miRNA) differential expression profile between the kidney tissues of db/db mice with diabetic nephropathy (DN) and the kidney tissues of db/m control mice, and to provide the evidence that miRNAs are involved in the molecular pathogensis of DN.Methods Twelve 9-week-old male db/db mice with hyperglycemia and significant elevation of urinary albumin excretion were used as animal model of the early stage of DN, and twelve 9-week-old male db/m mice were used as controls. Half of mice in each group were used in miRNA array, and the other half were used in real-time RT-PCR. mRNA was extracted from the kidney tissues of db/db mice and the db/m controls using Trizol reagent. MicroRNA differential expression profile in DN and normal controls were assayed by miRMouse₁0.0₀71620 miRNA array. Real-time RT-PCR was performed by TaqMan? MicroRNA Assays kits on the db/db DN mice and db/m controls. The relative expression was calculated using theΔΔCT method and normalized to the expression of snoRNA202. Results 9-week-old male db/db mice with hyperglycemia and significant elevation of urinary albumin excretion showed features similar to the early stage of diabetic nephropathy. We first examined the expression of 568 miRNA species based on version 10.0 of the Sanger miRBase (http://microrna.sanger.ac.uk/sequences) by miRNA microarrays. 480 miRNAs were expressed in DN group, and 214 miRNAs were expressed in control group. Sixty-six miRNAs were differentially expressed with P<0.01. Of these, 35 were overexpressed and 31 were underexpressed in DN. The changes with P<0.01 ranged from 1.1-fold to 10.2-fold. Three upregulated (miR-196a, miR-98 and miR-29c) and four down-regulated miRNA species (miR-21, miR-451, miR-709 and miR-187) were examined. As anticipated, real-time RT-PCR confirmed the differences in expression of these miRNAs in the kidney tissues of mice in groups DN and controls.Conclusion Some miRNAs expressed differently between DN and control groups, suggesting that they may be involved in the pathogensis of early DN. PART II IDENTIFICATION AND TARGET PREDICTION OF MIR-21Objective To identified the enkaryotic expression vector of miR-21 on the basis of the finding that miR-21 was a diabetic nephropathy related microRNA by microRNA microarray and real-time RT-PCR analysis. And to predicted the target gene of miR-21. To provide evidence for the potential role of miR-21 in DN.Methods Mir-21 was chemically synthesized and contained a Sac I restriction enzyme cut site. After annealing, the DNA segments were cloned into pGenesil-1 expression vectors and confirmed by DNA sequencing. bioinformatics approach was used to identify potential candidate genes of miR-21.Results The synthetic DNA was of the correct sequence and inserted into eukaryotic expession vector pGenesil-1 successfully. All known mRNA 3'-UTRs were scanned as potential targets for miR-21 by in silico analysis (Pictar, Targetscan, MiRbase, MiRanda, et al.). It was predicted phosphatase and a tensin homolog deleted from chromosome 10 (PTEN) gene, which contained the binding site of miR-21 in its 3'UTR, could be regulated by miR-21. With literature search, PTEN gene, a DN related gene was found a dirct target of miR-21 by luciferase report system. Therefore, PTEN gene was established the target gene of miR-21.Conclusion The identification of pGenesil-miR-21 clone plasmid and the primary establishment that PTEN gene being the target prediction of miR-21 provide basis for the study of the molecular mechanism of miR-21 in DN. PARTâ…¢EFFECT OF MIR-21 ON MESANGIAL CELL PROLIFERATION INDUCED BY HIGH GLUCOSE BY PTEN/PI3K SIGNAL PATHWAYObjective To explore the effect of miR-21, the kidney specific microRNA on the cell growth and proliferation of the mesangial cell cultured with high glucose, and to study the regulation of high-expressed miR-21 on PTEN/PI3K signal pathway in mesangial cell cultured with high glucose. To provide the evidence for the molecular mechanism of miR-21 in mesangial cell proliferation induced by high glucose.Methods Mesangial cells were maintained with high and low glucose to mimic diabetes mellitus and normal conditions. The expression of the enkaryotic expression vector of miR-21 (pGenesil-miR-21) was induced by transfection of the plasmid into the cells using Lipofectamine 2000, and the cells in the study was divided into 4 groups: cells in low glucose as the control group, a pGenesil-miR-21-transfected high glucose group, control empty plasmid transfected high glucose group, and untreated high glucose group. The cell proliferation was tested by MTT, the expression of miR-21 level was tested by real-time RT-PCR, the expression of PTEN, p-Akt (Ser 473) and PI3K p85αprotein levels were examined by western blot and immunohistochemistry.Results Real-time RT-PCR showed that miR-21 was inceased in the miR-21-transfected group, suggesting miR-21 can be overexpressed by Lipofectamine 2000 transfection. MTT showed that miR-21 prevents the cell proliferation of mesangial cells cultured with high glucose. By western blot and immunohistochemistry, we found PTEN protein was reduced in the cells in the miR-21-transfected group compared with the cells in the control empty plasmid transfected group and untreated group (all: p<0.01). Whereas PI3K p85αand phospho-Akt (Ser 473) were increased in the cells in miR-21-transfected group than the control empty plasmid group and the untreated group (all: p<0.01).Conclusion MiR-21 may prevents mesangial cells proliferation reduced by high glucose through decreasing PTEN protein expression and increasing PI3K p85αand phospho-Akt (Ser 473) protein expressions, and it may be a novel potential DN preventative and therapic target. PARTâ…£MIR-21 REGULATES EARLY DIABETIC NEPHROPATHY BY PTEN/PI3K PATHWAY IN DB/DB MICEObjective To explore the effect of miR-21, the kidney specific DN related microRNA on the mesangial hypertrophy of diabetic nephropathy db/db mice, and to study the regulation of high-expressed miR-21 on changes of 24-h urine albumin excretion (UAE), the morphology of glomeruli, PTEN/PI3K signal pathway in db/db mice with albuminuria. To provide the evidence for the molecular mechanism of miR-21 in DN.Methods 24 5-week-old male mice were randomized into 4 groups: control group (6 normal untreated db/m mice), miR-21-treated db/db group (6 mice), control empty plasmid treated db/db group (6 mice), or untreated db/db group (6 mice). Mice were injected intraperitoneally using a hydrodynamics-based procedure with plasmids (30mg/kg/d of miR-21 or 30mg/kg/d of control plasmid) until albuminuria was detected in the untreated db/db mice. The glomeruli were observed under the light and electron microscope, and measured by the software in the computer. The expression of miR-21 was tested by real-time RT-PCR, the expression of PTEN, p-Akt(Ser 473) and PI3K p85αprotein levels were examined by western blot, immunohistochemistry and immunofluorescence staining.Results 9-week-old male db/db mice with hyperglycemia and significant elevation of urinary albumin excretion showed features similar to the early stage of diabetic nephropathy. Real-time RT-PCR showed that miR-21 was inceased in the miR-21-treated group, suggesting miR-21 can be overexpressed by injected plasmids intraperitoneally using a hydrodynamics-based procedure. By measurement of glomeruli acreage, the glomeruli was smaller in miR-21-treated group than in the control empty plasmid treated and untreated groups (all: p<0.01). Additionally, miR-21 restrained mesangial hypertrophy under electron microscope. By western blot, immunohistochemistry, PTEN were reduced in the kidneys in the miR-21-treated group compared with the control empty plasmid treated group and untreated group (all: p<0.01). Whereas PI3K p85αand phospho-Akt (Ser 473) were increased in the kidneys in miR-21-treated group than the control plasmid treated and the untreated groups (all: p<0.01). Moreover, by dual-fluorescence staining, PI3K p85αand phospho-Akt (Ser 473) were found to be co-increased in miR-21-treated group.Conclusion MiR-21 protects from mesangial cell proliferation induced by diabetic nephropathy in db/db mice, and it may be a novel DN protecting factor.