1. Cell Culture and Osteogenic Differentiation
The human multiple myeloma cell lines RPMI8226, and MM1.S were cultured in RPMI1640 supplemented with 10% fetal bovine serum at 37 °C in a 5% CO2 atmosphere, the medium was renewed every three days. MC3T3-E1 were cultured in DMEM supplemented with 10% fetal bovine serum and 1% L-glutamine at 37 °C in a 5% CO2 atmosphere. 24-well multiwell insert System with 1.0 µm pore PET Membrane was used for establishing cell co-culture model, MC3T3-E1 were seeded in 12-well plates. MC3T3-E1 were seeded in the lower chamber and MM cells were seeded in the upper chamber, respectively. After co-culture for 24 h, the MM cells were removed and MC3T3-E1 were cultured with renewed medium for further study. For osteogenic differentiation, MC3T3-E1 were cultured in α-MEM containing 50uM ascorbic acid 2-phosphate, 5 mM β-glycerophosphate, and 100 nM dexamethasone.
2. Trantsient transfection
MiR-302b mimics and inhibitor were obtained from RiboBio. For the miR-302b mimic and inhibitor transfection, MM1.S and RPMI8226 cells at a concentration of 2 × 104 cells/well were incubated in 6-well plates for 24 h. Then, the cells were transfected with 50 nM miR-302b mimic or 100 nM inhibitor in Opti-MEM medium without FBS addition of Lipofectamine 3000, according to the manufacturer’s protocol.
3. MTT assay
MTT assay was used to detect the MM cell viability. Multiple myeloma cells(5000 cells per well) were seeded in 96-well plates in RPMI1640 medium. 24 h after the cells were seeded, trantsient transfection was performed according to the manufacturer’s protocol. After 24 h, 48 h and 72 h transfection, 10 µl of 5 mg/ml MTT solution was added to each well and then the cells were incubated for 4 h at 37 °C, 5% CO2. Cells in 96-well plates were centrifuged at 1,000 × g for 10 min and then the medium was removed. 100µL DMSO was added to each well and the plates were shaked on the oscillator dissolve for 10 min to dissolve the formazan crystals. Optical density (OD) of each well was measured at 570 nm using a microplate Reader(PE Enspire).
4. Flow cytometry detection:
Annexin V-FITC Apoptosis Detection Kit(CA1020) was obtained from Beijing Solarbio Science & Technology Co., Ltd. (China). Briefly, 5 × 105 sample cells were collected and washed with 4℃ PBS three times, and resuspended in 200 µl Binding Buffer. In the darkness, 10 µl Annexin V-FITC and 10 µl PI were mixed and added into the cell suspension to react for 15 minutes at room temperature. Then 300 µl Binding Buffer was added and the samples were detected by flow cytometry (Cytoflex, Beckman) within 1 hour.
5. Alizarin red staining and quantitative analysis of mineralization
After 10 days osteogenic differentiation, MC3T3-E1 cells were washed twice with PBS and then fixed with 4% paraformaldehyde for 5 min. Cell plates were washed with deionized water twice and then stained with 1% Alizarin (pH 4.1) Red S solution for 20 min. The deposited Alizarin Red S solution in each well was collected and mixed with 10% cetylpyridinium chloride (Sigma-Aldrich) and evaluated for absorbance at 560 nm. Then the plates were washed with water three times and were photographed under light microscope. The orange and red positions were identified as Calcium deposits.
6. Quantitative Real-time PCR(qPCR)
Total RNA was extracted from cells by using TRIzol Regent (Invitrogen). The RNA was reverse transcribed using a Transcriptor Universal cDNA Master (Roche) according to the manufacturer’s instructions. Real-time PCR was performed as manufacturer's instructions (Applied Biosystems). PCR amplification was carried out on CFX Connect Real-Time PCR Detection System(Bio-Rad, Richmond, CA). Primer sequences: miR-302b forward 5′-ATCCAGTGCGTGTCGTG-3′, reverse 5′-TGCTTAAGTGCTTCCATGTT-3′;. U6 was used as an internal control of miR-302b.U6 forward 5′-CTCGCTTCGGCAGCACATATACT-3′, reverse 5′- ACGCTTCACGAATTTGCGTGTC-3′. Other target genes used β‐actin as an internal control. All the experiments were repeated three times.
7. Dual-luciferase reporter assay
The wild-type (WT) sequence of DKK1 mRNA 3′UTR(WT-DKK1-3′UTR) and mutation (MUT) sequence of DKK1 mRNA 3′UTR with deletion of the miR‐302b binding sites(MUT-DKK1-3′UTR) were designed consequently. Luciferase report vectors were constructed. For dual luciferase reporter detection, miR-302b mimics was co-transfected with wide type or mutant pmirGLO-3’UTR vectors into RPMI 8226 or MM1.S cells, respectively. After 48 h of transfection, the fluorescence activity was detected using Dual‐Glo® Luciferase Assay Reagent (Promega, USA) following the standard protocol on GloMax® 20/20 Luminometer (Promega, USA).
8. Western blot detection
Cells were harvested and lysed in the RIPA buffer (Cell Signaling Technology, 9806), After the lysates were sonicated, the protein concentration was determined by the BCA assay. Proteins were separated by SDS–PAGE and transferred electrophoretically onto a PVDF membrane (Millipore, IPVH00010). Each antibody incubation was performed overnight at 4 °C followed by the secondary antibody treatment for 1 h at room temperature. We used primary antibodies against Bcl-2(Abcam,1:5000), Bax(Abcam༌1:3000), and DKK1(Abcam༌1:3000), Runx2(Abcam༌1:3000)༌ β-catenin(Abcam༌1:5000). We used β-actin(Abcam༌1:2000) as an internal control for all analyses. Immunoblotting was visualized using ECL system and analyzed using Image-Pro Plus 6.0.
9. MM bone destruction mouse model
The animal study was approved by the ethical committee of Zhongnan Hospital of Wuhan University. All of the procedures for the animals in this study were performed in accordance with the Declaration of Helsinki. MM1.S cells (0.5–1 × 106 cells/100 µL in PBS) were injected into the femur of NOD/ SCID(4 weeks, female) mice, PBS was injected into the contralateral side as a control. When the weight of mice droped significantly, 40 ul hsa-miR-302b agonist (10 mM) with multiple myeloma of NOD/SCID mice were injected in the femurs every 3d in 2 weeks, then execute mice and isolation of femurs in mice were used to detect bone destruction by Micro CT. All animal experiments were performed with approval from the Animal Study Committee of WuHan University and conformed to the relevant guidelines and legislations.
10. Micro CT detection
Micro-CT analysis was performed using R_mCT2 (SkyScan1278, Bruker) with an isometric resolution of 40 µm, or ScanXmateL090 with an isometric resolution of 12 µm. The micro-CT files were reconstructed as TIFF images and transferred to TRI/FCS-BON for the quantitative analysis. We measured the mineralized tissue volume using a calibration curve obtained from MicroCT Bar Pattern NANO Phantom.
11. Histology and Immunohistochemistry.
To perform histological analyses of induced osteoblastic lesions, H&E and von Kossa staining were performed using paraffin and undecalcified sections respectively. Immunohistochemistry was performed on frozen sections as previously mentioned. Sections were examined using a fluorescence microscope (TE2000U + NT88, NIKON) or a confocal microscope (µSurf, Nanofocus).
12. Statistical Analyses.
All the data were presented as the means ± SEM. We performed parametric statistics by Student’s t test, one-way ANOVA with Tukey’s HSD test, nonparametric statistics by the Mann–Whitney U test, or the Kruskal–Wallis test using R v3.3.1. The values were considered significant at P < 0.05. The results were representative of more than three individual experiments.
13. Clinical MM specimens
The study was approved by the Ethics Committee of Zhongnan Hospital of Wuhan University and conducted in accordance with the Declaration of Helsinki. Twelve pairs of MM patients and healthy individuals’ bone marrow were collected from the Department of Orthopedics of Zhongnan Hospital of Wuhan University from February 2019 to July 2020. All of the specimens were obtained with the patients' informed consent. All tumor samples were pathologically diagnosed as MM according to the World Health Organization (WHO) Classification of myeloid and lymphoid neoplasms.