Clinical samples
Bone tissues were collected from osteoporosis patients (n = 15) and healthy controls (n = 15) at the Affiliated Changzhou No.2 People’s Hospital of Nanjing Medical University between March 2016 and April 2018. Bone fragments extracted from the transcervical region of the femoral neck were dissected into smaller fragments, washed three times in PBS and stored at − 80 °C until further analysis. Written consents from all patients were collected before this work. This study was approved by the ethics committee of the Affiliated Changzhou No.2 People’s Hospital of Nanjing Medical University.
Cell culture and osteogenic differentiation induction
Human bone mesenchymal stem cells (hBMSCs) were obtained from the BeNa Culture Collection (BNCC, Beijing, China). The hBMSCs cultured in α-MEM supplemented with 10% fetal bovine serum (FBS), 100 mg/ml penicillin and 100 U/ml streptomycin in an incubator with 5% CO2 at 37 °C. To induce osteogenic differentiation, 10 µmol/L dexamethasone, 200 µM ascorbic acid and 10 mmol/L β-glycerophosphate were added, and the induction medium was changed every 3 days.
Cell transfection
The small short hair RNA (shRNA) targeting LINC00899 (shLINC00899) with its negative control (shNC), miR-374a mimics with its negative control (NC mimics), and miR-374a inhibitor with its negative control (NC inhibitor) were purchased from GenePharma (Shanghai, China). Cell transfection was carried out by Lipofectamine 2000 (Invitrogen).
Dual-luciferase reporter assay
Starbase (http://starbase.sysu.edu.cn/) was used to predict the binding sites between miR-374a and LINC00899 or RUNX2. The pmirGLO-LINC00899-WT/Mut and pmirGLO-RUNX2-WT/Mut reporters were obtained from GenePharma (Shanghai, China). Then miR-374a mimics or NC mimics was co-transfected with these above reporters into 293T cells. 48 h after transfection, the relative luciferase activity was detected using dual-luciferase reporter assay system (Promega).
RT-qPCR
Total RNAs were extracted from tissues and hBMSCs using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. The RNAs were reverse-transcribed to cDNAs through reverse transcriptase kit (Takara, Otsu, Japan) or the TaqMan® miRNA reverse transcription kit (Thermo Fisher Scientific). RT-qPCR was performed on the ABI 7900 Detection System (Applied Biosystems, USA) by using the SYBR-Green PCR Master Mix kit (Takara, Dalian, China). The primer sequences were as follows: LINC00899 forward, 5′-CAGTCAGCCTCAGTTTCCAA-3′ and reverse, 5′-AGGCAGGGCTGTGCTGAT-3′; miR-374a forward, 5′-GGTCACAGTGAACCGGTC-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′; RUNX2 forward, 5′-CTTATACAATGTCAACAGCC-3′ and reverse, 5′-TCCTTATGCTCTTTCTTCC-3′; GAPDH forward, 5′-CCACTCCTCCACCTTTGAC-3′ and reverse, 5′-ACCCTGTTGCTGTAGCCA-3′; and U6 forward, 5′-CTTCGGCAGCACATATACT-3′ and reverse, 5′-AAAATATGGAACGCTTCACG-3′.
RNA immunoprecipitation (RIP) assay
RIP assay was carried out using Magna RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA). Briefly, cell lysate was incubated in RIP buffer containing magnetic beads conjugating with the Ago2 antibody (Anti-Ago2, Abcam) or IgG antibody (Anti-IgG, Abcam). Subsequently, the enrichment of LINC00899 and miR-374a was determined by RT-qPCR.
Statistical Analysis
All experiments were repeated at least three times. The data were analyzed using Prism 6.0 (GraphPad Software, USA) and represented as mean ± standard deviation (SD). Student’s t-test was used to analyze the difference between the two groups. One-way ANOVA and Tukey's test were used to analyze the difference among multiple groups. P < 0.05 was considered statistically different.