We obtained IDD tissues (30 cases) and idiopathic scoliosis (IS) tissues (30 cases) from the Hospital Affiliated to Zhejiang University of Traditional Chinese Medicine (Hangzhou, China). All study procedures were approved by the Research Ethics Committee of the Guang Xing Hospital Affiliated to Zhejiang University of Traditional Chinese Medicine. Informed consent was given by all participants. All tissue samples were collected for analysis on obtaining informed consent from all patients. Both tissues were stored at − 80 °C.
Human NP cell culture (isolation and primary culture)
The isolation of human primary nucleus pulposus cells was described in the previous study . Firstly, after PBS washing, NP degenerated specimens were diced into small fragments of size 2–3 mm3. These fragments were incubated in 0.25% trypsin solution for 25 min, followed by treatment with 0.2% type II collagenase (Invitrogen, Carlsbad, CA) at 37 °C for 12 h in DMEM (Gibco, USA) containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, USA) at 37 °C in an atmosphere of 5% CO2. All experiments in this study used first- or second-generation cells.
The NP cells were seeded into 6-well plates at the density of 1 × 106 cells/well. When cell reached 70–80% confluence, cells were transfected with mimic control (miR-con) or miR-573 mimic (GenePharma Co., Ltd., Shanghai, China) at a concentration of 40 nM for transfection using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturers’ instructions. Successful transfection was determined by qRT-PCR. The sequence of miR-573 was 5′-CUGAAGUGAUGUGUAACUGAUCAG-3′, miR-573 mimic was 5′-CUGAAGUGAUGUGUAACUGAUCAG-3′, and mimic control was 5′-UCACAACCUCCUAGAAAGAGUAGA-3′. Then the cells were incubated for 24 h to continue the further analysis.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolated using TRIzol Reagent (Invitrogen, USA) in accordance with the protocol of the manufacturer. cDNA was synthesized using the QuantiTect reverse transcription kit (Qiagen). The Hairpin-itTM miR-573 qRT-PCR Primer Set (GenePharma Co., Ltd., Shanghai, China) was used to determine the relative quantity of miR-573, and the mRNA level of miR-573 was normalized to the expression of endogenous U6. Bax was measured by SYBR green qRT-PCR assay (Takara), and GAPDH was detected as an endogenous control. The primers used in our study were as follows: miR-573 forward, 5′-ACACTCCAGCTGGGCTGAAGTGATGTGTAA-3′ and reverse, 5′-TGGTGTCGTGGAGTCG-3′; Bax forward, 5′- CCCGAGAGGTCTTTTTCCGAG-3′ and reverse, 5′- CCAGCCCATGATGGTTCTGAT-3′; U6 forward, 5′- CCCCTGGATCTTATCAGGCTC-3′ and reverse, 5′- GCCATCTCCCCGGACAAAG-3′; GAPDH forward, 5′- GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′- GGCTGTTGTCATACTTCTCATGG-3′. Quantitative measurements were determined using the 2−-ΔΔCq method .
The MTT assay was applied for assessing cell viability. NP cells were divided into untreated (control), miR-con and miR-573 mimic groups. At 24 h after transfection, 5 × 103 cells/well were seeded into 96-well plates to incubate for 48 h. Then 20 μl of MTT was added into each well. Following incubation at 37 °C for 4 h, 200 μl of DMSO was added to the wells. The absorbance was measured with a plate reader at 490 nm.
Flow cytometry assay
NP cell apoptosis was performed with the Annexin V-allophycocyanin (APC) apoptosis detection kit (BD Pharmingen, San Diego, CA) according to the manufacturer’s protocol. Cells were collected and suspended with 500 μl of Annexin V binding buffer containing 5 μl of APC-labeled Annexin V at the density of 1 × 105 cells/ml. 5 μl of 7-aminoactinomycin-D (7-AAD) was added subsequently and incubated for 15 min in the dark at room temperature. The samples were detected using a flow cytometer (FACSCalibur, BD Biosciences). FlowJo software (Treestar, San Carlos, CA) was applied for analysis.
Western blot analysis
NP cells were collected and lysed for 25 min in cold RIPA lysis buffer (Beyotime biotechnology, China). The protein concentration was detected using the BCA protein assay (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Equal amounts of proteins were separated by 10% SDS-PAGE gels, and then transferred to PVDF membranes. After being blocked with 5% non-fat milk for 2 h at room temperature, these membranes were incubated overnight at 4 °C with primary antibodies. Then the membranes were incubated with the goat anti-rabbit horseradish peroxidase-conjugated IgG secondary antibodies (1:1000; cat. no. A0208; Beyotime Institute of Biotechnology, Haimen, China) at room temperature for 1 h. The protein bands were visualized using an ECL Chemiluminescence kit (Millipore, Billerica, MA, USA). Anti-Bax (1:1000; cat. no. 5023 T), anti-Bcl-2 (1:1000; cat. no. 4223 T), anti-cleaved caspase-3 (1:1000; cat. no. 9661 T), anti-cleaved caspase-9 (1:1000; cat. no. 9509 T), anti-caspase-3 (1:1000; cat. no. 9665 T), anti-caspase-9 (1:1000; cat. no. 9508 T) and anti-GAPDH (1:500; cat. no. 5174S) antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). GAPDH was considered as the internal control.
Luciferase reporter assay
NP cells were seeded in 96-well plates and incubated for 24 h at 37 °C. miR-573 bind sites Bax 3′-UTR-Luc vector with wild type (WT) or mutant (MUT) was established. Vectors were co-transfected with miR-573 mimics or mimic control using Lipofectamine 2000. After 48 h, NP cells were accessed and examined with a luciferase reporter assay system. The transfection efficiency was corrected by a Renilla luciferase vector.
All experiments were repeated at least three times. Data were presented as the mean ± standard deviation. Statistical analysis was carried out using the SPSS 20.0 software (SPSS, Chicago, IL, USA). Differences in mean values between groups were analyzed using Student’s t test or analysis of variance (ANOVA). P < 0.05 was considered to indicate a statistically significant difference.