1. Patient Materials
Non-degenerated IVD samples and degenerative IVD samples were abtained from idiopathic scoliosis patients and patients with IVDD undergoing disectomy, respectively. All patients performed routine magnetic resonance imaging (MRI) of spine before surgery. This study had been approved by the Ethics Committee of West China Hospital, Sichuan University and the Ethics Approvals have been provided. Informed consent was obtained from all subjects and all methods were carried out in accordance with relevant guidelines and regulations.
2. Clinical Evaluation of Disc Degeneration with Magnetic Resonance Imaging (MRI)
The disc degeneration degree was analyzed according to T2-weighted images by using the modified Pfirrmann classification.
3. Animals and Experimental Groups
The miR-17-92flox/flox mouse strain used in this study was supported by The Jackson Laboratory and the collagen II (Col2a1)-Cre mouse strain was supported by The Maine Medical Research Center in The United States. By hybridization of miR-17-92flox/flox with Col2a1-Cre mouse strain, miR-17-92-ccKO mice were obtained with specific knockout of miR-17-92 in chondrocytes, including NPCs. The mice of the control group in this study were C57BL/6 mice (WT), purchased from Beijing HFK Bioscience CO., LTD. All experimental protocols of the animal part of this study had been approved by the Animal Ethics Committee of West China Hospital, Sichuan University and the Ethics Approvals have been provided. All methods were carried out in accordance with relevant guidelines and regulations.
4. Histopathologic Analysis
Parts of mouse IVD tissues (Co6-Co8) were extracted for the histopathologic analysis. The target IVDs were excised, then fixed with 4% paraformaldehyde and decalcified with Ethylene Diamine Tetraacetic Acid (EDTA) solution, embedded with paraffin, and cut into 5um-thick section. The disc specimens were staind with hematoxylin-eosin (HE) and Safranin O staining.
5. Histological Evaluation of Intervertebral Disc Degeneration
Histological assessment of the degree of IVDD was based on Tam's report. The whole scoring system is divided into three parts to evaluate the histological appearance of NP, AF and NP/AF. The specific scoring system can be referred to Table-1.
6. Terminal-Deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL)
Apoptosis levels of NP tissues in each group were measured utilizing a TUNEL kit (Takara Bio Tech CO., LTD, Beijing, China) following the manufacturer’s instructions. Green fluroescence (FITC) indicated DNA fragments and the blue fluroescence (DAPI) indicated cell nucleus. The percentage cells undergoing apoptosis to the total number of NP cells was calculated and considered the apoptotic index.
7. Real-Time Fluorogenic Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from nucleus pulposus tissues using Animal Total RNA Isolation Kit (Foregene CO., LTD, Chengdu, China). The ratio of A260/A280 and concentration of RNA were determined using a Nanodrop ultraviolet spectrophotoeter (Thermo CO., LTD, Waltham, MA, USA), with RNA stored at -80℃. For the routine mRNA qRT-PCR, the total RNA (50ng) was reversely transcribed into cDNA with the PrimeScriptTM RT Reagent Kit (TaKaRa CO., LTD, Beijing, China). β-actin was used as an internal reference standard. After incubating at 95℃ for 30s, the qRT-PCR reaction conditions were set to 40 cycles of “95℃ for 10s + 60℃ for 30s”, and the incubation time at 95℃ for 5s. Primers of functional genes used in the study were listed in Table-2. For the miR qRT-PCR, the total RNA (50ng) was reversely transcribed into cDNA with the mix of 8 RT primers, including U6, miR-17-3p, miR-17-5p, miR-18a-5p, miR-19a-3p, miR-19b-3p, miR-20a-5p and miR-92a-3p (RiboBio CO., LTD, Guangzhou, China) and the PrimeScriptTM RT Reagent Kit (TaKaRa CO., LTD, Beijing, China). U6 was used as an internal reference standard. After incubating at 95℃ for 10min, the qRT-PCR reaction conditions were set to 40 cycles of “95℃ for 5s + 60℃ for 20s + 70℃ for 10s”, and the incubation time at 95℃ for 5s. qRT-PCR analyses were performed using TB Green Premix Kit (TaKaRa CO., LTD, Beijing, China) on the QuanStudioTM 12K Flex Real-Time PCR System (Thermo CO., LTD, Waltham, MA, USA). The 2-△△Ct method was applied to quantify the relative expression levels of targets genes.
8. Western Blot Analysis
Total protein of each group of NP tissue was extracted with RIPA buffer, and the BCA kit (EpiZyme, Shanghai, China.) was used to detect the protein concentration. Protein samples were separated by electrophoresis using a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). After processing in a 5% defatted milk in TBST, the blots were incubated with anti-β-actin, anti-GAPDH, anti-Akt, anti-p-Ser473-Akt, anti-Bcl-2, anti-Bax, anti-Caspase-3, anti-Cleaved-Caspase-3 (ProteinTech, Wuhan, China.), diuted from 1:500 to 1:1000, at 4℃, for over 12 hours. β-actin and GAPDH served as the internal reference. After washing with TBS containing Tween 20, the appropriate secondary antibody was incubated with the blots at 25℃ for 60min, treated with Chemiluminescence Kit (EpiZyme, Shanghai, China.) for 30s, and placed in a dark room for observation. Gray values were analyzed with Image J software v1.46.
9. Statistical Analysis
All statistical analyses were performed using SPSS 17.0 (SPSS, Chicago, Illinois). The results are presented as mean ± standard deviation. The differences between various groups were analyzed using Student T test. P＜0.05 was considered to indicate statistical significance.