| BACKGROUNDDiabetic nephropathy (DN) is one of the most common complications of diabetes. In 2013, a study published in the latest Journal of the American Medical Association showed that in Chinese adults the mortalityof diabetes and pre-diabetes has reached 11.6%and 50.1%, respectively. Edema and microalbuminuria are early symptoms of DN, The common pathological changes of DN are glomerular hypertrophy, glomerular basement membrane thickening, basement membrane-like material proliferation in mesangial area, and eventually developmentof glomerulosclerosis, fibrosis. Clinically in patients with DN, once persistent proteinuria occurred, the kidney injury is irreversible, until progression to end stage renal failure. DN has become a major cause of death of diabetic patients, especially young patients, therefore, it is urgent and valuable to study the pathogenesis and clinical treatment method for DN. However, the molecular mechanisms of DN is not yet fully understooddue to the complexity of the etiology.MicroRNAsare endogenous, non-coding small RNA molecules, with about 20-24 nucleotides in length, extensively existed for regulation of gene expression. Recent studies found that the development and occurrence of DN is closely related to the microRNA-directed gene expressionregulation. The study on the relationship of DN and microRNAs may generatenovel concept for understanding the development of DN, and may provide new biomarkers for early diagnosis, new targetsfor treatment and intervention of DN.The present studydevoted to the difficult and key spots in endocrine diseases,the molecular mechanisms of DN.Animal models of DN in mice and mouse mesangial cells cultivated with high-low level of sugar were used.The differential expression profile of microRNAs in DN,the molecular mechanisms of the interplay of miR-346 and TGF-β/Smad signaling pathway in the development of DN were investigatedin detail, using gene microarray, RT-PCR, Western-Blotting, immunohistochemistry methods, and etc..Exogenous miR-346 was used to disclosethe effects and possible mechanisms of microRNA on the progression of DN.The results of this study provided profoundtheoretical basisfor the early specific diagnosis and treatmentof DN.PART ONEMicroarray-based analysis of microRNAs in renal cortex of diabetic nephropathy mice(db/db mice)Objective To study the differential expression profile ofmicroRNAs (miRNAs) 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 Six 4-week-old male db/db mice with hyperglycemia and significant elevated urinary albumin excretion were used as animal model of the early stage of DN, and six 8-week-old male db/m mice were used as controls. Total RNA were extracted from the kidney tissues of mice using Trizol reagent. miRNAs differential expression profile in DN and normal controls were assayed by miRMouse miRNA array contained 1181 probes. Real-time RT-PCR was performed on the db/db DN mice and db/m controls for partial differential expression microRNAs. The relative levels of miRNA expression was normalized to the expression level of snoRNA202.Results 4-week-old male db/db mice with hyperglycemia and significant elevated urinary albumin excretion showed the features similar to the stage of diabetic nephropathy. Seventy-three miRNAs were differentially expressed more than 4 times between DN group and control group. Of these,39 were overexpressed and 34 were underexpressed in DN.Eight upregulated (miR-29c, miR-192, miR-216a, miR-17, miR-377, miR-196a, miR-166 and miR-98) and ten down-regulated miRNAs species (miR-215, miR-451, miR-200a, miR-200b, miR-200c, miR-141, miR-21, miR-346, miR-709, and miR-187) were examined by RT-PCR. As anticipated, RT-PCR confirmed the differences in expression of these miRNAs in the kidney tissues ofmice in groups DN and controls. All knownmRNA 3’-UTRs were scanned as potential targets for miR-346 by in silico analysis (Pictar,Targetscan, miRNA-base. miRNA-anda,et al.). We found that Smad 3/4 are target genes of miR-346.Conclusion Some miRNAs (including miR-346) expressed differently between DN and control groups. suggesting that they may be involved in the pathogensis of DN.PART TWOImpact of miR-346 on TGF-p-mediated Smad signaling pathway in the pathogenesis of diabetic nephropathyObjective To investigate the effect of miR-346 on TGF-β/P53 signal pathway in mouse model of diabetic nephropathy and mesangial cells.To explore the molecular mechanism of miR-346 in the occurrence and development of diabetic nephropathy.Methods Six 4-week-old male db/db mice with hyperglycemia and significant elevated urinary albumin excretion were used as animal model of the early stage of DN, and six 8-week-old male db/m mice were used as controls. Mesangial cells were treated with TGF-β anlone or incombination with P53 specific siRNA. The expression levels of TGF-β, P53, miR-346 and Smad3/4 were detected by RT-PCR and Western blotting.Results RT-PCR and Western blot analysis showed that the expression levels of TGF-β, P53 and Smad3/4 in db/db group were significantly higher than those in normal control group, while the expression level of miR-346 was significantly lower in DN group than that in the normal control group. TGF-P treatment led to up-regulation of P53 and Smad3/4 in mouse mesangial cells, on the contrary, interference on TGF-β led todown-regulation of P53 and Smad3/4. While the expression of miR-346 was significantly upregulated after interference of TGF-β. Transfectionof mouse mesangial cells with miR-346 plasmid led to significantly down-regulation of both the mRNA and protein levels of Smad3 and Smad 4, which could be blocked by treatment with recombinant TGF-β.Conclusion TGF-P can reduce the expression of miR-346 by upregulating the expression of P53, and indirectly upregulate the expression of intracellular Smad3/4, therefore contribute to the occurrence and development of diabetic nephropathy.PART THREETargeting treatment with miR-346for diabetic nephropathy in miceObjective To investigate the role of miR-346 inthe pathogenesis of diabetic nephropathy, and the effect ofmiR-346 on the morphology of glomeruli and TGF-β/P53 signal pathway in vivo. To explore the fesibility of miR-346 as a novel therapeutics for DN.Methods Eighteen 4-week-old male mice were randomized into 3 groups:miR-346 treated db/db group(6 mice), control empty plasmid treated db/db group (6 mice), or untreated db/db group (6mice). Mice were injected intravenously from tail vein using a hydrodynamics-based procedure with plasmids (30mg/kg/d of miR-346 or 30mg/kg/d of control plasmid) in 8-week-old mice for 4 weeks. The mRNA and protein levels of TGF-P, P53, and Smad3/4 were tested by real-time RT-PCR and Western blotting, respectively.Results There were no significant differents amoung treatment group, empty plasmid group, and the control group in the contex of average body weight and random blood sugar, at week 4,8,12, and 16. At week 12 and 16, the levels of UAE in the treatment group (108.96,82.14μg/24h) were much lower than those in control empty plasmid group and control group (P<0.05).At week 16, the kidney tissue from treatment group showed much less mesangioproliferation and glomerular injury than those from control empty plasmid group and control group. Compared with control empty plasmid treated db/db group and untreated db/db group, the mice in the miR-346 treated group showed much lower levels of mRNA and protein of Smad3/4 (P<0.05), with similar leves of mRNA and protein of TGF-β and P53 (P>0.05).Conclusion miR-346 could specifically inhibit mesangial proliferation through regulation the Smad3/4 signaling pathway, and maight be a novel potential therapeutic target for diabetic nephropathy.SUMMARYThis study took vast efforts to interpret the molecular mechanisms of DN, the microRNA differential expression profiles of DN were disclosed, the molecular mechanisms of the interplay of miR-346 and TGF-β/Smad signaling pathway in the development of DN were investigatedin detail.The results of this study provided profoundtheoretical basisfor the new treatmentconcept of DN by targeting at miR-346. In this study, the differential expression profiles of microRNAs, especially miR-346 in DN were uncovered by using gene microarray combined with quantitative real-time RT-PCR validation.The involvement of microRNAs in the molecular pathogenesis of DN was confirmed, and Smad3/4 were found as a target genes of miR-346 by bioinformatics prediction technology. The results of real-time quantitative RT-PCR and Western blotting indicated that the TGF-β may specifically down-regulate the expression of miR-346 through P53, indirectly up-regulate the intracellular expression of Smad3/4,therefore to promote the occurrence and development of DN.Thepresent study showed that administationDN mice with exogenous miR-346 can specifically regulatethe Smad3/4signaling, leading to the improvement of clinical symptoms and pathological injury, Therefore, miR-346 may be a new potential therapeutic target for DN,which provides a novelconcept for the specific treatment of DN. |
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