miR-21 deficiency inhibits osteoclast function and prevents bone loss in mice

MicroRNAs emerge as critical post-transcriptional regulators in bone metabolism. We have previously reported in vitro that miR-21 promotes osteogenesis, while studies have also revealed miR-21 as a regulator of osteoclastogenesis and a promoter of osteoclast differentiation in vitro. However, in vivo data are still lacking in identifying skeletal function of miR-21, particularly its effects on osteoporosis. Here, using miR-21 knockout (miR-21−/−) mice, we investigated effects of miR-21 on bone development, bone remodeling and bone loss. Unexpectedly, miR-21−/− mice demonstrated normal skeletal phenotype in development and maintained osteoblastogenesis in vivo. Besides, miR-21−/− mice showed increased receptor activator of nuclear factor κB ligand (RANKL) and decreased osteoprotegerin (OPG) through miR-21 targeting Sprouty 1 (Spry1). Nevertheless, interestingly, miR-21 deficiency promoted trabecular bone mass accrual physiologically. Furthermore, in pathological states, the protection of bone mass was prominent in miR-21−/− mice. These skeletal effects were attributed to inhibition of bone resorption and osteoclast function by miR-21 deficiency through miR-21 targeting programmed cell death 4 (PDCD4), despite the existence of RANKL. As far as we know, this is the first in vivo evidence of a pro-osteoclastic microRNA. Together, these findings clarified function of miR-21 in bone metabolism, particularly uncovering osteo-protective potential of miR-21 inactivation in osteoporosis.

To induce osteogenic differentiation, BMMSCs at the 1 st passage were cultured in osteogenic inducing media containing 100 μg/ml ascorbic acid (MP Biomedicals, USA), 2 mM β-glycerophosphate (Sigma-Aldrich, USA) and 10 nM dexamethasone (Sigma-Aldrich, USA). Cells were plated at 2 × 10 5 cells/well in 12-well plates. The media were changed every 3 days. After induction for 7 days, alkaline phosphatase (ALP) staining was performed to determine ALP activity in osteogenesis 2 . After induction for 14 days, alizarin red staining was performed to determine the mineralization in osteogenesis 1-3 , and total RNA was collected by direct addition of Trizol Reagent (Takara, Tokyo, Japan) to replicate wells. The quantitative parameter of the percentage of mineralized area was determined wit h the ImageJ 1.47 software. RNA was further purified by phenol-chloroform extraction and reversed transcribed for cDNA synthesis, as described 3,4 .
Colony forming efficiency (CFE) assay of BMMSCs was conducted as stated before 5 .
After treated with ACK lysis buffer (Lonza, Switzerland) to remove red blood cells, murine primary bone marrow cells were plated in 5-cm culture dishes at a density of 1 × 10 5 cells/cm 2 and cultured. The formation of colonies was evaluated after 14 days of culture. The colonies were fixed with 4% paraformaldehyde for 30 min and stained with crystal violet for Page 4 5 min. Colonies with over 50 cells were counted using the ImageJ 1.47 software.
For proliferation analysis, BMMSCs at the 1 st passage were plated at 2 × 10 3 cells/well in 96-well plates. At the same time in Day-1, 4, 7 and 10 (3 wells per time point), cells were incubated with 20-μl 5 mg/ml methyl thiazolyl tetrazolium (MTT) (MP Biomedicals, USA) for 4 h. The precipitates were extracted with 180-μl DMSO and the cell viability was measured at the optical density (OD) of 490 nm.
Culture and analysis of murine osteoblasts. Osteoblasts from adult murine long bone were isolated and cultured according to the published protocol 6 . Briefly, long bones without bone marrow were harvested, cut into pieces, washed with PBS, and incubated with collagenase II shaking at 37℃ for 2 h. The bone pieces were then rinsed with PBS, plated, and cultured with α-MEM (Invitrogen, USA) supplemented with 20% FBS (Invitrogen, USA), 2 mM L-glutamine (Invitrogen, USA), 100 U/ml penicillin (Invitrogen, USA), and 100 g/ml streptomycin (Invitrogen, USA) in a humidified atmosphere of 5% CO 2 at 37℃. The media were changed every 3 days. At confluence, primary osteoblasts were passaged with 0.25% trypsin (MP Biomedicals, USA).
For receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) examinations, osteoblasts at the 1 st passage were plated at 2 × 10 5 cells/well in 12-well plates and cultured in 500-μl normal media for 24 h. siRNA transfections were performed as stated below, the media were then changed, and cells were then cultured in normal media for 24 h.
For extracellular signal-regulated kinase (ERK) signaling inhibition, the pharmacological inhibitor PD98059 was added into the culture media at a concentration of 10 μM 7,8 for 24 h.

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The conditional media were collected by centrifuging at 3000 rpm 10 min at 4℃ followed by 12000 rpm 10 min at 4℃ 9 . Total RNA was collected by direct addition of Trizol Reagent (Takara, Tokyo, Japan) to the culture plate. RNA was further purified by phenol-chloroform extraction and reversed transcribed for cDNA synthesis, as described 3,4 .
Culture and analysis of murine osteoclasts. Osteoclasts were cultured and differentiated according to standard protocols 4,10 . Briefly, after 24-h incubation of total bone marrow cells, nonadherent cells were collected, seeded at 1 × 10 6 cells/well in 24-well plates, and cultured with 20 ng/ml macrophage colony-stimulating factor (M-CSF) in α-MEM (Invitrogen, USA) supplemented with 10% FBS (Invitrogen, USA), 2 mM L-glutamine (Invitrogen, USA), 100 U/ml penicillin (Invitrogen, USA), and 100 g/ml streptomycin (Invitrogen, USA) in a humidified atmosphere of 5% CO 2 at 37℃. After 3 days, the adherent cells were used as bone marrow macrophages (BMMs). BMMs were further cultured with 20 ng/ml M-CSF in the presence or absence of 50 ng/ml RANKL. siRNA transfections were performed as stated below. Tartrate resistant acid phosphotase (TRAP) staining was performed to determine mature osteoclasts, and TRAP + multinucleated cells with over 3 nuclei were identified as mature osteoclasts 4 . Total RNA of mature osteoclasts was collected by direct addition of Trizol Reagent (Takara, Tokyo, Japan) to replicate wells. RNA was further purified by phenol-chloroform extraction and reversed transcribed for cDNA synthesis, as described 3,4 .
For resorption assay, as reported 11,12 , dentine slices with 4-mm length, 4-mm width and 200-μm thick were prepared by cutting human premolars with a low-speed cutting machine (SYJ-150, Kejing Instrument, China). Human healthy premolars extracted for orthodontic Page 6 needs were kindly provided by the Department of Orthodontics, School of Stomatology, the Fourth Military Medical University. Dentine slices were cleaned by ultrasonication in distilled water, sterilized using 75% ethanol, and plated in 24-well plates overnight under ultraviolet light. BMMs were harvested, seeded on dentine slices, and further cultured with 20 ng/ml M-CSF in the presence or absence of 50 ng/ml RANKL. siRNA transfections were performed as stated below. At the end of the test, cells were removed from dentine slices by wiping the surfaces, and the resorption pits were stained with 1 μg/ml toluidine blue 13 .
Percentages of pit area over total area were determined using the ImageJ 1.47 software.
siRNA transfection. siRNA transfections were performed according to our previous protocol 14 . siRNAs for Sprouty 1 (Spry1) and programmed cell death 4 (PDCD4) and their respective negative controls (NCs) were purchased from RiboBio (Guangzhou, China). After seeding of osteoblasts and BMMs and at 50%-70% confluent, the diluted siRNAs or NCs were combined with the supplied transfection reagent and added to each well according to the standard protocol. The siRNAs or NCs were transfected at final concentrations of 100 nM.
Cells were then incubated for 24 hours before further assays. Transfection efficacy tests were also performed after 24 h incubation, by direct addition of Trizol Reagent (Takara, Tokyo, Japan) to replicate wells. RNA was further purified by phenol-chloroform extraction and reversed transcribed for cDNA synthesis, as described 3,4 .
Quantitative real-time polymerase chain reaction (qRT-PCR) analysis. qRT-PCR analysis of microRNAs and mRNAs were according to previous studies 3, 15 . For microRNAs, qRT-PCR primers from the Bulge-loop TM miRNA qRT-PCR Primer Sets were designed by RiboBio (Guangzhou, China). For mRNAs, RNA was further purified by phenol-chloroform extraction and reversed transcribed for cDNA synthesis, and the primer sequences were listed in Supplementary Table S3. qRT-PCR were performed using the SYBR Premix Ex Taq II Kit (Takara, Japan) and detected by a Real-Time System (CFX96, Bio-Rad, USA). The relative expression level of each gene was obtained by the cycle number after normalizing against RNU6 (for miR-21) and ACTIN (for mRNAs) abundances using the 2 -ΔΔCT method.
Western blotting. Western blotting was performed as previously described 3 . Whole-cell lysates of BMMSCs underwent osteogenic induction for 14 days, osteoblasts after siSPRY1 transfection for 24 h, and mature osteoclasts were prepared using the Cell Lysis Buffer