TGF-β1 stimulation and miR-133b transfection
The HK2 cells were cultured in Dulbecco’s modified Eagle’s 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. In the transfection experiment, miR-133b mimic and miRNA mimic control (GenePharma, China) were transfected into HK2 cells for 6 h, as per the instructions of jetPRIME® transfection reagent (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 TGF-β1 (PeproTech, USA) for 48 h.
RNA extraction and real-time polymerase chain reaction (PCR)
Total RNA was extracted from HK2 cells and kidney tissues of each group by Trizol, and used to synthesize miR-133b and U6 cDNA using miScript II RT Kit (QIAGEN, China). Primers specific for target genes were designed with reference to their mRNA-coding regions in GenBank using Primer 5.0 software. The primer sequences were verified on BLAST. The total RNA was used to synthesize cDNA of target genes using ReverTra Ace qPCR RT Master Mix kit (TOYOBO, Japan). The expression of the 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 a ABI-prism-7500 sequence detection system (Applied Biosystems, USA) using miScript SYBR Green PCR Kit (QIAGEN). The relative expression level was calculated using U6 or GAPDH as internal control.
hsa-miR-133b 5’TTTGGTCCCCTTCAACCAGCTA 3’;
mmu-miR-133b 5’ TTTGGTCCCCTTCAACCAGCTA 3’;
hsa-CTGF 5’ GGCCTCTTCTGTGACTTCG3 ’and 5’ ATGCAGGGAGCACCATCT 3’;
has-α-SMA 5’ ACTGCCGCATCCTCATCC3’ and 5’CCCATCAGGCAACTCGTAA3’;
has-Ecad 5’ CTGAGAACGAGGCTAACG3’ and 5’GTCCACCATCATCATTCAATAT3’;
has-FN 5’ GTGCCACCTACAACATCA3’ and 5’CCACGGTAACAACCTCTT3’;
has-Col3A1 5’ CTTCTCGCTCTGCTTCAT3’ and 5’CTATCCGCATAGGACTGAC3’;
has-GAPDH 5’ AGCCACATCGCTCAGACA3’ and 5’CCCAATACGACCAAATCC3’;
mmu-CTGF 5’ CAAATCCCTGTTGGTGAA3 ’and 5’TAGGAATCGGACCTTACC3’;
mmu-αSMA 5’ CAGGGAGTAATGGTTGGA3’ and 5’GATGATGCCGTGTTCTAT3’;
mmu-Ecad 5’ GCCAAGCAGCAATACATC3’ and 5’AAAGGGTTCCTCGTTCTC3’;
mmu-FN 5’ GCCGAATGTAGATGAGGA3’ and 5’ATGAGGATAGAGGTGGTAGTC3’;
mmu-Col3A1 5’ CCCACAGCCTTCTACACCT3’ and 5’CCAGGGTCACCATTTCTC3’;
mmu-GAPDH 5’ GACAGCCGCATCTTCTTG3’ and 5’ACACCGACCTTCACCATT3’.
Western blot analysis
After lysis and denaturation of HK2 cells or kidney tissues from each group using radioimmunoprecipitation assay (RIPA) buffer, the proteins (50 µg) were separated on 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto nitrocellulose (NC) membranes. After blocking with 1 × casein for 1 h to prevent non-specific binding, NC membranes were incubated with the following primary antibodies at 4 °C for overnight: (a) Rabbit monoclonal antibody E-cadherin (BD Bioscience, USA) diluted to 1:100; (b) rabbit monoclonal antibody against α-SMA (Abcam, UK) diluted to 1:300; (c) mouse monoclonal antibody against CTGF (Abcam) diluted to 1:200; (d) rabbit polyclonal antibody (Proteintech, USA) against fibronectin diluted to 1:500; (e) rabbit polyclonal Col3A1 antibody (Proteintech) diluted to 1:500; (f) mouse monoclonal β-actin antibody (Beyotime, China) diluted to 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 probed with 1:1000 diluted secondary antibodies at room temperature (25℃) for 2 h. Enhanced chemiluminescence (ECL) western blotting kit (APPLYGEN, China) was used to detect target bands, and β-actin was used as internal reference to calculate the relative expression of protein 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 PiTar (http://genie.weizmann.ac.il/index.html).
Dual-luciferase reporter assay
The seed sequence for the binding between CTGF and miR-133b was searched using the bioinformatic 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), which was 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 logarithmic growth phase were digested with pancreatin and seeded in 48-well plates for 24 h. After reaching 80% confluency, the cells were transfected the cell fusion reagent. According to the instructions of jetPRIME® transfection reagent, the synthesized miR-133b and miRNA mimic control (NC-miR) were respectively co-transfected with miRGLO-Wt-CTGF or miRGLO-Mut-CTGF into 293T cells. After 48 h, the cells were lysed using a passive lysis buffer (Promega, USA) and the cell lysate collected. The luciferase activity of the lysate was detected according to the steps indicated in the dual-luciferase reporter assay system (Promega).
Immunofluorescence staining
HK2 cells were seeded at about 105 cells/well in six-well plates with glass cover slips and disinfected at high temperature and high pressure. The plates were placed in an incubator at 37 °C with 5% carbon dioxide 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 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 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) and α-SMA antibody (1:100 dilution) diluted in 5% BSA at 4 °C for 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 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 (100×) with random fields of vision.
Experimental animals and establishment of a 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 (Beijing) Biotechnology Co., Ltd. Mice were randomly divided into sham (n = 8 ), UUO + NS-miR-133b (n = 10), and UUO + miR-133b groups (n = 10).
To establish a 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.
miR-133 or NC-miR (3 mg/kg) was diluted to 1 µg/µL concentration using endotoxin-free purified water. The transfection reagent Entranster-in vivo (Engreen, China) was diluted in a 10% glucose solution at a final glucose concentration of 5%. The transfection complex was prepared by mixing the two agents and incubating for 15 min. The UUO + miR-133b and UUO + NC-miR groups were administered with miR-133b and NC-miRNA transfection complex (100 µL/animal), respectively, by a caudal vein injection at 24 h before operation and once every 3 days thereafter. The sham group was given the same amount of normal saline by caudal vein injection. The mice were sacrificed at 7 and 14 days after UUO (4 mice from the sham group and 5 mice from the UUO group were sacrificed at each time point). The kidney tissue was collected from the obstructed side for western blotting, real-time PCR, and pathological analysis.
Kidney tissue pathological examination
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 Masson’s trichrome, 10 fields of vision were selected under a light microscope (400×). The area of each field of vision and area of green collagen fibers were measured using the Image-Pro Plus software. The relative area of collagen deposition was calculated was follows: area of green collagen fiber/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 the one-way analysis of variance (ANOVA) with completely random design. P < 0.05 was considered statistically significant.