Establish osteoporosis mice model
Male C57BL/6J mice (6 months old) were provided by VitalRiver Laboratory Animal Co.Ltd (Beijing, China). These animals were maintained under the condition of 21°C, with 12 h light and 12 h dark cycle. To establish unloading model, the mice were suspended by the tail at an angle of approximately 30° with their forelimbs touching the floor which allowed them to move and access food and water for free. The tail suspension lasted for 3 weeks. Finally, these mice were sacrificed using overdose anesthesia (100mg/kg pentobarbital). The bilateral femurs were dissected for Micro-computed tomography (µCT) analysis. Animal studies were all approved by the Ethics Committee of China-Japan Union Hospital of Jilin University and were performed according to the approved guidelines. Then the bone tissues were collected and analyzed using Micro-CT scanner (Scanco Medical AG, Switzerland).
Simulated microgravity treatment
2D Rotating Wall Vessel Bioreactor (RWVB) clinostat was used to simulate microgravity. 1 × 105 of MC3T3-E1 cells were seededon cell climbing pieces. 1×105 MC3T3-E1 cells were seeded on coverslips. After culture for 24 h, the climbing pieces were placed in a box 12.5 mm away from therotational axis. After the air bubbles were removed, the chambers were fixed in the clinostat and rotated around a horizontal axis at 28 rpm for 15 min. The vertical rotation groups were used as controls. The rotation process was taken at 37°C under 5% CO2.
The mouse pre-osteoblast cell line MC3T3-E1 was purchase from American Type Culture Collection (ATCC, USA). Cells were cultured in α-MEM medium supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, 100 µg/mL streptomycin and 110 mg/mL sodium pyruvate under 5% CO2 at 37°C in a humidified atmosphere. For osteogenic differentiation, a specific cultu was purchase from Cell bank of Chinese Academy of Sciences and cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, 100 µg/mL streptomycin and 110 mg/mL sodium pyruvate under 5% CO2 at 37°C in a humidified atmosphere. For osteogenic differentiation, a specific culture medium containing 100 nM dexamethasone, 50 µM ascorbic acid (Sigma), and 10 mM β-glycerophosphate was used.
Empty vector (pcDNA3.1), MALAT1 (pcDNA3.1-MALAT1), siRNA (si)-nc, si-MALAT1, mimic negative control (nc), miR-485-5p mimic, inhibitor nc, miR-485-5p inhibitor, si-WNT7B were synthesized by Hanbio (Shanghai, China). MC3T3-E1 cells were seed in 6-well plates and transiently transfected using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA) according to the instructions. After incubation of 6 h, the medium was replaced by a complete medium.
Total RNA was isolated via TRIzol reagent (Invitrogen, Carlsbad, CA, USA). After the concentration determined the absorbance (A)260/A280 ratio, RNA was reverse transcribed to cDNA using SuperScript reverse transcriptases (for mRNA; Thermo Fisher Scientific, Waltham, MA, USA) and One Step PrimeScript miRNA cDNA Synthesis Kit (for miRNA; Takara, Tokyo, Japan). qPCR for mRNA was conducted by SYBR PCR Master Mix (GenePharma, Shanghai, China) with the conditions of 95℃ for 3 min; 40 cycles of 95℃ for 12 s and 62℃ for 40 s. For miRNA, qPCR was performed using SYBR Premix Ex Taq II (Perfect Real Time) (Takara, Tokyo, Japan) with the condition of 95℃ for 10 s, 40 cycles of 95℃ for 5 s and 60℃ for 20 s. All samples were repeated three times. The expression level of genes was calculated using 2−ΔΔCT method. GAPDH and U6 were used as the internal control for mRNA and miRNA respectively.
Luciferase reporter assay
The luciferase reporter vector pGL3.1 carrying wild type binding cite (MALAT1-wt or WNT7B-wt) or mutant type binding cite (MALAT1-mut or WNT7B-mut) were constructed by RiboBio (Guangzhou, China). 293T cell was transfected with the MALAT1-wt or MALAT1-mut along with miR-485-5p mimic or mimic control. Renilla luciferase plasmid was transfected as a internal control. 48 h later, according to the instructions of the dual luciferase detection kit, a microplate reader was used to assess the firefly and Renilla luciferase activity in each group. The ratio of firefly fluorescence intensity of Renilla fluorescence intensity reflects the relative fluorescence intensity of each group.
The biotinylated probe of miR-485-5p and the control probe were synthesized by Shenggong Biotech (Shanghai, China). Then the probe were incubated the probe with streptavidin-coated beads (Invitrogen, Carlsbad, CA, USA) at 25°C for 2h to generate the probe-coated beads. The streptomycin beads were capable of binding to biotin. Cells were lysed to extract total RNA. After pretreatment of magnetic beads, RNA and beads were mixed. After separation, qPCR was used to quantify the relative expression of MALAT1 or WNT7B.
The total proteins were extracted with lysis buffer containing protease inhibitor and phosphatase inhibitor cocktail (Sigma, CA, USA). 40 µg proteins were separated in 10% SDS-PAGE gel and then transferring onto a polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Subsequently, the membranes were blocked in 5% non-fat dry milk in TBST buffer for 1 h at room temperature. Thereafter, the blots were incubated with the primary antibodies 4°C overnight followed by incubating with the horseradish peroxidase conjugated secondary antibody for another 2 h at room temperature. Finally, the blots were visualized using an enhanced chemiluminescence kit (Beyotime, Shanghai, China). The bands were quantified using ImageJ software.
Alkaline phosphatase (ALP) activity
The ALP activity of cells was evaluated using a Alkaline Phosphatase Assay Kit (Beyotime, Shanghai, China) according to the manufacture’s instructios. Briefly, lysed cell were incubated with test buffer at 37°C for 10 min. 100 µl reaction stopping solution was used to terminate the reaction. A microreader (Biorad, USA) was used to assess the OD value at 490 nm.
The ALP staining was conducted in the dark and assayed with the BCIP/NBT Alkaline Phosphatase Color Development according to the manufacture’s instructions (Beyotime, Shanghai, China). The images were taken under a light microscope.
Alizarin red staining
After transfection, cells were collected and fixed with 4% paraformaldehyde for 15 min. After washed with PBS thrice, cells were cultured with osteogenic induction medium for 3 weeks and then stained with alizarin red regents for 30 min. Calcified nodules were captured using a microscope and calculated using spectrophotometric wavelength of 570 nm.
After transfection and treatment, cells were washed with PBS thrice. Then the cells were stained with 2.5 µg/ML of Annexin V-FITC and propidium iodide and incubate at room temperature in the dark for 15 minutes. Finally, the cells were captured with a fluorescent microscope and evaluated by a FACSVerse flow cytometer system (BD, USA)with FlowJo software.
All the data are presented as means ± standard deviation (SD). The statistical analyses were performed using SPSS version 17. Data were analyzed with one-way ANOVA and Student's test. P < 0.05 was considered statistically significant. Pearson analysis was employed to evaluate the correlations between MALAT1 and miR-485-5p as well as between miR-485-5p and WNT7B.