Characterization of patients. The present study was approved by The Fifth Affiliated Hospital, Southern Medical University, Shanghai Tenth People’s Hospital, Tongji University and Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (approval no. XHEC-D-2015-112). Spinal ligament tissues were obtained during surgery using intraoperative aseptic techniques. Patients who underwent anterior cervical decompression surgery were enrolled in this study. A total of 37 posterior longitudinal ligament specimens were obtained from The Fifth Affiliated Hospital, Southern Medical University and Shanghai Tenth People's Hospital, Tongji University. The OPLL group included patients with a radiographic diagnosis of OPLL involving the cervical spine, as well as symptoms, such as neck pain and numbness in the extremities. By contrast, the non-OPLL group included patients with cervical spinal fractures, who had undergone anterior decompression surgery. Non-OPLL patients did not have OPLL, cervical spondylosis or stenosis. The inclusion and exclusion criteria for patients referenced previous method[19, 20]. Demographics of these patients are provided in Table I. All experiments were performed in The Fifth Affiliated Hospital, Southern Medical University and Shanghai Tenth People’s Hospital, Tongji University. All patients provided written informed consent between January 2015 and June 2017, and the research was approved by The Fifth Affiliated Hospital, Southern Medical University Ethics Committee.
Reagents and antibodies. Monodansylcadaverine (MDC) was purchased from Sigma-Aldrich; Merck KGaA. Polyclonal antibodies against Beclin1, and LC3 were obtained from Cell Signaling Technologies, Inc. β-actin, vimentin and keratin antibodies were obtained from Abcam.
Measurement of cell viability. Cell viability was examined using 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo-phenyl)-2H-tetrazolium (Beyotime Institute of Biotechnology), according to the manufacturer’s protocols. Briefly, the ligament fibroblasts were plated in 96-well culture plates and cultured in osteogenic differentiating medium for 1, 2, 3, 4, 5, 6, 7 or 8 days. Cell viability assays were performed and the absorbance of optical densities were measured at each time point and detected by a microplate spectrophotometer at 450 nm.
Specimen processing and cell isolation. During the anterior cervical decompression surgery, 37 posterior longitudinal ligament specimens were obtained. To avoid contamination with osteoblasts or osteocytes, the ligaments were extracted carefully, cut up into 1 mm2 pieces and washed with PBS several times. Subsequently, the specimens were divided into two parts. One was stored in liquid nitrogen for RT-qPCR analysis. The remaining were washed with normal saline, plated in 35-mm culture dishes, and maintained in Dulbecco’s modified Eagle’s media supplemented with 10% fetal bovine serum (Gibco, USA). All assays were carried out on fifth passage cell cultures.
RT-qPCR. Total RNA was obtained from the posterior longitudinal ligament specimens or the ligament fibroblasts, according to our previous study design. Expression of Beclin1, LC3, ULK1, COL I, OCN and ALP were examined by RT-qPCR, according to our previous method [16]. For PCR amplification, specific oligonucleotide primers of rat sequences were designed on the basis of sequences in GenBank (Table II).
Western blotting. The protein expression of Beclin1 and LC3-II/I was detected by western blotting, according to our previous paper [15]. Briefly, total protein was extracted using a western blot kit (Beyotime Institute of Biotechnology). Each sample (50 µg/well) was separated on a 10% sodium dodecyl sulfate-polyacrylamide gel for Beclin1 protein and 12% sodium dodecyl sulfate-polyacrylamide gel for LC3 protein. Next, separated proteins were transferred to polyvinylidene difluoride membranes, followed by blocking with 5% non-fat milk for 1 h. Next, the polyvinylidene difluoride membranes were incubated with anti-Beclin1(Cat.#3495; CST, USA) (dilution 1:500) or anti-LC3 antibodies (Cat. #83506; CST, USA) (dilution 1:200) and detected using the ECL detection kit (Beyotime Institute of Biotechnology). The blots were quantified by densitometry using Image Lab version 2.1 software (Bio-Rad Laboratories, Inc.).
Knockdown of Beclin1 in ligament fibroblasts. siRNAs specifically targeting Beclin1 were constructed and designed by superbiotek (Shanghai, China). To target Beclin1, the following sequences were used: Forward, 5’- GAGCGAUGGUAGUUCUGGAGG-3’ and reverse, 3’-UCCAGAACUACCAUCGCUCUG-5’. A missense siRNA vector (lack of complementary sequences) served as a non-silencing control (siControl). All transfections were carried out using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions.
Levels of autophagy and apoptosis quantified by flow cytometry. The rates of apoptosis were analyzed by flow cytometry in the present study, according to our previous method [16]. The rates of autophagy were analyzed by flow cytometry according to the method of Bursch et al [21] and Shen et al [22]. Briefly, the cells were incubated with 0.05 mM MDC (Cat. #30432; Sigma-Aldrich, USA) in PBS at 37 °C for 10 minutes and then the intracellular MDC was measured by flow cytometry within 30 minutes. The autophagy incidence was determined as the percent of MDC positive cells automatically analyzed using FlowJo software (Tree Star, San Carlos, CA) and the unstained cells were used as control.
Immunofluorescence for vimentin and keratin. The ligament fibroblasts were seeded on 6-well plates with sterile glass cover slip, fixed with 4% paraformaldehyde and permeabilized in 0.1% Triton X-100 for 20 min. Subsequently, the ligament fibroblasts were incubated with primary anti-vimentin (Cat.#5741; CST, USA) (dilution 1:200) or anti-keratin antibodies (Cat.#13063; CST, USA) (dilution 1:200) for 2 h. This was followed by incubation with an Alexa Fluor® 488-conjugated secondary antibody (Cat. #4408; CST, USA) (dilution 1:200) for 1 h, and images were taken with a fluorescence microscope (Olympus Corporation).
Immunofluorescence for LC3 and Beclin1 protein. The ligament fibroblasts were seeded on 6-well plates with sterile glass cover slips and transfection of LC3 protein fused with green fluorescent protein plasmid (GFP-LC3) was carried out using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), as previously described [23]. Subsequently, cells were incubated for 72 h and then fixed with 4% paraformaldehyde for 15 min, followed by incubation with anti-Beclin1(Cat.#3495; CST, USA) (dilution 1:200) in 5% bovine serum albumin (Cat. # ST023; Beyotime, China) overnight at 4˚C. Subsequently, the ligament fibroblasts were incubated with Alexa Fluor® 594-conjugated secondary antibody (Cat.#8889; CST, USA) (1:100) and then observed by fluorescence microscopy (Olympus Corporation).
Statistical analysis. All data were from at least three independent experiments and presented as the mean ± SD. Differences in mRNA expression among the OPLL and non-OPLL groups were analyzed using a t-test, with SPSS 13 (SPSS, Inc.). Multiple comparisons of data among the groups were determined by the one-way ANOVA followed by Dunnett’s test. Pearson correlation coefficients were used to analyze the correlation between parameters of autophagy level and osteogenic makers. P < 0.05 was considered to indicate a statistically significant difference.