Tissue collection
Nucleus pulposus (NP) tissues were collected from patients with IDD and spinal cord injury (n=30 per group). Our study was approved by the Ethic Committee of the First Affiliated Hospital of Jinan University. Written informed consent was obtained from each subject.
Cell isolation and culture
These NP tissues were rinsed twice with PBS and sliced into 1 mm3 pieces followed by digestion with trypsin. The isolated NPCs were grown in DMEM (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum, 100 mg/ml streptomycin, 100 U/ml penicillin, and 1% L-glutamine at 37°C.
Cell transfection
Cell transfection was performed as mentioned previously (11) using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions after the confluence reached 30-50%.
Luciferase reporter assay
NPCs were transfected with the luciferase reporter vectors (Promega, Madison, WI, USA) containing SUMO2 3'UTR wild or mutant types and miR-25 mimic. The luciferase activities were measured after transfection.
RNA pull down assay
Biotin-labeled RNAs were reversely transcribed and treated with RNase-free DNase I. The enrichment of SUMO2 mRNA in co-precipitated RNAs was determined by real-time PCR.
Real-time PCR (RT-PCR)
To quantify the expression of miR-25, SUMO2, and p53, RT-PCR was performed using SYBR-Green Premix Ex Taq (Takara, Shiga, Japan). The thermocycling conditions: 95˚C for 10 min; 40 cycles, 95˚C for 15 sec; 60˚C for 30 sec. The primers used in this study were as follows: miR-25, forward: 5′‐ACTTTGTTCGTTCGGCTC‐3′, reverse: 5′‐GAGCAGGGTCGGAGGT‐3′; SUMO2, forward: 5’-GGCAACCAATCAACGAAACAG-3’, reverse: 5’-TGCTGGAACACATCAATCGTATC-3’; p53, forward: 5’-GACGCTGCCCCCACCATGAG-3’, reverse: 5’-ACCACCACGCTGTGCCGAA
A-3’; U6, forward: 5’-CTCGCTTCGGCAGCACA, reverse: 5’-AACGCTTCACGAA
TTTGCGT-3’; GAPDH, forward: 5’-CCACGAAACTACCTTCAACTC-3’, reverse: 5’-TCATACTCCTGCTGCTTGCTGATCC-3’.
Western blot
Protein quantification was determined by the BCA method. The protein levels of indicated genes were detected by substrate coloration following electrophoresis separation.
Cell proliferation assay
For CCK-8 assay, CCK-8 dyes (10µL) were added at 24, 48 and 72 h after transfection. Following incubation for 4 h, the number of surviving cells was assessed at 450 nm. For 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, transfected cells were exposed to EdU solution (500 μl; RiboBio, Guangzhou, China). Following incubation for 2 h, cells were fixed with 4% formaldehyde for 30 min, permeabilized with 0.5% Triton X-100 for 10 min and visualized by fluorescence microscopy.
Cell apoptosis assay
Cells were incubated with Annexin V-FITC Apoptosis Detection kit (BD Biosciences, San Diego, CA, USA) in the dark for 15 min after digestion, centrifugation and PBS washing. The apoptotic rate was analyzed by flow cytometry.
Statistical analysis
All data were expressed as mean ± standard deviation (SD). Correlation analysis was performed using the Spearman's rank test. Statistical significance was compared by analysis of variance test. P < 0.05 indicated a statistically significant difference.