| BackgroundDiabetic kidney disease(DKD) is commonly recognized as a leading cause of chronic kidney disease worldwide which is a common chronic microvascular complication in patients with diabetes. DKD has become the leading cause of end-stage renal disease and the prevention of DKD become a serious challenge of endocrinologists and nephrologist. The pathogenesis of DKD has not yet been fully elucidated. DKD is characterized by excessive extracellular matrix (ECM) deposition in the renal tubulointerstitium and glomerulus, and this deposition can develop into interstitial fibrosis and glomerulosclerosis. Tubulointerstitial fibrosis has been shown to be the best histological predictor of DKD progression.It is well known that myofibroblasts are important for ECM synthesis and secretion(including FN and Col-I). The EMT is a complex pathological process involving four pivotal steps:(1) loss of epithelial cell adhesion; (2) de novo a-SMA expression and actin reorganization; (3) disruption of the tubular basement membrane; and (4) enhanced cell migration and invasion. Numerous studies have demonstrated that the epithelial-to-mesenchymal transition (EMT) contributes to matrix generation in kidney disease and tubular epithelial cells and that EMT is crucial for tubulointerstitial fibrosis. Inhibiting interstitial fibrosis and renal injury would be an important way to prevent DKD and protect renal function. miRNAs are endogenously produced, small, noncoding RNAs of ～22 nucleotides, which play key roles in the posttranscriptional repression of target gene expression by binding to the 3’ untranslated regions (3’UTRs) of mRNAs. In humans, it has been found that more than 2000 miRNAs and more than 60% of human protein-coding genes could be regulated by miRNAs. miRNAs participate in the development of many diseases.Recent studies show that miR-26 and miR-30 family play an important role in multiple organ tissue fibrosis, apoptosis and other physiological and pathological processes. In idiopathic pulmonary fibrosis, miR-26a which target Lin28B then regulate expression of let-7d, inhibit pulmonary fibrosis. In cardiac fibroblast, inhibition of NF-κB restores miR-26a expression, attenuating Col-I and CTGF gene expression in the presence of Ang Ⅱ, which confer a feedback regulatory mechanism. Fluorescent in situ hybridization staining showed that miR-26a was predominantly expressed in tubular in the porcine kidney which was significantly decreased in the stenotic kidney. Decreased tubular miR-26a expression in the poststenotic kidney may be responsible for tubular cell apoptosis and renal dysfunction. miR-133 and miR-30c directly downregulate CTGF in the myocardium. miR-30 protects podocytes by targeting Notch 1 and p53 and that the loss of miR-30 facilitates podocyte injury. However, few studies describe the roles miR-26a and miR-30c in DKD, especially in renal tubular epithelial cells EMT. Here we study the roles of miR-26a and miR-30c in tubular epithelial cells.Due to their imperfect base complementarity, miRNAs can target multiple genes. In addition, a single mRNA can be targeted by one or more miRNAs. miR-143 and miR-145, which coordinately target ERBB3, inhibit cell proliferation and invasion in breast cancer. miR-455 targets Necdin, Runxltl and HIFlan signaling molecules, thereby regulating brown fat cell differentiation. Many studies have explored the role of single novel miRNAs in the pathogenesis of DKD, but few have examined the synergistic effects of miRNAs in the regulation of EMT in DKD. We hypothesized that key miRNAs may synergistically target multiple key genes to form a network for regulating the DKD. We observed that miR-26a and miR-30c were significantly decreased treated by TGFβ1 in renal tubular epithelial cells and the kidney cortex of 40-week OLETF rats. Interestingly, using computational miRNA target-site prediction algorithms, we supposed that miR-26a and miR-30c may co-target CTGF. In addition, we identified Snail1 as another potential target for miR-30c. Next, we investigated the potential role of miR-26a/30c in TGFβ1-treated NRK-52E cells and explored whether miR-26a and miR-30 could co-suppress TGFβ1-induced ERK1/2 and p38 MAPK activation. We further explored whether CTGF and Snail1 could synergistically inhibit TGFβ1-induced fibrogenesis. Herein, we show that miR-26a and miR-30c coordinate the inhibition of CTGF and Snail1 expression and consequently suppress matrix proteins in NRK-52E cells.Methods1. Cell cultureNRK-52E cells were grown in 10% FBS-DMEM with 4.5 g/L glucose. Cells were cultured in a 5% CO2 incubator at 37 ℃. Medium was replaced every 2 to 3 days.2. cell transfection1) miRNA overexpression or knockdownThe day before transfection, NRK-52E cells were seeded at a density of 3*104 cells per well in 12-well plates.50 nM miR-26a/30c mimic and 150 nM inhibitor were transfected into NRK-52E cells using Lipofectamine(?)3000. For the miRNA overexpression experiment,50 nM of miR-26a mimic,50 nM of miR-30c mimic or 25 nM each of miR-26a and miR-30c mimic in combination were used. For the miRNA silencing experiment,150 nM of miR-26a inhibitor,150 nM of miR-30c inhibitor or 75 nM each of miR-26a and miR-30c inhibitor in combination were used.2) siRNA transfectionThe day before transfection, NRK-52E cells were seeded at a density of 3*104 cells per well in 12-well plates. For this experiment,50 nM siRNA were transfected into NRK-52E cells using Lipofectamine(?) 3000. For the gene knockdown experiment,50 nM of CTGF siRNA,50 nM of Snail 1 siRNA or 25nM each of CTGF and Snail 1 siRNA in combination were used.3) plasmid and miRNA mimic cotransfection For the luciferase reporter assays, NRK-52E cells were seeded in 24-well plates, and1μg of pMIR-REPORT plasmid,0.2 μg of β-gal plasmid, and 50 nmol miR-26a or miR-30c mimic alone,25 nmol of each of miR-26a and miR-30c mimic in combination, or 50 nmol scrambled negative control RNA were co-transfected using Lipofectamine(?) 3000 in OptiMEM medium with β-gal as a transfection control.3. RNA extraction and qPCRTotal RNA was extracted from NRK-52E cells and rat kidney cortices with TRIzol. Reverse transcription of the total RNA was performed with an M-MLV kit. The gene expression levels of FN, Col-Ⅰ, E-cadherin, a-SMA, CTGF, Snail 1 and β-actin were determined using a Roche LightCycler480 Real-Time PCR System. The relative values for each gene were calculated by the comparative 2-ΔΔCt method with β-actin as an internal control.4. Western BlotEqual amount of proteins (50 μg), which were extracted from NRK-52E cells and rat kidney cortices, were electrophoresed using 8-12% SDS-PAGE gels and transferred to PVDF and blocked and then incubated with primary antibodies at 4℃ overnight. A fluorescent secondary antibody was then added and incubated with the blots at room temperature for 1 h. The images were acquired using an Odyssey infrared imaging system. Quantity One software (Bio-Rad, USA) was used for the densitometry analysis.5. In vivo studiesThe 40-week OLETF rats with spontaneous type 2 diabetes and healthy LETO control rats (n=3 for each group) used in this study were obtained from Otsuka Pharmaceutical, Tokyo, Japan. All of the experiments were approved by the local institutional review board at the authors’affiliated institution, and the animal studies were performed in accordance with the Institutional Animal Care Guidelines. Animals were housed in specific pathogen-free conditions. The animals were killed when they were 40-weeks old. Kidneys were extracted and frozen in liquid nitrogen.6. Renal histopathologyRenal tissues were collected and fixed in 4% paraformaldehyde for 24 h, and then samples were paraffin-embedded and sectioned. Tissue sections were stained with Masson’s trichrome stain. Tissue sections were also stained with anti-CTGF polyclonal antibody (1:100) or anti-Snaill polyclonal antibody (1:100), followed by HRP-conjugated secondary antibody. Finally, slices were stained with hematoxylin. All images were obtained using an Olympus microscope.7. Luciferase reporter assayCTGF 3’UTR-luciferase and Snail1 3’UTR-luciferase reporter plasmid were constructed using the pMIR-REPORT vector. Seed sequences were mutated using fast site-directed mutagenesis kit. The cells were harvested and analyzed using luciferase assay kits.8. Statistical AnalysisAll data were analyzed using SPSS 21.0. All results are presented as mean±SEM. A two-tailed Student’s t-test was used for comparisons of independent groups. One-way ANOVA was used to compare three or more independent groups, the least significant difference (LSD) test was used for multiple comparisons, and Dunnett’s T3 procedure was used for heterogeneous variances, p values less than 0.05 were considered significant.Results1. miR-26a and miR-30c expression decreased in TGFβ1-treated NRK-52E and renal cortices of diabetic OLETF ratsFirst, fibrotic markers were analyzed by employing qRT-PCR and western blotting in NRK-52E cells exposed to TGFβ1 (10 ng/ml). TGFβ1treatment for 72 h significantly augmented the expression of the myofibroblast markers a-SMA, Col-I, FN, CTGF, and Snail 1 but reduced the expression of E-cadherin, an epithelial marker compared with those in the control group. These data demonstrate that TGFβ1has the capability to induce the EMT in NRK-52E cells.We then sought to identify miRNAs that were differentially regulated in TGFβ1-treated (diabetic condition) NRK-52E cells. When examining several candidate miRNAs, we observed that miR-26a/30c was consistently down-regulated compared to the control conditions.To explore the in vivo correlation between miR-26a/30c and diabetic nephropathy, we compared the expression of CTGF/Snaill and miR-26a/30c in the renal cortices of 40-week OLETF rats (diabetic) and LETO rats (non-diabetic). Masson staining revealed more extensive tubular interstitial collagen deposition in the OLETF group, which confirmed that the 40-week OLETF rats exhibited tubulointerstitial fibrosis. Immunohistochemistry and WB results showed that CTGF and Snail1 expression significantly increased in renal tubules and interstitial areas in OLETF rats compared with LETO rats. These changes paralleled the decrease in the expression of miR-26a and miR-30c in the kidney cortex of OLETF rats compared with the levels observed in non-diabetic LETO rat controls.2. miR-26a and miR-30c target CTGF and Snail1 and synergistically regulated the TGFp 1-induced EMT1) miR-26a and miR-30c directly target CTGF and miR-30c targets Snail 1a) A computational program from TargetScan was used to show that the 3’UTRs of CTGF have two predicted binding sites for miR-26a and miR-30c. In addition, the 3’UTRs of Snail 1 has one predicted binding site for miR-30c. Luciferase reporter gene assay was used to comfirm miR-26a and miR-30c directly target the 3’UTRs of CTGF and miR-30c directly target the 3’UTRs of Snail 1.b) In the presence or absence of TGFβ1, CTGF mRNA and protein levels were markedly reduced in the treatment with either miR-26a or miR-30c mimic alone; treatment with a combination of miR-26a and miR-30c mimics strengthened the suppressive effects on CTGF compared with the treatment with either miR-26a or miR-30c mimic alone. Furthermore, for the transfection with a miR-30c mimic alone, we observed a decrease in both Snail 1 mRNA and protein expression.2) miR-26a and miR-30c synergistically regulated the TGFβ1-induced EMTa) We investigate the synergistic effect of miR-26a and miR-30c overexpression in NRK-52E cells treated with TGFβ1. After TGFβ1 treatment, FN, Col-I and a-SMA expression was markedly reduced in the treatment with either miR-26a or miR-30c mimic alone; moreover, co-transfection of NRK-52E cells with miR-26a and miR-30c mimic in combination significantly inhibited the EMT compared with transfection with either miR-26a or miR-30c alone, as determined by mRNA and protein levels. E-cadherin showed the opposite trend.b) We performed a loss-of-function study by knocking down either miR-26a or miR-30c alone or in combination using miRNA inhibitors. However, the TGFβ1-mediated EMT was enhanced by miR-26a and miR-30c inhibitor alone; furthermore, co-inhibition with both miR-26a and miR-30c inhibitors showed a stronger pro-fibrotic effect.3. The potential mechanism of the miR-26a and miR-30c co-regulation of TGFβ1-induced EMT in NRK-52E cells.1) Recent studies have proposed that CTGF activates the ERK1/2 and p38 MAPK signaling pathways. Therefore, miR-26a and miR-30c might synergistically inhibit CTGF and then restrain the phosphorylation activity of the ERK1/2 and p38 MAPK signaling pathways.2) Furthermore, as expected, activation of the ERK1/2 and p38 MAPK signaling pathways due to the TGFβ1 treatment was markedly attenuated by both miR-26a and miR-30c mimic alone. Furthermore, co-treatment with the combination of miR-26a and miR-30c mimics further enhanced the inhibitory effect. Hence, miR-26a and miR-30c effectively and synergistically inhibited the TGFβ1-triggered activation of MAPKs in TGFp1-treated NRK-52E cells.In addition, CTGF and Snail 1 are important transcription factors that trigger the EMT in DKD. Previous studies reported that CTGF and Snail 1 silencing were able to inhibit EMT and fibrosis in the kidney. Thus, determining whether CTGF and Snail 1 collaboratively suppress the TGFβ1-induced EMT was also important for explaining the potential mechanism of the miR-26a/30c co-regulation. First, the role of the interaction was investigated. Gene expression analysis showed that silencing of CTGF or Snail 1 alone could not prevent their interaction.Furthermore, silencing of CTGF or Snail 1 alone could markedly suppress TGFβ1-induced EMT, as determined by the mRNA levels. However, compared with treatment with either siCTGF or siSnaill alone, co-treatment with both siCTGF and siSnaill (each at half dose) did not markedly inhibit EMT at the mRNA level. These data suggest that CTGF and Snail 1 are independent transcription factors mediating EMT in NRK-52E cells.Conclusion1. miR-26a and miR-30c expression decreased in TGFβ1-treated NRK-52E cells and the renal cortices of 40-week OLETF rats2. Luciferase reporter gene assay demonstrated that miR-26a and miR-30c target CTGF and miR-30c targets Snail 1. Treating cells with a combination of miR-26a and miR-30c mimic strengthened the suppressive effects on CTGF mRNA and protein levels compared with that for the treatment with either miR-26a or miR-30c alone. Furthermore, we observed a decrease in both Snail 1 mRNA and protein expression when treated with a miR-30c mimic.3. miR-26a and miR-30c mimic synergistically suppressed the TGFβ-1-stimulated EMT in NRK-52E cells. miR-26a and miR-30c inhibitor coordinately enhanced the TGFp 1-induced EMT in NRK-52E cells.4. miR-26a and miR-30 co-suppress TGFβ1-induced ERK1/2 and p38 MAPK activation.5. CTGF and Snail1 are independent transcription factors that they could not influence each other. Treatment with either siCTGF or siSnaill alone, significantly inhibited the EMT. Compared with either siCTGF or siSnaill alone, co-treatment of cells with both siCTGF and siSnaill could not significantly inhibited the EMT in NRK-52E cell. |
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