Human blood sample
Five pairs of human blood samples were obtained from patients who underwent Surgical treatment between 2017 and 2018 and were diagnosed with Neurofibromatosis type I (NF1) with scoliosis based on a computed tomography(CT),Magnetic Resonance Imaging(MRI), CT Mylography(CTM) or histopathological evaluation. The matched control blood was obtained from volunteer. The samples were snap‑frozen in liquid nitrogen and stored at ‑80˚C until use. No local or systemic treatment was conducted on these patients prior to surgery.
The use of human blood samples followed internationally recognized guidelines, as well as local and national regulations. All research carried out on human participants followed international and national regulations. Ethics approval for this study was obtained from the Medical Ethics Committee and all participants provided written informed consent prior to enrollment.
Antibodies
Primary antibodies were obtained from the following sources: GPR56 (ab77515, 1:1000), IGFBP3 (ab77635, 1:1000), Ras(ab52939, 1:1000), Raf1(ab137435, 1:1000), Phospho Raf1(ab173539, 1:1000), ALP(ab83259, 1:1000), OCN(ab13420, 1:1000), OPN(ab8448, 1:1000), Runx2(ab192256, 1:1000), NFATc1(ab2796, 1:1000), C-Fos(ab190289, 1:1000) were obtained from Abcam Inc. MEK1/2(#4694, 1:1000), Phospho MEK1/2(#9127, 1:1000), ERK1/2(#9102, 1:1000), Phospho ERK1/2(9101, 1:1000), anti-mouse secondary IgG anti-goat and anti-rabbit secondary IgG anti-goat antibodies were received from Cell Signaling Technology, Inc. (1:2000).
Cell culture
The rat Schwann cells(RSC96), mouse embryonic osteoblasts cells(MC3T3) and the mouse mononuclear macrophage leukemia cells(RAW264.7) were obtained from the National Infrastructure of Cell Line Resource (Cell resource center, institute of basic medicine, Chinese academy of medical sciences) and were cultured in Dulbecco's Modified Eagle's Medium (DMEM-H, Hyclone, USA) supplemented with 10% fetal bovine serum (Gibco, USA) at 37˚C with 5% CO2. The medium was changed every two days.
Osteoclast differentiation and TRAP staining
Raw264.7 cells were seeded in 24-well plates at a density of 2.5 ×104 cells/well. After (4-6)h culturing, media were added recombinant mouse RANKL (Sigma, USA) to 50 ng/mL of the concentration ; the cultures were incubated at 37℃for 7 days. Cells were fixed with 4% paraformaldehyde and stained for TRAP activity as described previously[34]; the plates were then scanned using an Inverted microscope (CKX31) (Olympus, Japan).
RSC96, MC3T3 and Osteoclast were transfected with ADGRG1-siRNA
The Rat/Mouse-siRNA pool against ADGRG1(ADGRG-R-siRNA & ADGRG-M-siRNA) was synthesized from Guangzhou Ruibo Biotechnology Co., Ltd. (Guangzhou, China). Transfection was conducted using RiboFECTTM Transfection Reagent (Ruibo, Guangzhou, China) according to the instructions. After 72h, the transfected cells were collected for experimental determination.
RNA Sequence
Total RNAs isolated from blood samples (NF-1 with scoliosis patients and healthy people) using TRIzol reagent (Invitrogen, USA). RNA integrity and concentration were determined using Nanodrop and agarose gel electrophoresis. The purified RNA samples, with RIN (RNA Integrity Number) over 8.0, determined by Agilent 2100 Bioanalyzer (Agilent, Waldbroon, Germany) were sequenced at NovoGene corporation(Beijing, China), who constructed their digital gene expression libraries and sequenced by means of Illumina HiSeq™ 2500 platform to obtain the expression libraries of 150-nt read length. Independent triplicate cultures were sampled. The clean reads were mapped against human genome hg19 with less than two-base mismatching, using Tophat (version 2.1.1b). The normalized expression values for each gene were calculated by FPKM (expected number of Fragments per Kilo base of transcript sequence per Million base pairs sequenced). Differential expression gene was determined by Cuffdiff package (v2.2.1). The transcript levels of genes having a P-value of less than 0.05 were significantly differential between two groups. Then, we found the gene (P≤ 0.05, logFC≥1 or logFC≤-1) to further research by the NCBI PubMed(https://www.ncbi.nlm.nih.gov/pubmed/).
Quantitative real-time PCR
According to the manufacturer's instructions, TRIzol reagent (Invitrogen, Carlsbad, California) was using for isolating the total RNA of cells. First-strand cDNA was synthesized from 1 μg of total RNA by incubation for 1 hour at 42°C with Superscript III reverse transcriptase (Invitrogen) following oligo (dT) priming. After the reverse transcription reaction, an ABI PRISMR500 system (ABI, USA) was applied to measure the real-time polymerase chain reaction (RT-PCR) according to the manufacturer's instructions. The conditions for RT-PCR were as follows: denaturation at 95°C for 10 seconds and 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. A dissociation stage was added to the end of the amplification procedure. No nonspecific amplification was observed, as determined using the dissociation curve. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The data were analyzed using the comparison Ct (2‐ΔΔCt) method and expressed as the fold change relative to the respective control. Each sample was analyzed in triplicate.
Western blot analysis
We collected the cells from the plates and then used lysis buffer supplemented with protease inhibitors (10 mg/mL leupeptin, 10 mg/mL pepstatin A, and 10 mg/mL aprotinin) for extracting total protein about 30 minutes. Protein samples were collected by centrifugation at 12 000g at 4°C for 15 minutes. Then the Micro Bicinchoninic acid (BCA) Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) was using for determining protein concentrations. In addition, 20 μg of total protein extract was then subjected to 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to poly vinylidene fluoride(PVDF) membranes (Millipore, USA). After being blocked with 5% BSA, the membranes were incubated with according antibodies overnight at 4°C. A horseradish peroxidase-conjugated secondary antibody was added and visualized using an enhanced chemiluminescence detection system (Millipore, Billerica, Massachusetts) as recommended by the manufacturer.
CCK8 assay
Cell proliferation was measured by CCK8 dye reduction assay. Briefly, the transfected cells were transfected (blank control group, siRNA NC group, the siRNA - 1 group, 2 group and siRNA- 3 groups) with high-DMEM containing 10% FBS culture solution, formed cell suspension, and respectively inoculated into 96- well plates, 100μl /holes, each group of five hole. Then, at 24 h, 48 h, 72 h and 96 h, samples were token, 10μl per hole CCK8 solution was added avoid light, continuing to culture 1 h. The absorbance was measured at 450 nm using a microplate reader.
Flow cytometric analysis
After 48 h of transfection, the cells cultured in 12-well plates were collected and fixed in 70% ethanol at −20 °C overnight. The flow cytometric analysis was carried on a BD AccuriC6 flow cytometer (BD Biosciences, San Jose, CA, USA) with a Cell Cycle Analysis Kit (Nanjing kaiji biotechnology co., LTD, China). The data were processed using the FlowJo7.6 software (Treestar Incorporated, Ashland, OR, USA).
Statistical analysis
Statistic comparisons between results from multiple groups were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett's test. For experiments involving two groups, an unpaired Student's t-test was performed. A value of P < 0.05 was considered statistically significant. Data are expressed as mean ± standard deviation (SD). A P-value < 0.05 was considered statistically significant.