| Background and objectiveDiabetic nephropathy (DN) is a serious microvascular complication of diabetes, aswell as a major cause of end stage renal disease (ESRD). So far because the mechanismsinvolved in the process of DN remain unknown and the effective treatment methods arelacking, the prevention and cure of DN have become the urgent need. Increasing studieshave shown that, multifaceted metabolic and signaling events following excessivechanneling of glucose might contribute to the pathogenesis of DN, however, existingclinical treatment could not effectively inhibited these events. MicroRNAs (miRNAs) are aclass of endogenous non-coding small RNAs that modulate gene expression via binding tothe3’-untranslated region (3’-UTR) of target mRNAs and thereby induce mRNAdegradation or block protein translation at the post-transcriptional level. miRNAs arewidely involved in diverse physiological and pathological processes and are emerging asimportant mediators in kidney health and disease, Moreover, numerous reports havedescribed that dysregulation of miRNAs is closely linked to progressive fibrosis of DN,however, the mechanism by which miRNA contribute to the pathology of diabeticnephropathy is not fully defined. On these grounds, the present study propose to investigatethe renal expression changes of the kidney specific miRNA microRNA-215(miR-215) andits target gene CTNNBIP1in DN of type2diabetic db/db mice. Moreover, a luciferasereporter assay was used to determine CTNNBIP1was a direct target of miR-215. Finally,the study explores the role of miR-215in the development and progression of DN.Subjects and methods1.Determination of miR-215expression changes in db/db mice in vivo and MMCscultured with high glucose in vitro1) Male4-week-old C57BL/KsJ db/db mice, an animal model representative of type2diabetes, and age-matched nondiabetic db/m littermates as normal controls group. At theage of4,8,12, and16weeks, body weight (BW), blood glucose(Glu), urinary albumin excretion (UAE) of the two groups of mice were detected; Renal pathological changes ofmices were observed by using special staining of periodic acid-schiff (PAS). These resultswere used to evaluate whether the db/db mice developed kidney disease in the setting ofsevere hyperglycemia.2) The expression changes of miR-215in db/db mice at the age of8,12and16weeksand in MMCs cultured with high glucose (30mmol/L) for6，12，24，48hours were detectedby real-time quantitative PCR (qRT-PCR).2. Determination of the target gene of miR-2151) To investigate the role of miR-215in the pathogenesis of DN, we focused ourattention on the potential mRNA targets of miR-215. The computational target predictiontools TargetScan (http://www.targetscan.org), PicTar (http://pictar.mdc-berlin.de) andmiRanda (http://www.microrna.org) were used to assess potential targets sites for miR-215,and the results were compared. Target gene annotations available in the Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were utilized toillustrate the most statistically significant target gene that may be regulated by miR-215wascatenin beta interacting protein1(CTNNBIP1).The RNA Hybrid program was used topredict the secondary structure of the CTNNBIP13’-UTR/miR-215duplex.2) To test whether CTNNBIP1is a potential target gene of miR-215, we firstdetermined the mRNA and protein expression levels of CTNNBIP1in renal glomeruli fromdb/db mice at the age of8,12, and16weeks by qRT-PCR, western blot andimmunohistochemistry, respectively.3) To determine whether miR-215directly regulates CTNNBIP1expression bytargeting the3’-UTR of CTNNBIP1mRNA, luciferase reporter constructs were generated,and luciferase assays were performed to identify that miR-215can negatively regulateCTNNBIP1gene by targeting its3’-UTR sequence.4) Gain-and loss-of-function approaches were used to demonstrate that miR-215actsas a regulator of the CTNNBIP1/β-catenin pathway by transfecting MMCs with an miR-215mimic or an miR-215inhibitor.Results1. Dynamic alteration of BW, Glu and UAE levels in db/db miceAt the age of4weeks, the blood glucose concentration and UAE were not significantly different between the two groups of mice. The BW, Glu and UAE of the db/db mice weresignificantly increased compared with db/m mice at the age of8weeks, suggesting that DNin db/db mice begins at8weeks of age. In addition, with the progressive hyperglycemia ofthe db/db mice from the ages of8to16weeks, the degree of albuminuria also consistentlyincreased. These results indicated that DN in db/db mice showed a trend of rapiddevelopment as time go on.2. Dynamic alteration of renal pathology in db/db miceGlomerular and tubular lesions not detected in db/m mice at any age. At the age of4weeks, the glomerular lesions were not obvious in db/db mice. At the age of8weeks,glomerular hypertrophy began to emerge. At the age of12weeks, mesangial cellsproliferation was prominent in the renal glomeruli of db/db mice. Furthermore, typical renalpathological changes, including glomerular basement membrane thickening, mesangialmatrix expansion and diffuse nodular mesangial sclerosis lesions similar to theKimmelstein-Wilson nodules described in advanced human DN were present in tissuestions obtained from16-week-old db/db mice. These results further determined that DN indb/db mice showed a trend of rapid development as time go on.3. Dynamic alteration of renal miR-215in db/db miceCompared with the db/m mice, renal special miR-215was highly expressed in db/dbmice at8,12and16weeks of age, furthermore, with the growth of db/db mice, theexpression levels of miR-215was gradually increasing and with a time-dependent manner.This indicated that the renal miR-215expression showed a trend of progressive increasingas the development of DN in db/db mice. Moreover, we also tested the effect of highglucose on miR-215expression in cultured MMCs. We observed a striking induction ofmiR-2156hours after stimulation, with levels gradually increasing and peaking48hoursafter stimulation. These data convincingly showed that miR-215is highly upregulated indiabetic conditions，and plays an important role in the pathogenesis of DN.4. miR-215target prediction and functional annotationThe computational target prediction tools TargetScan (http://www.targetscan.org),PicTar (http://pictar.mdc-berlin.de) and miRanda (http://www.microrna.org) were used toassess the targets for miR-215，selected the target genes that three calculations can predictas the potential targets of miR-215，including Alcam，Blcap，Bhlhe22，Ctnnbip1，Cdc6， Pkp4，Wnk1，Msn；Gene annotations available in the Gene Ontology (GO) and KyotoEncyclopedia of Genes and Genomes (KEGG) databases revealed that CTNNBIP1(cateninbeta interacting protein1) is a novel β-catenin interacting protein, which can negativelyregulate wnt signaling via inhibition of the interaction between β-catenin and TCF/LEFfamily members，suggesting that CTNNBIP1may be a renal specific target of miR-215. Asdetermined by RNA Hybrid analysis, we found that miR-215and its binding site inCTNNBIP13’-UTR could potentially form a very stable secondary structure.The minimumfree energy predicted for hybridization with the CTNNBIP13’-UTR and miR-215at thissite was Δ G=17.3kcalmol1, consistent with an authentic miRNA targeting. Moreover，the3’-UTR of the CTNNBIP1gene is perfectly complementary to the seed region ofmiR-215. Thus, we hypothesized that CTNNBIP1might serve as a target for miR-215inthe diabetic milieu.5. Dynamic alteration of renal CTNNBIP1in db/db miceCompared with the db/m mice, renal mRNA expression of CTNNBIP1was significantdecreased in db/db mice at8,12and16weeks of age. With the growth of db/db mice, theexpression levels of CTNNBIP1was consistently down-regulated and with atime-dependent manner. Consistent with the mRNA expression, a parallel decrease inCTNNBIP1protein levels in the glomeruli of diabetic mice at8,12and16weeks of age.We observed that CTNNBIP1was markedly expressed in the mesangial area of renalglomeruli, moreover, a significant reduction in CTNNBIP1protein levels in renal glomeruliof db/db mice at12weeks of age relative to the control group. These results indicated thatthe renal CTNNBIP1expression showed a trend of progressive decreasing as thedevelopment of DN in db/db mice, the expression of miR-215has a negative correlationwith the CTNNBIP1level, suggesting that high expression levels of miR-215downregulates the expression of CTNNBIP1in the progression of DN.6. The direct target gene of miR-215is CTNNBIP1To verify CTNNBIP1is a direct target gene of miR-215, the mouse CTNNBIP13’-UTR was amplified by PCR from mouse genomic DNA, digested with SpeI and HindIIIrestriction enzymes, and ligated into the multiple cloning site of the pMIR-REPORTluciferase vector to yield pMIR-CTNNBIP13’-UTR, pRL-TK as a control vector.Luciferase activity was measured using the Dual-Luciferase Reporter Assay System and was normalized to the expression of the control TK Renilla construct (pRL-TK). weobserved a marked decrease in the luciferase activity of the CTNNBIP13’-UTR reporter inMMCs transfected with an miR-215mimic relative to the negative miR-control. Theseresults demonstrated that the CTNNBIP13’-UTR sequence is recognized by miR-215andthat CTNNBIP1is a direct target of miR-215.7. The changes of CTNNBIP1protein expression and β-catenin activity in MMCstransfected with miR-215mimic/inhibitorTo determine the regulated functions of the renal specific miR-215in theCTNNBIP1/β-catenin pathway, the protein expression of CTNNBIP1and β-catenin activitywere examined by western blot in MMCs transfected with an miR-215mimic or anmiR-215inhibitor. Compared with the miR-control group, CTNNBIP1protein productionwas significantly downregulated and β-catenin activity was markedly elevated in MMCstransfected with the miR-215mimic, In contrast, the miR-215inhibitor significantlyupregulated the level of CTNNBIP1and downregulated the level of β-catenin activity.These results indicated that miR-215negatively regulates β-catenin pathway via targetingCTNNBIP1, and participates in the pathogenesis of DN.8. The effect of miR-215on mouse mesangial cellular proliferationTo determine the effect of the renal specific miR-215on the cellular proliferation, theproliferative activity was examined by MMT in MMCs transfected with an miR-215mimicor an miR-215inhibitor. Compared with the miR-control group, the proliferation activitywas significantly increased in MMCs transfected with the miR-215mimic. In contrast, themiR-215inhibitor markedly downregulated the cellular proliferation activity.These resultsindicated that miR-215could promote the proliferation activity of MMCs by regulating theCTNNBIP1/β-catenin pathway．Conclusions1. The kidney specific miR-215was highly expressed in db/db mice and highglucose-cultured MMCs with a time-dependent manner, and participates in thepathogenesis of diabetic nephropathy.2. Luciferase reporter assays further revealed that CTNNBIP1is a direct target ofmiR-215as predicted by the bioinformatic analysis. Moreover，CTNNBIP1is a novelβ-catenin interacting protein, which can negatively regulate wnt signaling via inhibition of the interaction between β-catenin and TCF/LEF family members.3. CTNNBIP1expression in the glomeruli of diabetic mice at both the mRNA andprotein levels was consistently downregulated and with a time-dependent manner,suggesting that the expression of miR-215has a negative correlation with CTNNBIP1level,miR-215overexpression in MMCs can significantly downregulate the expression ofCTNNBIP1and promote the level of β-catenin activity, in contrast, attenuation of miR-215can markedly upregulate the expression of CTNNBIP1and inhibit the level of β-cateninactivity in MMCs.Accordingly, our data implicated the potential role for high expressionlevels of miR-215in the initiation and progression of DN by downregulating the expressionof CTNNBIP1．... |
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