NP samples were collected from 76 patients with degenerative disc degeneration who underwent discectomy (mean age 56.8 ± 4.3 years). Surgery is indicated when conservative therapy fails and progressive neurological impairments develop, such as progressive motor weakness or cauda equina syndrome. Patients with lumbar spine stenosis, ankylosing spondylitis, isthmus or degenerative spondylolisthesis, or generalized idiopathic skeletal hypertrophy were excluded from this study. A total of 78 patients were recruited to serve as age- and sex-matched controls. Control NP tissue samples were obtained from patients who underwent anterior decompressive surgery for traumatic lumbar fractures complicated with neurological disorders. These patients received a typical lumbar spine MRI scan prior to surgery. The degree of IDD was rated using the Pfirrmann classification. The research protocol was approved by our hospital’s Ethics Committee, and each participant provided written informed permission.
Primary nucleus pulposus cell culture and transfection
The human NP tissues were washed three times with phosphate-buffered saline (PBS; Gibco, Grand Island, NY), minced into small fragments, digested with 0.25% (w/v) trypsin and 0.2% (w/v) type collagenase (Gibco), and then placed in PBS for approximately 3 h at 37 °C in a gyratory shaker. The cells were filtered using a 70-μm mesh filter (BD, Franklin Lakes, NJ, USA). Primary NP cells were grown in 100-mm culture dishes with growth medium (Dulbecco’s modified Eagle’s medium and Ham’s F-12 nutrient mixture (DMEM-F12; Gibco), 20% (v/v) foetal bovine serum (FBS; Gibco), 50 U/mL penicillin, and 50 g/mL streptomycin (Gibco). Trypsin was used to passage the cells at approximately 80% (v/v) confluence, and the cells were subcultured in a 60-mm culture dish (2.5 × 105 cells/well). The subsequent studies employed cells that had been passaged no more than twice.
Prior to transfection, NP cell culture was incubated for 24 h after controlling the initial concentration to approximately 2×104 cells per well. The samples were sorted into groups and transfected with either one of several miR-217 mimics (mirVana miRNA mimics, Thermo Fisher Scientific, Waltham, MA, USA), miR-217 inhibitor (mirVana miRNA inhibitors, Thermo Fisher Scientific), mimic control (Thermo Fisher Scientific), or inhibitor control (Thermo Fisher Scientific). The transfection relied on Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). MiR-217 mimics or inhibitors were labelled with Cy3 using the Silencer® siRNA Labelling Kit (#AM1632, Invitrogen).
First, total RNA was extracted using the TRIzol technique from NP cells kept at a final concentration of 1 mg/mL. After purifying the miRNA component of the total RNA with the miRNA isolation kit (Ambion), the extracted miRNA samples were evaluated on the chip using the Agilent Company’s human miRNA chip (v.12.0). GeneSpring GX v12.1 software was used to process the data (Agilent Technologies).
TRIzol reagent was used to extract total RNA from transfected cell lines (Takara, Japan). For reverse transcription, a one-step PrimeScript miRNA cDNA Synthesis Kit (Takara) was used. On the ABI 7300 system, we used SYBR Green Real-Time PCR Master Mix (Takara) to execute qRT–PCR and synthesize the data (ABI). Additionally, U6 snRNA was used as an internal control.
Prediction of CpG islands and bisulfite sequencing PCR (BSP)
Promoter Inspector (http://www.genomatix.ed) was used to estimate the promoter area. CpG islands linked with the promoter were predicted using the CpG prediction algorithm. The Qiagen DNeasy Blood and Tissue Kit was used to isolate genomic DNA from NP, which was then placed in bisulfite. Then, using BSP primers, the genomic DNA was amplified and cloned into the pGEMT Easy vector (Promega, WI, USA). Then, the samples were sequenced, and the data were examined using a BIQ analyser.
The 3′UTR of the FBXO21 gene fragment, containing potential binding sites of miR-217, was amplified by PCR. Mutations were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). Either the wild-type or mutant FBXO21-3′UTR fragment was inserted into the psi-CHECKTM-2 vector (Promega, Madison, WI) downstream of the firefly luciferase gene with XhoI and NotI (Thermo Fisher Scientific). For the luciferase assay, cultured primary human NP cells were seeded at 3000 cells per well in a 96-well plate. Cells were cotransfected with WT- or mutant-type FBXO21 3′UTR-Luc reporter plasmid and miR-control or miR-217 using Lipofectamine PLUSTM reagent (Invitrogen). Cell lysates were harvested 48 h after transfection, and luciferase activity was assayed with the Dual-Glo Luciferase Assay system (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) after 48 h. Experiments were performed in triplicate and repeated at least three times independently.
After processing the cells according to the treatment conditions and time specified for each group, each plate was replenished with 50 μmol/L EdU (Sigma–Aldrich) medium. For 2 h, the cells were grown in a 37 °C, 5% CO2 incubator and then rinsed twice with PBS. The cells were fixed with 4% paraformaldehyde, 0.2% glycine was added, and the cells were rinsed with PBS for 5 min. The membranes were ruptured for 10 min with 0.5% Triton-100 and rinsed with PBS, and each well was replenished with 100 μL of Apollo staining reaction solution. The plate was incubated at room temperature in the dark for 30 min on a shaker and then rinsed with 0.5% Triton-100 and 100 μL methanol and PBS. The cells were stained with DAPI for 20 min before being washed with PBS and examined under a fluorescence microscope.
Fluorescence in situ hybridization
Complementary locked nucleic acid (LNA) probes for miR-217 were tagged with 5′ and 3′-digoxigenin (Exiqon, Woburn, MA, USA). FISH detection was performed on NP tissue from IDD patients. After the removal of a section, the gene break probe was added dropwise. The section was then placed on the hybridization instrument for 10 min at 75 °C and then incubated overnight at 42 °C. It was removed the next day and rinsed for 5 min at room temperature and 3 min at 72 °C. It was then dried, and DAPI was added dropwise to completely cover the slide. To examine and capture the image, a fluorescence microscope (Olympus IX-81; Olympus, Tokyo, Japan) was used. The percentages of miR-217 cells in three representative high-power fields from a single sample were then analysed.
Using the DAVID bioinformatics program (https://david.ncifcrf.gov/tools.jsp), GO analysis was conducted to predict the effects of downregulated mRNAs on intervertebral discs. Cytoscape (v.3.6.1) was used to design and analyse a miRNA-hub gene network to investigate the relationship between possible target genes and DE-miRNA candidates. TargetScan (http://www.targetscan.org/), miRanda (http://www.microrna.org/), PicTar (https://pictar.mdc-berlin.de/), PITA (https://genie.weizmann.ac.il/), and RNA22 (https://cm.jefferson.edu/rna22/) were used to determine the target genes of miR-217.
Flow cytometry (FCM)
Apoptosis was determined using FITC-Annexin V and ethidium iodide (PI, 556547, BD Biosciences). Referring to the kit instructions for the operating technique, the percentage of apoptosis based on the fluorescence intensity was calculated.
Protein lysates were prepared from cultured primary human NP cells using RIPA buffer supplemented with protease and phosphatase inhibitors. The protein concentration of each group of cells was determined using the BCA technique, and 20 μg/well of protein was electrophoresed at 6 to 12% SDS–PAGE. The electrophoresis-separated proteins were deposited onto PVDF membranes. After incubation with the first antibody, a second antibody against rabbit immunoglobulin G (ab99697, Abcam, Cambridge, USA) was added, incubated at room temperature for 1 h, and developed with ECL reagent (Thermo Fisher Scientific, Inc.). The grey value of each protein band was analysed using ImageJ software (National Institutes of Health) to calculate the optical density.
The primary antibody information is as follows: anti-Col II antibody (ab34712, Abcam, Cambridge, USA), anti-aggrecan antibody (ab36861, Abcam, Cambridge, USA), anti-ADAMTS5 antibody (ab41037, Abcam, Cambridge, USA), anti-MMP13 antibody (ab39012, Abcam, Cambridge, USA), anti-MMP3 antibody (ab52915, Abcam, Cambridge, USA), anti-FBXO21 antibody (A16107, ABclonal, Wuhan, China), anti-ERK antibody (ab72100, Abcam, Cambridge, USA), anti-pERK antibody (ab229912, Abcam, Cambridge, USA), and anti-GAPDH antibody (ab8245, Abcam, Cambridge, USA).
Protease and phosphatase inhibitors were added before use. Cells were collected in lysis buffer (25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40). The lysate was centrifuged and immunoprecipitated using protein agarose beads and a primary antibody. Precipitated proteins and initial whole-cell lysates were boiled in an SDS loading buffer, separated on an SDS-polyacrylamide gel, transferred to a PVDF membrane, and incubated with primary and secondary antibodies.
After the cells were seeded on glass slides in a 6-well culture plate, they were transfected the following day, and 4% paraformaldehyde was applied to the glass slides 48 h later to fix the cells on the glass slides. This was followed by incubation at 4 °C for an additional 20 min with 0.5% Triton X-100, 30 min of blocking with normal goat serum, and finally the addition of the primary antibody. These cells were treated with FITC-labelled secondary antibody at room temperature for 1 h in the dark before being washed. The nuclei were counterstained with DAPI, washed, mounted, and viewed under a fluorescence microscope to determine their structure. Detailed information on the primary antibodies is provided below: anti-Col II antibody (ab34712, Abcam, Cambridge, USA) and anti-MMP13 antibody (ab39012, Abcam, Cambridge, USA). GAPHA anti-rabbit IgG (ab150077, Abcam, Cambridge, USA) was used as a secondary antibody.
An IDD model was generated in this work using AF needle puncture on mice (12-week-old, male, C57BL/6, Bar Harbour Jackson Laboratory, USA) [8, 9]. The tail disc was chosen as the IDD model because of its anatomical accessibility and low surgical morbidity [10,11,12]. Ketamine (100 mg/kg) was chosen as the anaesthetic for mice undergoing surgery and was administered via intraperitoneal injection. After achieving general anaesthesia, the mouse was positioned on the left side, and the model surgery was performed. A short longitudinal skin incision from Co6 to Co8 was made to assist in locating the disc position for needle insertion in the tail. Next, a syringe needle was used to puncture the Co6–Co7 coccygeal discs. The syringe needle was introduced vertically into the Co6–Co7 disc and then rotated 180° in the axial direction and held for 10 s. The puncture was made parallel to the endplates through the AF into the NP using a 31-G needle, which was inserted 1.5 mm into the disc to depressurize the nucleus. The other segments were left undisturbed for contrast. All procedures were approved by the ethics committee of Wuhan University Renmin Hospital.
We separated 48 male mice that had undergone IDD surgery into four groups for treatment tests (12 mice in each group). On days 3, 7, 14, and 21, a total of 10 μL of a solution containing agomiR-217/antagomiR-217 or its negative control (RiboBio, Guangzhou, China) was slowly injected into the discs multiple times. The treatments were performed as follows: (1) IDD + agomiR NC group, (2) IDD + agomiR-217 group, (3) IDD + antagomiR NC group, and (4) IDD + antagomiR-217 group. To determine the transfection efficiency of agomiR-217/antagomiR-217 or their negative controls labelled with Cy3, in vivo fluorescence imaging was performed 24 and 72 h after injection using an IVIS 200 Imaging system (Xenogen, Calliper Life Science, MA, USA). At 12 weeks after IDD surgery, the intervertebral discs of each group were collected for radiographic and histological examination.
Histological and radiographic evaluation
Mouse intervertebral discs were fixed in 10% neutral formalin buffer for 1 week, decalcified in EDTA decalcification solution, and then sectioned. Then, using an Olympus BX51 microscope, the histological images were evaluated using haematoxylin, eosin, and saffron O-type green staining (Olympus Centre Valley, PA, USA). An improved histological grading system for intervertebral disc degeneration was created following a review of the literature on the subject [13,14,15,16,17,18,19]. More specifically, the cellularity and morphology of the AF, NP, and the border between the two structures were examined. The scale was based on 5 categories of degenerative changes with scores ranging from 0 points (0 in each category) for a normal disc to 15 points (3 in each category) for a severely degenerated disc. For the morphology of the NP, score 0: round shape and the NP constitutes >75% of the disc area, score 1: round shape and the NP constitutes 50–75% of the disc area, score 2: round shape and the NP constitutes 25–50% of the disc area, and score 3: round shape and the NP constitutes <25% of the disc area. For the cellularity of the NP, score 0: stellar-shaped cells with a proteoglycan matrix located at the periphery, evenly distributed; score 1: partially stellar and partially round cells, more stellar than round; score 2: mostly large, round cells, separated by dense areas of proteoglycan matrix; and score 3: large, round cells, separated by dense areas of proteoglycan matrix. For the morphology of the AF, score 0: well-organized collagen lamellae with no ruptures; score 1: inward bulging, ruptured, or serpentine fibres constitute <25% of the AF; score 2: inward bulging, ruptured, or serpentine fibres constitute 25−50% of the AF; and score 3: inward bulging, ruptured, or serpentine fibres constitute >50% of the AF. For the cellularity of the AF, score 0: fibroblasts comprise >90% of the cells, score 1: fibroblasts comprise >75–90% of the cells, score 2: intermediate, and score 3: chondrocytes comprise >75% of the cells. For the border between the NP and AF, score 0: normal, without any interruption, score 1: minimal interruption, score 2: moderate interruption, and score 3: severe interruption.
Twelve weeks after the injection, radiographs were collected. The disc height index (DHI) was used to determine the change in IVD height. The DHI was calculated by dividing the mean of the three measurements from the midline to the boundary of the middle 50% of disc width by the mean of the two neighbouring vertebral body heights. The percentage change in the DHI of punctured discs was calculated as follows: % DHI=post-punctured DHI/prepunctured DHI × 100.
All statistical analyses were performed using SPSS 19.0 (SPSS Inc., Chicago, IL), and graphs were generated using GraphPad Prism 5 Software (Graph Pad Software, Inc., La Jolla, CA, USA). For the qRT–PCR results, we employed the Mann–Whitney U test. Unpaired two-tailed Student’s t tests were applied to evaluate the difference between two sets of data. Among multiple groups, one-way analysis of variance (ANOVA) coupled with Tukey’s post hoc test was applied to evaluate the difference. Pearson’s correlation test was employed to evaluate the associations between the expression of miR-217 and the disc degeneration grade of patients (Pfirrmann scores). A P value of <0.05 was considered statistically significant.