The present study aimed to investigate the effects of microRNA (miR)-21 on osteoclastogenesis and its underlying molecular mechanisms. The expression levels of tartrate-resistant acid phosphatase (TRAP) and miR-21 were detected during osteoclastogenesis in receptor activator of NF-κB ligand (RANKL)-induced RAW264.7 cells via reverse transcription-quantitative PCR. Bioinformatics and dual luciferase reporter assays were performed to analyze the association between miR-21 and Pten. RANKL-induced RAW264.7 cells were divided into the following groups: MiR-negative control (NC), miR-21 mimic, miR-21 inhibitor and miR-21 mimic + LY294002. The effects of miR-21 on osteoclastogenesis and bone resorption were then detected using TRAP staining and a bone resorption assay. Pten, phosphorylated-Akt and nuclear factor of activated T cell (NFATc1) expression levels were measured by western blotting to analyze the effects of miR-21 on the PI3K/Akt signaling pathway. The present study revealed that miR-21 was upregulated during osteoclastogenesis in RANKL-induced RAW264.7 cells. Furthermore, miR-21 negatively regulated Pten. Compared with the miR-negative control (NC) group, the number of osteoclasts and the percentage of bone resorption were increased in the miR-21 mimic group, whereas they were decreased in the miR-21 inhibitor group. The number of osteoclasts and the percentage of bone resorption in the miR-21 mimic + LY294002 group were lower than in the miR-21 mimic group. Compared with the miR-NC group, the protein expression levels of Pten were decreased, whereas p-Akt and NFATc1 were increased in the miR-21 mimic group. Conversely, Pten protein expression was increased, whereas p-Akt and NFATc1 were decreased in the miR-21 inhibitor group. In the miR-21 mimic + LY294002 group, Pten protein expression was higher, and p-Akt and NFATc1 were lower than in the miR-21 mimic group. In conclusion, miR-21 is upregulated during osteoclastogenesis, and may promote osteoclastogenesis and bone resorption through activating the PI3K/Akt signaling pathway via targeting Pten.
Osteoclasts (OCs) are primarily derived from mononuclear macrophages that arise from pluripotent hematopoietic stem cells; as the only cell with bone resorption function, OCs coordinate the dynamic balance between bone resorption and osteoblast formation (
Numerous studies have reported that microRNAs (miRNAs/miRs) serve important roles during the process of osteoclastogenesis, including miR-503 (
In the present study, bioinformatics analysis predicted that Pten was a target gene of miR-21. Pten is a negative regulator of PI3K; Pten dephosphorylates PI3K, resulting in reduced PI3K/Akt signaling activity (
The murine macrophage cell line RAW264.7 was purchased from the American Type Culture Collection and was cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin (100 U/ml)/streptomycin (100 µg/ml) (Hyclone; GE Healthcare Life Sciences) in an incubator containing 5% CO2 at 37°C. RAW264.7 cells were cultured with 50 ng/ml RANKL (Thermo Fisher Scientific, Inc.) for 3–5 days to induce osteoclastogenesis.
TargetScan (version 7.2;
miR-NC (5′-CAGUACUUUUGUGUAGUACAA-3), miR-21 mimic and miR-21 inhibitor (5′-UCAACAUCAGUCUGAUAAGCUA-3′) were synthesized and obtained from Guangzhou RiboBio Co., Ltd. RAW264.7 cells at the logarithmic phase were seeded into 6-well plates and separated into the following groups: MiR-NC (cells transfected with 25 µM miR-NC), miR-21 mimic (cells transfected with 25 µM miR-21 mimic), miR-21 inhibitor (cells transfected with 25 µM miR-21 inhibitor) and miR-21 mimic + LY294002 [cells transfected with miR-21 mimic and subsequently treated with the PI3K inhibitor LY294002 (10 µM; APExBio Technology) and incubated at 37°C for 48 h]. At 70% confluence, the cells were transfected using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. At 48 h post-transfection at 37°C with 5% CO2, >60% cells were successfully transfected. Prior to subsequent reverse transcription-quantitative PCR (RT-qPCR), TRAP staining and western blotting experiments, the cells were cultured with 50 ng/ml RANKL for 3 days at 37°C with 5% CO2 to induce osteoclastogenesis.
Total RNA was extracted from cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol and was reverse transcribed using the SuperScript First Strand cDNA system (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. PCR amplification was performed using the SYBR Green PCR master mix (Thermo Fisher Scientific, Inc.). Stem-loop RT-qPCR and conventional RT-qPCR were used for quantification of miR-21, and tartrate-resistant acid phosphatase (TRAP; a specific marker of OCs and bone resorption) and Pten, respectively. The primers used in the present study were synthesized by Sangon Biotech Co., Ltd. (
RANKL-induced RAW264.7 cells were seeded at a density of 5×105 cells/well into 24-well plates and cultured for 24 h at 37°C with 5% CO2. Subsequently, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature. TRAP staining solution was added and incubated at 37°C for 1 h in the dark. The procedure was performed according to the TRAP staining kit protocol (Sigma Aldrich; Merck KGaA). TRAP-positive multi-nucleated cells (TRAP+ MNCs) containing >3 nuclei were considered OCs and were observed under an inverted light microscope (magnification, ×200). The number of TRAP+ MNCs/well was independently counted under the inverted microscope by two individuals. The average of the three experiments was taken as the number of OCs present.
A pit assay was performed to observe the bone resorption of OCs. Bovine bone slices were generated in the laboratory and were obtained from fresh bovine cortical bone. Bone was purchased from cows that were sacrificed for commercial/consumer purposes. Notably, cows are not an endangered species, and the bones were obtained from a licensed source and did not contain potentially harmful agents (biological and chemical or genetically modified material). Bovine cortical bone was dried, rough-cut and sliced using a low speed diamond saw into pieces ~50 µm thick and 6 mm in diameter. Subsequently, the bone slices were treated in distilled water for ultrasonic cleaning prior to use. At 48 h post-transfection and prior to treatment with RANKL, RAW264.7 cells were seeded onto the bovine bone slices in 24-well plates at 5×105 cells/well and were cultured in routine medium for 24 h at 37°C with 5% CO2. Subsequently, the medium was changed to routine medium supplemented with 50 ng/ml RANKL for 3 days, and then the bovine bone slices were ultrasonicated at 30 Hz for 10 min at room temperature in 1 mol/l NH4OH to remove adherent cells. Bone slices were stained with 0.1% toluidine blue at room temperature for 3–5 min and the bone resorption pits were subsequently observed under a light microscope (magnification, ×400). Bone resorption was calculated as pit area/total bone area of each slice, and was calculated using ImageJ software (version 1.8.0; National Institutes of Health).
Total protein was extracted from cells using a Total Protein Extraction kit (Beiing Solarbio Science and Technology Co., Ltd.), according to the manufacturer's protocol. The bicinchoninic acid method was used to measure protein concentration and 40 µg protein/lane was separated by SDS-PAGE on 10% gels and transferred to PVDF membranes (EMD Millipore). PVDF membranes were blocked in TBS containing 0.25% Tween and 5% skimmed milk for 1 h at room temperature. Membranes were incubated with primary antibodies targeted against: Pten (cat. no. ab32199; 1:1,000), Akt (cat. no. ab18785; 1:5,000), phosphorylated (p)-Akt (cat. no. ab38449; 1:1,000), NFATc1 (cat. no. ab25916; 1:500) and GAPDH (cat. no. ab9485; 1:5,000) at 4°C overnight. All primary antibodies were purchased from Abcam. Membranes were then incubated with horseradish peroxidase-conjugated IgG secondary antibodies (cat. no. ab6721; 1:10,000; Abcam) for 1 h at room temperature. ECL detection reagent (Beiing Solarbio Science and Technology Co., Ltd.) was used to observe protein bands and relative protein expression levels were calculated using ImageJ software (version 1.8.0; National Institutes of Health) with GAPDH as an internal reference.
Each experiment was repeated at least 3 times. SPSS software (version 19.0; IBM Corp.) was used to analyze all data. Kolmogorov-Smirnov test was used to confirm that data were normally distributed and data are presented as the mean ± SD. Comparisons among multiple groups were assessed by one-way ANOVA followed by Tukey's post hoc test. An unpaired Student's t-test was used to assess the differences between the two groups in the dual-luciferase assay. P<0.05 was considered to indicate a statistically significant difference.
The mRNA expression levels of the OC marker TRAP were detected in RANKL-induced macrophage RAW264.7 cells at different time points by RT-qPCR, in order to analyze osteoclastogenesis (
Pten promotes the proliferation of T cells and tumor cells, and has a critical role in RANKL-induced OC differentiation (
In order to verify the effects of miR-21 on Pten, the expression levels of miR-21 and Pten were detected in RANKL-induced RAW264.7 cells post-transfection by RT-qPCR and western blotting (
TRAP staining was used to detect osteoclastogenesis of RAW264.7 cells transfected with miR-21 mimic and miR-21 inhibitor, in order to assess the effect of miR-21 on OC differentiation (
The bone resorption assay was used to detect the percentage of bone resorption (
It has previously been indicated that Pten regulates osteoclastogenesis in RANKL-induced RAW264.7 OC precursors via activating the PI3K/Akt signaling pathway (
Kagiya and Nakamura (
As a carcinogenic miRNA, miR-21 is upregulated in almost all types of cancer, and is responsible for promoting their occurrence and development, including gastric cancer (
At present, the molecular mechanism underlying the effects of miR-21 on the regulation of osteoclastogenesis remains unclear. Sugatani
In conclusion, the present study revealed that miR-21 was upregulated in osteoclastogenesis, and promoted osteoclastogenesis and bone resorption via activating the PI3K/Akt signaling pathway through targeting Pten. These results provided preliminary evidence towards elucidating the molecular mechanism underlying how miR-21 promotes osteoclastogenesis, laying a theoretical foundation for the clinical treatment of diseases associated with bone metabolism, including osteoporosis and osteomalacia.
Not applicable.
The present study was funded by the Natural Science Foundation of China (grant. no. 81471558).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request, and in the TargetScan (
SW and XW designed the study. SW drafted the manuscript. ZL performed TRAP staining and the bone resorption assay. JW analyzed and verified the target gene of miR-21. XJ performed western blotting. ZY acquired, analyzed and interpreted the data. XW revised the manuscript. All authors approved the final published version of this manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Expression of miR-21 and TRAP during osteoclastogenesis. Relative expression levels of (A) TRAP and (B) miR-21 in RANKL-induced RAW264.7 cells. *P<0.05 vs. day 1; #P<0.05 vs. day 3. miR-21, microRNA-21; RANKL, receptor activator of NF-κB ligand; TRAP, tartrate-resistant acid phosphatase.
miR-21 targets Pten. (A) Sequence alignment of miR-21 with the 3′-untranslated region of Pten and results of a dual luciferase reporter gene assay. Relative expression levels of (B) miR-21, (C) Pten protein and (D) Pten mRNA post-transfection. *P<0.05 vs. miR-NC. miR, microRNA; MT, mutant; NC, negative control; WT, wild type.
TRAP staining and number of osteoclasts. (A) miR-NC group, (B) miR-21 mimic group, (C) miR-21 inhibitor group and (D) miR-21 mimic + LY294002 group. Arrows indicate osteoclasts (TRAP+ multi-nucleated cells). *P<0.05 vs. miR-NC; #P<0.05 vs. miR-21 mimic. Scale bars, 50 µm. miR, microRNA; NC, negative control; TRAP, tartrate resistant acid phosphatase.
Bone resorption areas on bovine bone slices. (A) miR-NC group, (B) miR-21 mimic group, (C) miR-21 inhibitor group and (D) miR-21 mimic + LY294002 group. Arrows indicate bone resorption areas. *P<0.05 vs. miR-NC; #P<0.05 vs. miR-21 mimic. Scale bars, 100 µm. miR, microRNA; NC, negative control.
Relative protein expression levels of Pten, p-Akt, Akt and NFATc1. *P<0.05 vs. miR-NC group; #P<0.05 vs. miR-21 mimic group. miR, microRNA; NC, negative control; NFATc1, nuclear factor of activated T-cell; p, phosphorylated.
Effect of miR-21 on osteoclastogenesis. miR-21 regulated the PI3K/Akt signaling pathway in osteoclast differentiation by targeting Pten. miR, microRNA; RANK, receptor activator of NF-κB; RANKL, RANK ligand; IKB, IκB kinase; NFATc1, nuclear factor of activated T cell.
Primers sequence for reverse transcription-quantitative PCR.
Sequence (5′→3′) | ||
---|---|---|
Gene | Forward | Reverse |
miR-21 | 5′-CTCGCTTCGGCAGCACA-3′ | 5′-GCCGCTAGCTTATCAGACTCAACA-3′ |
U6 | 5′-CTCGCTTCGGCAGCACA-3′ | 5′-AACGCTTCACGAATTTGCGT-3′ |
TRAP | 5′-ACACAGTGATGCTGTGTGGCAACTC-3′ | 5′-CCAGAGGCTTCCACATATATGATGG-3′ |
Pten | 5′-AATTCCCAGTCAGAGGCGCTATGT-3′ | 5′-GATTGCAAGTTCCGCCACTGAACA-3′ |
GAPDH | 5′-AACATCAAATGGGGTGAGGCC-3′ | 5′-GTTGTCATGGATGACCTTGGC-3′ |
miR, microRNA; TRAP, tartrate-resistant acid phosphatase.