Animal models
C57BL/6 mice (female, 6-week-old) were purchased from Vital River Laboratory Animal (Beijing, China) and housed in pathogen-free conditions. These mice were divided into two groups (n = 6/group), control and diabetic mice (DM). Mice in the DM group were administered with high-fat and high-glucose diets. Then, mice were intraperitoneally injected with streptozotocin (45 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) to increase the burden on the kidney for consecutive 7 days. Streptozotocin was dissolved in 0.1 M citrate acid solution. Mice were considered to have DN until fasting blood-glucose reached 16.5 mmol/L, urinary albumin level over 30 mg/24 h. Mice in the control group were administered with standard laboratory chow and injected with citrate acid solution (45 mg/kg) for consecutive 7 days. Mice were sacrificed after 0, 1, 2 and 4 months, and kidney tissues were excised for further analysis. Animal studies were performed in agreement with the guidelines of the Animal Care and Use Committee of Affiliated Hospital of Inner Mongolia University for Nationalities.
Cells and cell treatment
Mouse glomerulus mesangial cells (SV40-MES13; Procell, Wuhan, China) were cultured in 71.25% DMEM + 23.75% Ham’s F-12 medium + 5% FBS. Cells were administered with normal glucose (NG; Sigma-Aldrich; 5.5 mmol/mL), high glucose (HG; 25 mmol/mL) or mannitol (20 mmol/L; Sigma-Aldrich) and then cultured for 24 h. Cells were cultured at 37 °C conditions containing 5% CO2.
Cell transfection
CircSMAD4 overexpression vector (circSMAD4), blank pCD-ciR expression vector (vector), short hairpin RNA targeting circSMAD4 (sh-circSMAD4), and its negative control (sh-NC) were all obtained from Geneseed (Guangzhou, China). MiR-377-3p mimic (miR-377-3p), miR-377-3p inhibitor (anti-miR-377-3p) and their corresponding negative controls (miR-NC and anti-NC) were bought from Ribobio (Guangzhou, China). BMP7 overexpression vector (BMP7) and blank pcDNA expression vector (pcDNA) were provided by Genepharma (Shanghai, China). SV40-MES13 cells were transfected with oligos or vectors using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA).
ELISA
24 h mouse urine was collected to determine albumin and 8-OH-dG using the Mouse Albumin Detection ELISA Kit (Chondrex, Redmond, WA, USA) and QuickDetect 8-OH-dG (Mouse) ELISA Kit (BioVision, Milpitas, CA, USA) according to the protocols. The releases of inflammatory factors, including IFN-γ, MCP-1, IL-6 and TNF-α, were determined using the Mouse IFN-γ ELISA Kit (Sigma-Aldrich), MCP-1 (Mouse) ELISA Kit (BioVision), IL-6 (Mouse) ELISA Kit (BioVision) and TNF-α (Mouse) ELISA Kit (BioVision) in line with the instructions.
Western blot
Total protein was extracted using RIPA lysis reagent (Cwbio, Beijing, China) and quantified by BCA kit (Cwbio). Equal amount of protein was loaded in 10% SDS-PAGE and then transferred on PVDF membranes, followed by blocking using 5% skim milk. The primary antibodies (Abcam, Cambridge, MA, USA), including anti-fibronectin (ab268021; 1/1000 dilution), anti-collagen IV (ab227616; 1/1000 dilution), anti-Bcl-2 (ab182858; 1/2000 dilution), anti-Bax (ab32503; 1/5000 dilution), anti-BMP7 (ab129156; 1/5000 dilution) and anti-β-actin (ab8226; 1/2000 dilution), were used to incubate the membranes overnight at 4 °C Next, the membranes were incubated with the secondary antibody. The blots on the membranes were presented using the ECL kit (Cwbio).
Flow cytometry assay
SV40-MES13 cells were collected at 48 h post-transfection to determine the number of apoptotic cells using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA). According to the protocol, cells were resuspended in Annexin V-FITC binding buffer and next stained with Annexin V-FITC and propidium iodide (PI). The apoptotic cells were sorted and distinguished using a flow cytometer (BD Biosciences).
Quantitative real-time PCR (qPCR)
Trizol reagent (Cwbio) was applied for total RNA isolation. Subsequently, cDNA was assembled using the PrimeScript 1st strand cDNA Synthesis Kit (Takara, Dalian, China) or microScript microRNA cDNA Synthesis Kit (Norgen Biotek, Thorold, Canada), followed by qPCR amplification using the SYBR reagent (Sigma-Aldrich). Using β-actin or U6 as the internal reference, the fold-change of relative expression was calculated using the 2−ΔΔCt method. The primer sequences were shown as below:
circSMAD4 (mmu), F: 5′- CACTATGAGCGGGTTGTCTCA-3′ and R: 5′-AGCAGGATGACCATTACTCTGC-3′; miR-377-3p (mmu), F: 5′-CGCGATCACACAAAGGCAAC-3′ and R: 5′-AGTGCAGGGTCCGAGGTATT-3′; BMP7 (mmu), F: 5′-CCTATGGCCATGTCGCATCT-3′ and R: 5′-GCAGCCCAAGCTACTGAAGA-3′; U6 (mmu), F: 5′-CTCGCTTCGGCAGCACATATACT-3′ and R: 5′-ACGCTTCACGAATTTGCGTGTC-3′; β-actin (mmu), F: 5′-CACTGTCGAGTCGCGTCC-3′and R: 5′-CGCAGCGATATCGTCATCCA-3′.
Dual-luciferase reporter assay
MiR-377-3p was predicted as a target of circSMAD4 by starbase (http://starbase.sysu.edu.cn/). BMP7 was predicted as a target of miR-377-3p by DIANA tools (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=site/index).
According to the wild-type (WT) sequence of circSMAD4 or BMP7, the mutant-type (MUT) sequence fragment of circSMAD4 or BMP7 containing mutated miR-377-3p binding site was designed and synthesized. Then, the WT and MUT reporter vectors of circSMAD4 and BMP7 were constructed, named WT-circSMAD4, MUT-circSMAD4, WT-BMP7 3′UTR and MUT-BMP7 3′UTR. Then, these reporter vectors were separately transfected into SV40-MES13 cells transfected with miR-377-3p or miR-NC. At 48 h post-transfection, luciferase activity in cells was tested using the dual-luciferase assay kit (Promega, Madison, WI, USA).
RNA pull-down assay
SV40-MES13 cells were transfected with biotinylated miR-377-3p (Bio-miR-377-3p; 50 nM; Ribobio) or biotinylated miR-NC (Bio-miR-NC) and maintained for 24 h. The cells were then harvested and lysed in lysis buffer (Invitrogen). Cell lysates were incubated with streptavidin magnetic beads (Invitrogen) for 4 h. The beads were washed, and RNA compounds on beads were eluted and isolated for qPCR analysis.
Statistical analysis
Data were collected from three independent experiments for each assay and operated using GraphPad Prism 7 (GraphPad, La Jolla, CA, USA). The differences in different groups were determined using Student’s t-test or using analysis of variance followed by the Tukey post-test. The data were displayed as the mean ± standard deviation (SD). P < 0.05 was considered to be statistically significant.