Patient sample
Blood samples were drawn from two patients for gene sequencing.
Cell lines and reagents
The human bone marrow-derived mesenchymal stem cells (hBMSCs) used in the experiment were obtained from the stem cell bank at the Chinese Academy of Sciences (No. SCSP-405). The hBMSC cells were maintained in Dulbecco's Modified Eagle Medium (Gobco, Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco) in a cell incubator (37 °C, 5% CO2).
The osteogenic induction fluid formula was as follows: 10% fetal bovine serum Dulbecco's Modified Eagle Medium (low glucose), 1 × 10-8 mol/L dexamethasone, ascorbic acid 2 × 10-4 mol/L, and beta-phosphate glycerol 10 mM/L.
Real-time PCR
The hBMSCs were osteogenically induced for 0, 7, 14, and 21 days. Total RNA was extracted using the TRIzol method; reverse transcription was performed using Prime ScriptTM RT Master Mix (Perfect Real Time; Takara, Otsu, Japan), and gene expression was detected via TB GreenTM Premix Ex TaqTM (Takara) following manufacturer’s protocols. The primers used in the experiment were all synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China).
Plasmid construction and cell transfection
The NBPF1 and NBPF15 plasmids used in the experiment were constructed by Hanyin Biotechnology (Shanghai) Co., Ltd. (Shanghai, China) using the following steps. The synthetic primers were designed to amplify the target fragment and ligated into the enzyme through the restriction sites at both ends. After excision overexpressing the lentiviral vector; the ligation product was transferred into the prepared bacterial competent cells, and the grown monoclonal colonies were identified by PCR. The positive colonies were identified by PCR and used for sequencing and identification by comparing with the correct clones. A successful target gene was constructed to overexpress a lentiviral vector. The vector used was CMV-MCS-3XFlag-PGK-Puro, restriction site 1: XhoI, restriction site 2: EcoRI. This vector was transfected into hBMSCs using the virus solution prepared by the company, with polybrene as a transfection reagent, and the expression levels of NBPF1 and NBPF15 were detected using real-time PCR (Fig. 1a). The constructed overexpressing cells were used in the experiment, and the control group was transfected with empty plasmid. Gene expression of the osteogenic indicators alkaline phosphatase (ALP), runt-related transcription factor (RUNX2), collagen type I alpha 1 chain (COL1a), and osteopontin (OPN) was detected via real-time PCR (Fig. 2).
Small interfering RNA construction and cell transfection
The NBPF1 and NBPF15 small interfering RNAs (siRNAs) were synthesized by Guangzhou Ruibo Biotechnology Co., Ltd. (Guangdong, China) and used to transfect hBMSCs to silence NBPF1 and NBPF15 genes. Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used as a transfection reagent. The specific steps were as follows: When the cells were fused at 70–80% cell confluency, the siRNA was dissolved in 50 μL of Opti-mem serum-free medium. In addition, 2 μL of Lipofectamine 2000 was dissolved in another tube with 50 μL of Opti-mem serum-free medium, mixed, and allowed to stand at room temperature for 5 min. The two tubes were mixed and left to stand for 20 min. The medium was changed to serum-free medium during transfection, and the mixture was added to the corresponding wells (the final concentration of siRNA was 100 nm). After 6 hours, the medium was replaced with serum medium. Total RNA was extracted at 48 hours, and siRNA silencing efficiency was detected by real-time PCR (Fig. 1b). The siRNA with the best silencing effect was used in subsequent experiments. The control group was transfected with a negative control provided by the company. Osteoblasts were induced for 0, 7, 14, and 21 days. Real-time quantitative polymerase chain reaction was used to detect the expression of the osteogenic indicators ALP, RUNX2, and OPN (Fig. 3). AR staining and ALP staining results counted using Image J[13].
Statistical analysis
Whole-exome sequencing data analysis
The sequencing readings generated by the Hiseq series were mapped to the human genome (hg19) using BWA software [14]. The resulting bam files were then imported to VarScan2 software 9[15], and germline variants were called using the following parameters: min-coverage: 20 min-var-freq: 0.08, and p value: 0.05. The fpfilter module of VARSCAN was applied to identify false positive variations. SnpEff and Variant Effect Predictors 10 and 11 were used to annotate the filtered variants. Further, we applied the following criteria to filter germline variants: (1) depth of alternative alleles: >5; (2) allele frequency of the alternative allele in the population: <0.001; (3) variant ratio of the alternative allele: >0.3; (4) impact severity = “HIGH” or “MED”; and (5) the prediction result of POLEPHEN2 was not “benign,” and the prediction result of SIFT was not “tolerated.”
Real-time PCR
The real-time PCR data were analyzed using the 2-ΔΔCT method normalized to the mean Ct values of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase. Two-fold changes in gene expression (both decreases and increases) were considered statistically significant. The ∆Ct value for the control group was set at 1. The expression level of each indicator gene was calculated.
Staining statistics
The image was counted stained area using Image J. Three fields were selected for each well to calculate the average value. Three replicate wells were used in the statistical analysis; P < 0.05 was considered statistically significant.