TGF-β1 stimulation and miR-133b transfection
The HK2 cells were cultured in the Dulbecco’s modified Eagle medium (DMEM)/F12 (Corning, USA) supplemented with 5% fetal bovine serum. After reaching 50% confluency, the cells were synchronized in serum-free DMEM/F12 for 18 h and then stimulated with TGF-β1 at 6, 8, and 10 ng/mL concentrations for 24, 48, and 72 h. miR-133b mimic and miRNA mimic control (GenePharma, China) were transfected into HK2 cells for 6 h using the jetPRIME® transfection reagent according to the manufacturer’s instructions (Polyplus-transfection, France). Following transfection, the cells were cultured in DMEM/F12 with 5% serum for 18 h and then incubated with DMEM/F12 with 5% serum and 8 ng/mL of TGF-β1 (PeproTech, USA) for 48 h.
RNA extraction and real-time polymerase chain reaction (RT-PCR)
Total RNA was extracted from HK2 cells and kidney tissues of each group using Trizol, and subsequently used to synthesize miR-133b and U6 cDNA using the miScript II RT Kit (QIAGEN, China). Primers specific for target genes were designed with reference to their mRNA-coding regions in GenBank using the Primer 5.0 software. The primer sequences were verified using BLAST. Total RNA was used to synthesize cDNA of target genes using the ReverTra Ace qPCR RT Master Mix kit (TOYOBO, Japan). The expression of genes encoding miR-133b, CTGF, E-cadherin, α-smooth muscle actin (SMA), fibronectin, collagen 3A 1 (Col3A1), U6, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected on the ABI-prism-7500 sequence detection system (Applied Biosystems, USA) using the miScript SYBR Green PCR Kit (QIAGEN). The relative expression levels were calculated using U6 or GAPDH as internal control.
Western blot analysis
After lysis and denaturation of HK2 cells or kidney tissues from each group using the radioimmunoprecipitation assay (RIPA) buffer, proteins (50 μg) were separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose (NC) membranes. After blocking with 1x casein for 1 h to prevent non-specific binding, NC membranes were incubated with the following primary antibodies overnight at 4°C : (a) rabbit monoclonal E-cadherin antibody (BD Bioscience, USA) diluted 1:100; (b) rabbit monoclonal anti-α-SMA antibody (Abcam, UK) diluted 1:300; (c) mouse monoclonal anti-CTGF antibody (Abcam) diluted 1:200; (d) rabbit polyclonal anti-fibronectin antibody (Proteintech, USA) diluted 1:500; (e) rabbit polyclonal Col3A1 antibody (Proteintech) diluted 1:500; (f) mouse monoclonal β-actin antibody (Beyotime, China) diluted 1:10000. The membranes were washed with TBST (Tris-buffered saline with Tween-20, 20 mM of Tris, 140 mM of NaCl, and 0.1% Tween-20) and then probed with 1:1000 diluted secondary antibodies at room temperature (25ºC) for 2 h. Enhanced chemiluminescence (ECL) western blotting kit (APPLYGEN, China) was used to detect the target bands, and β-actin was used as an internal reference to calculate the relative expression levels of proteins in each experimental group.
Prediction of target genes of miR-133b
The target genes of miR-133b were predicted using three commonly employed target gene prediction software, namely TargetScan (http://www.targetscan.org/), miRBase (http://www.mirbase.org/), and PicTar (https://pictar.mdc-berlin.de/).
Dual-luciferase reporter assay
The seed sequence for the binding of CTGF and miR-133b was searched using the bioinformatics software TargetScan. The sequence 5¢-AUUUGUUGAGUGUGACCAAAA-3¢ containing the 3¢-untranslated region (UTR) of CTGF was synthesized and cloned into a luciferase reporter vector GP-miRGLO (GenePharma), and termed as miRGLO-Wt-CTGF. A mutant sequence 5¢-AUUUGUUGAGUGUUGGAUUAA-3¢ of the target was also synthesized and cloned into the plasmid to obtain miRGLO-Mut-CTGF, which was used as the negative control.
293T cells from the logarithmic growth phase were lysed with pancreatin and seeded in 48-well plates for 24 h. After reaching 80% confluency, the cells were transfected using the cell fusion reagent. The synthesized miR-133b mimic and miRNA mimic control (NC-miR) were co-transfected with miRGLO-Wt-CTGF or miRGLO-Mut-CTGF, respectively, into 293T cells using the jetPRIME® transfection reagent according to the manufacturer’s instructions. After 48 h, the cells were lysed using a passive lysis buffer (Promega, USA) and the cell lysate was collected. The luciferase activity of the lysate was detected according to the instructions indicated in the dual-luciferase reporter assay system kit (Promega).
Immunofluorescence staining
HK2 cells were seeded at a density of approximately 105 cells/well in 6-well plates with sterile glass coverslips which were disinfected by autoclaving. The plates were placed in an incubator at 37°C with 5% carbondioxide for 6 h to allow the cells to adhere to the glass cover slips. After synchronization and transfection as mentioned above, the cells were incubated with DMEM/F12 containing 5% serum with or without 8 ng/mL of TGF-β1 for 48 h. The cells were then fixed with 4% paraformaldehyde at room temperature for 20 min and treated with 0.2% Triton X-100 for 2 min for permeabilization. The cells were then blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h and then treated with anti-E-cadherin rabbit monoclonal primary antibody (1:100 dilution), α-SMA antibody (1:100 dilution), rabbit polyclonal Col3A1 antibody (1:100 dilution), fibronectin antibody (1:100 dilution) and mouse monoclonal anti-CTGF antibody (1:100 dilution) diluted in 5% BSA at 4°C overnight. After washing with phosphate-buffered saline (PBS), the cells were probed with an anti-rabbit fluorescein isothiocyanate (FITC)-conjugated fluorescent secondary antibody (Beyotime) (1:400 dilution) at room temperature (25ºC) in the dark for 2 h. The slides were then washed with PBS and treated with 4¢,6-diamidino-2-phenylindole (DAPI) (ZSGB-BIO, China) fluorescence nuclear staining mounting medium. The expression of α-SMA and E-cadherin in HK2 cells from each group was observed under a fluorescence microscope (100x) with random fields of vision.
Experimental animals and establishment of UUO model
Animal care and experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee of Chinese PLA General Hospital. A total of 28 female C5BL/6J7 mice (SPF grade), aged 24 months, weighing 20±2 g, were provided by SPF Biotechnology Co., Ltd (Beijing, China). Mice were randomly divided into sham (n=8), UUO+NC-miR-133b (n=10), and UUO+miR-133b (n=10) groups.
To establish the UUO model, each mouse was anesthetized by an intraperitoneal injection of pentobarbital (50 mg/kg) and the abdominal cavity was opened under sterile conditions. The left ureter was dissociated, double-ligated, and disconnected at 15 mm below the renal pelvis with 4-0 thread. The abdominal cavity was closed by layered suture. In the control group, the abdominal cavity was closed immediately after the ureter was dissociated.
The miR-133b mimic or NC-miR (3 mg/kg) was diluted to 1 mg/mL concentration using endotoxin-free purified water. The transfection reagent Entranster-in vivo (Engreen, China) was diluted in a 10% glucose solution to a final glucose concentration of 5%. The transfection complex was prepared by mixing the two agents and by incubating for 15 min. The UUO+miR-133b and UUO+NC-miR groups were administered with miR-133b and the NC-miRNA transfection complex (100 mL/animal), respectively, by caudal vein injection at 24 h before surgery and once every 3 days thereafter. The sham group was administered with the same volume of normal saline by caudal vein injection. The mice were euthanized at 7 and 14 days after the UUO procedure (four mice from the sham group and five mice from the UUO group were euthanized at each time point). The kidney tissues were collected from the obstructed side for western blotting, real-time PCR, and pathological analyses.
Pathological examination of the kidney tissue
The kidney tissue was fixed in 10% neutral formaldehyde, dehydrated with ethanol, embedded in paraffin, and cut into 2-μm sections. Morphological changes in the kidney tissue were observed by periodic acid-Schiff (PAS) and Masson’s trichrome staining. After staining with the Masson’s trichrome, 10 fields of vision were selected under a light microscope (400x). The area of each field of vision and area of collagen fibers stained in green were measured using the Image-Pro Plus software. The relative area of collagen deposition was calculated as follows: area of collagen fibers stained in green /area of field of vision × 100%.
Statistical analysis
The data were analyzed using the SPSS 17.0 software, and the results were expressed as mean ± standard error of mean (SEM). The differences among the experimental groups were analyzed using one-way analysis of variance (ANOVA) with completely random design. Results with P<0.05 was considered as statistically significant.