Animal
A total of 10 SPF DN mice (4-week-old, Strain: BKS.Cg-+Leprdb/Leprdb/J, db/db, 20-30 g) and 10 normal mice (4-week-old, db/m, 20-30 g) were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). Mice were fed in a standalone environment at 22°C and 50% relative humidity under the alternating day and night of 12 h/12 h. After 4 days of feeding with standard diet (acclimatization), mice were anesthetized by intraperitoneal injection of 50 mg/kg sodium pentobarbital, and then sacrificed by cervical dislocation. The kidney tissues were stripped and frozen in liquid nitrogen. This study was approved by the local ethics committee of our hospital. All animal experiments were performed in accordance with the care and use of laboratory animals (National Institutes of Health, USA).
Cell culture
MMCs (SV40 MES13, established from the kidney of a mouse transgenic for the SV40 early region) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MMCs were cultured in a mixture of DMEM containing 10% FBS and Ham's F12 medium (3: 1) containing 14 mM HEPES, and maintained in a humidified incubator (MCO-15AC, SANYO) with 5% CO2 at 37°C. The medium was refreshed every 48 h, and cells were passaged at 80% confluence. Logarithmic growth phase cells at the third passage were used for transfection.
Cell treatment and transfection
PVT1 siRNA (si-PVT1) and siRNA negative control (si-NC) were synthesized by Shenggong Bioengineering Co., Ltd. (Shanghai, China) (Table 1). MiR-93-5p mimics, miR-93-5p inhibitor, mimics negative control (mimics-NC) and inhibitor negative control (INC) were purchased from Jima Pharmaceutical Technology Co., Ltd. (Shanghai, China). Cells were transfected with the above agents by using a Lipofectamine kit (Invitrogen). Cells without transfection were considered as the control. The transfection efficiency was identified by qRT-PCR. After the transfection for 48 h, cells were incubated in DMEM containing 5.55 mmol/L (normal physiological environment, NG group) or 30 mmol/L D-Glucose (diabetic physiological environment, HG group) for another 48 h. Cells were collected for the following assay.
qRT-PCR
Total RNA was extracted from MMCs by using TRIZOL reagent (Invitrogen), and then reverse-transcribed on a SimpliAmp PCR instrument (Applied Biosystems, Foster City, CA, USA). The qRT-PCR was performed on an ABI7500 Sequence Detection System (Applied Biosystems). The PCR program included 40 cycles of 95°C for 10 min, 95°C for 10 s, 60°C for 20 s and 72°C for 34 s. The relative mRNA expression level was analyzed by the 2-ΔΔCt method [26]. Oligonucleotide primers were synthesized by Biotechnology Bioengineering Co., Ltd. (Shanghai, China), and the primer sequences were shown in Table 1.
Western blot
Total proteins were extracted from cells using lysis buffer, and quantified using a BCA kit (Invitrogen). Then, 20 μg protein samples were run in a 10% SDS-PAGE, and transferred onto PVDF membrane. Afterwards, the membrane was blocked with 5% skim milk in TBST solution for 2 h, and incubated with primary antibody overnight at 4°C. The antibodies included E-cadherin (1:1000, 3195, CST), N-cadherin (1:1000,13116, CST), Vimentin (1:1000, 5741, CST), GAPDH (1: 1000, 5174, CST), Col. Ⅳ(1:10000, ab6586, Abcam), FN (1:10000, ab2413, Abcam), TGF-β1 (1:10000, ab92486, Abcam), PAI-1 (1:10000, ab66705, Abcam), Bcl-2 (1:1000, ab196495, Abcam), Bax (1:1000, ab199677, Abcam), cleaved caspase-3 (1:1000, ab49822, Abcam), cleaved PARP (1:1000, ab32064, Abcam), CyclinD1 (1:5000, ab226977, Abcam), CDK4 (1:3000, ab137675, Abcam), PI3K (1:1000, 4292, CST), p-PI3K (1:1000, 4228, CST), Akt (1:1000, 9272, CST), p-Akt (1:1000, 4060, CST), mTOR (1:1000,2972,CST), and p-mTOR(1:1000, 2971, CST). GAPDH was used as the internal control. After 3 times of washing with TBST, the membrane was incubated with HRP-labelled secondary antibody (1:1000, Sigma) for 2 h at 25°C. An enhanced chemiluminescence Plus (Thermo Fisher) was used to visualize the protein bands. The gray value was measured by using a Lab Works 4.5 software. Western blot was performed in three independent repetitions, and the representative images were shown.
EdU and Colony formation assay
An EdU cell proliferation detection kit (Ruibo Biotechnology Co., Ltd., Guangzhou, China) was used for EdU assay (cell proliferation). Briefly, cells were labeled with EdU, fixed with 4% paraformaldehyde, and then stained with DAPI. The EdU fluorescence (red) was detected by a laser confocal microscope (NIKON A1, Japan).
For Colony formation assay, cells were seeded into 6-well plates (300 cells/well) and cultured for 2 weeks. Cells were then fixed with 4% paraformaldehyde for 10 min and stained with crystal violet for 15 min. The stained colonies were observed under an inverted microscope (Olympus Ckx53, Japan), and counted using an ImageJ (1.48 V) software.
Annexin V-PI assay
An Annexin V-PI kit (Invitrogen) was used for the detection of cell apoptosis. Briefly, cells were stained with Annexin V-EGFP and PI for 15 min under darkness at 25°C. The apoptosis rate was then analyzed on a MUSETM cytometer (EMD Millipore, USA).
Cell cycle analysis
A MuseTM Cell Cycle Reagent (Invitrogen) was used for the detection of cell cycle. Briefly, cells were fixed in 70% ethanol, and incubated with MuseTM Reagent in the dark for 30 min at 25°C. The cell cycle was then analyzed on a MUSETM cytometer (EMD Millipore).
Scratch assay
Cells were seeded into 6-well plate, and cultured in serum-free DMEM until 90% confluence. A scratch wound was then made in each well with a pipet tip. Cell debris was removed by washing with PBS. After 48 h of culturing in serum-free DMEM, the scratch width was photographed under an inverted microscope (Olympus Ckx53). The migration rate = (1 - scratch width at measurement/initial scratch width) × 100%.
Transwell assay
The cell invasion was detected by using transwell chambers (Corning Corporation, Midland, MI, USA). Briefly, cells were seeded into Matrigel-coated upper chamber. The lower chamber was filled with DMEM. After 48 h of incubation, cells on the lower chamber were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 20 min. The stained cells were observed under an inverted microscope (Olympus Ckx53), and counted randomly at five fields of views at 200 × magnifications.
ELISA for fibrosis factors
Cells were cultured for 48 h after transfection, and the cell-culture supernatants were collected. The contents of Col. IV, PAI-1, FN and TGF-β1 in the supernatants were detected by using ELISA kits (BioSource International, Camarillo, CA, USA) in accordance with the manufacturers' instructions.
Dual-Luciferase reporter gene (DLR) assay
The binging site of PVT1 in miR-93-5p was predicted by StarBase3.0 software. A DLR kit (Promega, USA) was used to identify the relationship between PVT1 and miR-93-5p. The mutant sequence (MT) and the wild sequence (WT) of PVT1 were amplificated according to the binging sequences, and then cloned into the pmirGLO luciferase vector. HEK-293T cells were then co-transfected with WT/MT and miR-93-5p mimics/miR-93-5p mimics NC (Shanghai Jima Pharmaceutical Technology Co., Ltd.), and grouped as MT + mimics, MT + NC, WT + mimics, and WT + NC group. After 48 h of transfection, the fluorescence was measured on a Microplate Reader (Invitrogen).
Statistical analysis
All assays were performed in triplicate. Data were expressed as mean ± standard deviation (SD). All statistical analyses were performed by SPSS 20.0 and GraphPad.Prism 5.01 statistical software. The data among multi-groups were analyzed by One-Way ANOVA, followed by Tukey's test (between two groups). P < 0.05 represented statistically significant.