Tissue samples
The Ethics Committee of the Traditional Chinese Medical Hospital of Lianyungang approved this study in patients with IDD. All participants signed the written informed consent. IDD tissues and control tissues were respectively collected from two groups of patients and next frozen in liquid nitrogen at -80°C. According to the MRI scans, IDD degree was classified utilizing the modified Pfirrmann classification.
Cell culture and treatment
Human degenerative NPCs used in this paper were extracted from NP tissues of IDD patients. The isolated NP tissues were rinsed utilizing PBS and subsequently minced into pieces, followed by digestion via 0.2% collagenase II (Crescent Chemical, Islandia, NY, USA) and 0.25% trypsin (Gibco, Carlsbad, CA, USA). Thereafter, NPCs were grown in DMEM/F12 (Gibco) with 10% FBS (Gibco), 100 U/mL penicillin as well as 100 mg/mL streptomycin under the condition of 37°C with 5% CO2. NPCs at the third passage were used for later experiments. 1 µg/ml of LPS (cat. no. L2630; Sigma-Aldrich) was utilized to stimulate NPCs in serum-free DMEM for 1 d.
Cell transfection
Short harpin RNA targeting OIP5-AS1 (sh-OIP5-AS1; 5’-CCGGGCTCCTAGGATTCCAGTTATCCTCGAGGCAGAAGGCTGAGTTTCATTTTTTTTG-3’) with the negative control sh-NC (5’-CACCGTTCTCCGAACGTGTCACGTCAAGAGATTACGTG ACACGTTCGGAGAATTTTTTG-3’), miR-25-3p mimic (Sense 5’-UCC CUG AGA CCC UAA CUU GUG A-3’; antisense 5’-ACA AGU UAG GGU CUC AGG GAU U-3’) with mimic NC were obtained from GenePharma (Shanghai, China). Transfection in NPCs was conducted via Lipofectamine 2000 (Invitrogen) as per the instructions from the manufacturer.
RT-PCR
Tissues or NPCs were prepared for isolation of total RNA using TRIzol reagent (Invitrogen) as per the manufacturer’s guidebook. Then reverse transcription of 1-µg RNA into cDNA was carried out with the use of the 1st Strand cDNA Synthesis Kit (TAKARA). RT-PCR was conducted on the ABI 7500 real-time PCR system (Applied Biosystems) with the SYBR-Green PCR Master Mix (Roche) following the introductions. The parameters for PCR were: 10-min pre-treatment at 95°C; 40 cycles at 95˚C for 15 sec; at 60°C for 1 min; and at 72˚C for 5 min. The 2−ΔΔCt method was applied for calculation of gene expression that was normalized to GAPDH or U6. Utilized primers were as follows: OIP5-AS1 sense, 5’-GGTCGTGAAACACCGTCG-3’ and antisense, 5’-GTGGGGCATCCAGGGT-3’; Collagen II sense, 5’-CTGGTGATGATGGTGAAG-3’ and antisense, 5’-CCTGGATAACCTCTGTGA-3’; Aggrecan sense, 5’-CAGATGGCACCCTCCGATAC-3’ and antisense, 5’-GACACACCTCGGAAGCAGAA-3’; GAPDH sense, 5’-AATCCCATCACCATCTTCCAG-3’ and antisense, 5’-TGATGACCCTTTTGGCTCCC-3’; miR-25-3p sense, 5’-CATTGCACTTGTCTCGGTCTGA-3’ and antisense 5’-GCTGTCAACGATACGCTACGTAACG-3’; U6 sense, 5’-CTCGCTTCGGCAGCACA-3’ and antisense, 5’-AACGCTTCACGAATTTGCGT-3’.
Immunofluorescence
NPCs were fastened utilizing 4% paraformaldehyde for just 0.5 h, cultured with 0.1% Triton X-100 for another 15 min, and subsequently blocked via applying 2% bovine serum albumin (Sigma) for extra 1 h. After that, NPCs were cultivated with primary antibody against collagen II (1:200; Santa Cruz Biotechnology) for whole night at 4°C, followed by exposure to secondary antibodies labelled with Alexa Fluor® 594 (1:100; Life Technologies) at indoor temperature for 1 h. After thrice washes by PBS, NPCs were stained via DAPI. Photos were observed by laser confocal microscopy (OLYMPUS).
CCK-8 experiment
To assess cell viability, a commercial Cell Counting Kit-8 (CCK-8; Dojindo) was utilized. NPCs (5×103 cells/well) were placed in 96-pore dishes and 10-µl CCK-8 solution was supplemented for extra culture of 2 h. Absorbance in each hole was estimated at 450 nm.
EdU assay
96-well plates (1 × 104 cells/well) were prepared for culture of NPCs. For this assay, Cell-LightTM EDU Apollo 488 kit (Ruibio, China) was employed. Cells were dyed with Hoechst 33342. To assess cell proliferation rate, the ratio of the EdU positive cells/Hoechest staining cells selected randomly at three fields was calculated. Fluorescence microscope (Leica Microsystems) was utilized for observation.
Flow cytometry
Apoptosis ability was evaluated via Annexin V-FITC-propidium iodide (PI) apoptosis detection reagent (BD Biosciences) as the manufacturer guided. Cells were first rinsed thrice utilizing PBS, digested using trypsin (1 ml) and next adjusted to the density of 1×105 cells/100 µl for resuspension in 1X Annexin binding buffer. Next, cells were collected and dyed via applying the above reagent for 15 min. The apoptosis rate was quantified with flow cytometry 10.0 (FlowJo, FACS CaliburTM, BD Biosciences).
Western blot
To extract total protein, an RIPA lysis buffer was employed. Also, a BCA Protein Assay Kit (Thermo Scientific) was utilized for protein concentration. Equivalent protein (20 µg) was separated by employing 10% SDS-PAGE, which was transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). Blocking of mixture was implemented by use of 5% fat-free milk for extra 1 h at room temperature. Next, primary antibodies against Collagen II, Aggrecan and GAPDH (1:1,000; Abcam) were adopted at 4 °C for one night. Secondary culture was implemented with HRP-labelled goat anti-rabbit antibody (Boster, Wuhan, China). ECL Plus reagent (Millipore, USA) was adopted for visualization of signals. The intensity of bands was quantified via Image Lab 3.0 software (Bio-Rad, CA, USA).
ELISA
The contents of IL-6, TNF-α, IL-10, and IL-1β were estimated through relative ELISA kits (eBioscience) following the producer's instructions. Under the wavelength at 450 nm, all samples were assessed with a microplate reader (SpectraMax M5, Molecular Devices). Next, a standard curve was plotted with the help of computer software based on the absorbance value.
Luciferase reporter assay
The wild- and mutant-type OIP5-AS1 with or without putative binding sites for miR-25-3p (OIP5-AS1-WT/MUT) were constructed into pmiRGLO (Invitrogen). After that, NPCs were co-transfected with miR-25-3p or miR-NC with OIP5-AS1-WT or OIP5-AS1-MUT, the procedures of which were implemented utilizing Lipofectamine 2000 (Invitrogen). 48 h post transfection, the luciferase activity in each group was analyzed using a dual-luciferase reporter system (Promega). Renilla luciferase activity acted as the normalization.
Statistical analysis
The gained data of triplicated experiments were dissected by SPSS v.18.0 software (SPSS Inc., Chicago, IL, USA) and represented as the mean ± standard deviation (SD). Comparisons between or among groups were analyzed via Student’s t-test or one-way ANOVA. The statistics were significant upon P < 0.05.