Animal
A total of 20 SPF diabetic model mice (4 weeks, Strain: BKS.Cg-+Leprdb/Leprdb/J, db/db, 20-30 g) and normal mice (4 weeks, db/m, 20-30g) were obtained from Model Animal Research Center of Nanjing University. 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 weeks of feeding, the anesthesia with sodium pentobarbital (50mg/kg) was performed on mice by intraperitoneal injection. Then, all mice were sacrificed by using cervical dislocation, followed by the kidneys stripped and frozen in liquid nitrogen. This study was approved by the ethics committee of our hospital, and all experiments were in accordance with the guide for the care and use of laboratory animals established by United States National Institutes of Health (Bethesda, MD, USA).
Cell culture
Mouse mesangial cells (MMCs) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. MMCs were cultured in DMEM complete medium containing 10% FBS (Gibco) and penicillin (Gibco). All cells were cultured in a humidified humidity incubator (MCO-15AC, SANYO) with 5% CO2 at 37 °C. Cells were digested with 0.25% trypsin when the cells reached 80% confluency, followed by subculture every other day.
Cell transfection and grouping
The PVT1 gene was silenced by RNA interference technology, and primer sequences such as PVT1 siRNA (si-PVT1) (synthesized by Shanghai Shenggong Bioengineering Co., Ltd.) were shown in Table 1. Transient co-transfection of MMCs was performed using the liposome Lipofectamine RNAiMAX kit (Invitrogen). Transfected cells were divided into three groups including blank control group (Mock), negative control group (si-NC), and si-PVT1 experimental group (siPVT1). After transfection, the cells were replaced with 10% FBS no-anti-DMEM medium for 4 hours, and then incubated for 12 hours in serum-free DMEM medium to synchronize the cells in G0 phase. Cells cultured in DMEM full-medium containing 5.55 mmol/L D-Glucose simulated normal physiological environment were named as control group, while cells cultured in DMEM full-medium containing 30 mmol/L D-Glucose simulated diabetic physiological environment were named as high-sugar group. Thus, there were finally 4 groups in current study including control group (NG), high glucose group (HG), high glucose negative control group (HG + siNC) and high glucose siPVT1 group (HG + siPVT1). After 48 hours of high glucose culture, the cells and the supernatant of each group were collected and stored at -80 °C. The qRT-PCR was used to detect RNA interference efficiency.
MiR-93-5p mimics, mim-93-5p inhibitor, mim-NC and Inhibitor NC (Shanghai Jima Pharmaceutical Technology Co., Ltd.) were added to MMCs (80% confluency) respectively, followed by cultivation in DMEM medium containing 30 mmol/L D-Glucose. The transfected cells were divided into five groups: Mock, mim-NC, mimics, INC and inhibitor. Moreover, siNC, siPVT1, miR-93-5p inhibitor and inhibitor NC were co-transfected with MMCs (60% confluency), and named as siNC+INC, siNC+ miR-93-5p inhibitor, siPVT1+ INC and siPVT1+ miR-93-5p inhibitor group respectively.
The qRT-PCR assay
The expression level of genes including miR-93-5p was detected of by qRT-PCR. Briefly, cells cultured for 48 h were collected to extract total RNA by using TRIZOL reagent (Invitrogen), and reverse-transcribed by PCR amplification instrument. The qRT-PCR was performed on A ABI7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). 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 data was analyzed by the 2-ΔΔCt method [20], and all oligonucleotide primers were designed and synthesized by Biotechnology Bioengineering (Shanghai) Co., Ltd. The experiment was repeated for 3 times. All primer sequences for samples and internal references were listed in Table 1.
Western blot analysis
Proteins expression was measured by Western blot. Briefly, a total of 20 μg total protein was extracted by lysis buffer, followed by quantifying using a BCA kit (ThermoFisher Co., Ltd.). Then, samples were subjected to 10 % SDS-PAGE, and transferred onto PVDF membrane. Afterwards, the membrane was blocked with 5 % skim milk in TBST solution. Subsequently, membrane was sequentially incubated with primary antibodies including 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), p-mTOR(1:1000, 2971, CST) and secondary antibodies (goat anti-rabbit IgG, 1:10000, Sigma). GAPDH was used as the internal reference. At last, blots were visualized by enhanced chemiluminescence Plus, and integrate optical density was measured by software Lab Works 4.5.
EdU and colony formation assay
Cells in each group were cultured for 48 hours after transfection, and EdU cell proliferation assay and colony formation assay were performed. For EdU assay, cells in all groups were performed with EdU labeling, fixation, Apollo staining and DNA staining based on EdU cell proliferation detection kit (Guangzhou Ruibo Biotechnology Co., Ltd.). All images were acquired by laser confocal microscope (NIKON A1). For colony formation assay, cells were washed with PBS and digested with 1% trypsin. All the cells were primarily added into 6-well plates (300 cells/well) with 2.5 mL medium in each well. Two weeks later, cells were fixed with 4 % paraformaldehyde solution and then stained with Swiss-Gimsa for 15 min. Then, photographs were taken using an inverted phase contrast microscope (Olympus Ckx53), and the number of clones was automatically counted using ImageJ (1.48 V) software. Cell colony formation rate was calculated as (number of colonies/total number of cells seeded) ×100%.
AnnexinV-PI assay
Cells of each group were stained with Annexin V-PI kit (Invitrogen), and the apoptosis of each group was detected by MUSETM cytometer (EMD Millipore, USA). Briefly, after 48 hours of transfection, cells were centrifuged, washed twice with PBS, and collected (1-5×105 cells). A total of 500μl Binding Buffer, 5μl Annexin V-EGFP and 500μl Propidium Iodide were added to the cell samples (37°C, dark environment, 5-15 min). Finally, results were detected by flow cytometry (Ex = 488 nm, Em = 530 nm), followed by the apoptotic ratio calculation.
Cell cycle analysis
The flow cytometry was used to detect the cell cycle. Simply, at 48 hours after transfection, cells were mixed with 1 ml trypsin and 1-2 ml DMEM medium containing FBS, followed by centrifugation and fix with 70% ethanol. Subsequently, MuseTM Cell Cycle Reagent was added to the cell precipitation and incubated at room temperature for 3 0min. Finally, the cell cycle was measured by MUSETM cytometer (Merck Milliber, USA). Cell Quest software was used for data analysis (results were expressed as cell percentage).
Scratch assay
Cell scratch assay was used to detect cell migration in current study. Simply, after adjusting the cell density of each group, the cells were inoculated into the 6-well plate. After drawed a line across the surface of culture medium, washed by PBS and added fresh culture medium, cells were continuous culture for 48 h. Then, these cells were observed and photographed under inverted microscope (Olympus Ckx53) to calculate the cell migration rate. Cell mobility was calculated as (1 - scratch width at measurement / initial scratch width) × 100%.
Transwell assay
After transaction for 48 h, cells suspension was added in transwell chamber (Corning Corporation, Midland, MI, USA) and matrigel (BD Biosciences) - coated transwell chamber, respectively. Then, the medium with 10% serum was added to the lower compartment of transwell chamber, followed by incubtation with 5% CO2 at 37 °C for 48 h. Then, the upper compartment of transwell cell was washed by PBS, fixed by 4% paraformaldehyde and stained with 0.25% Coomassie blue. Finally, five visual fields (´ 200) were randomly selected for observation under an inverted microscope (Olympus Ckx53).
ELISA for inflammatory factors
MMCs cells were cultured for 48 hours after transfection, and the supernatants of each group were collected. The concentration of Col. IV (Sigma), PAI-1 (CST company) FN protein (Assay pro) and TGF-β1 (CST) in cells of each group were detected by ELISA kit (BioSource International, Camarillo, CA, USA). All the operations were carried out according to the kit instructions.
Luciferase reporter gene assay
Prediction of PVT1 interacting miRNAs was performed based on StarBase 3.0 database. Then, the wild-type or mutant for miR-93-5p and PVT1 were designed respectively according to the predicted results. The PVT1 mutant sequence and the wild sequence fragment were cloned to the pmirGLO luciferase control reporting vector (Promega, USA). Then, HEK-293T cells were co-transfected with the mutant sequence in combination with miR-93-5p mimics or miR-93-5p negative control (Shanghai Jima Pharmaceutical Technology Co., Ltd.), which named as MT + mimics group and MT + NC group respectively. In addition, the negative control of wild-type sequence combined with miR-93-5p mimics (WT + mimics group) or miR-93-5p (WT + NC group) were co-transfected with HEK-293T. After transfection for 48 h, luciferase assay was determined by dual luciferase reporter assay kit (Promega).
Statistical analysis
All statistical analyses were performed using SPSS (version 20.0) and GraphPad.Prism (version: 5.01) statistical software. The results were presented in the form of mean ± standard deviation (SD). The data between two groups or among groups were analyzed by the Student t test or One-Way ANOVA respectively. P < 0.05 was considered to be statistically significant.