Silencing of LncRNA-ANCR Promotes the Osteogenesis of Osteoblast Cells in Postmenopausal Osteoporosis via Targeting EZH2 and RUNX2

Abstract

Purpose: This study aimed to explore the effects and mechanisms of long non-coding RNA (lncRNA) anti-differentiation non-coding RNA (ANCR) on the osteogenesis of osteoblast cells in postmenopausal osteoporosis (PMOP).

Materials and methods: Mice models of PMOP were established. ANCR expression and intracellular calcium ions were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and laser confocal microscopy, respectively. ANCR was silenced in osteoblast cells from PMOP mice by the transfection of siRNA-ANCR (si-ANCR). The proliferation and apoptosis of osteoblast cells was analyzed by MTT and flow cytometry, respectively. Alkaline phosphatase (ALP) activity and calcium nodules were examined by ALP and alizarin red staining assay, respectively. The expression of enhancer of zeste homolog 2 (EZH2), runt related transcription factor 2 (RUNX2), and OSTERIX was detected by qRT-PCR and Western blot. Furthermore, an osteogenesis model was constructed in mice, and osteoid formation was observed by hematoxylin-eosin (HE) staining. The interaction between lncRNA-ANCR and EZH2 was further identified by RNA pull-down assay.

Results: ANCR expression and intracellular calcium ions were increased in PMOP mice. Si-ANCR significantly increased the proliferation, ALP activity, calcium deposition of osteoblast cells and decreased apoptosis. ANCR and EZH2 were down-regulated by si-ANCR, while RUNX2 and OSTERIX were upregulated. Si-ANCR also promoted osteoid formation in mice treated with hydroxyapatite-tricalcium phosphate. In addition, ANCR specifically bound to EZH2.

Conclusion: Silencing ANCR promotes the osteogenesis of PMOP osteoblast cells. The specific binding of ANCR with EZH2 suppressed RUNX2, thereby inhibiting osteogenesis.

Keywords: EZH2; Postmenopausal osteoporosis; RUNX2; lncRNA-ANCR; osteoblast; siRNA.

Fig. 1. Long non-coding RNA anti-differentiation noncoding RNA (lncRNA-ANCR) expression analysis based on a mouse postmenopausal osteoporosis (PMOP) model. The PMOP model was constructed using three groups: osteoporosis group, control group, and sham group (n=15 each group). (A) Bone mineral density comparison using microcomputed tomography. (B) Laser confocal microscopy detection for calcium level (×100, Fluo8 fluorescent probes are labeled). (C) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis for lncRNA-ANCR mRNA and protein expression in osteoblast cells of the PMOP model. X-axis in C represents lncRNA-ANCR relative mRNA expression levels; Y-axis presents different groups in the PMOP model. ^*p<0.001 vs. Control group and Sham group.

Fig. 2. mRNA expression of long non-coding RNA anti-differentiation noncoding RNA (lncRNA-ANCR) in osteoblast cells transfected with siRNA-ANCR (si-ANCR). X-axis presents relative lncRNA-ANCR expression; Y-axis presents the three groups in postmenopausal osteoporosis model. ^*p<0.001 vs. NC-ANCR (negative control group, cell line was transfected with lncRNA-ANCR negative control) group and BLANK group (blank control group, cells without treatment).

Fig. 3. Effects of long non-coding RNA anti-differentiation noncoding RNA (lncRNA-ANCR) transfected with siRNA-ANCR (si-ANCR). (A) The effect of lncRNA-ANCR on proliferation evaluated by MTT assay. (B) The effect of lncRNA-ANCR on apoptosis determined by flow cytometry. (C) The effect of lncRNA-ANCR on alkaline phosphatase (ALP) activity detected by ALP staining. X-axis represents the relative lncRNA-ANCR expression; Y-axis presents the three groups in the postmenopausal osteoporosis model. (D) The effect of lncRNA-ANCR on calcium deposition upon alizarin red staining. ^*p<0.05, ^†p<0.01, ^‡p<0.001 vs. NC-ANCR group (negative control group, cell line was transfected with lncRNA-ANCR negative control) and BLANK group (blank control group, cells without treatment). PI, propidium iodide; FITC, fluoresceine isothiocyanate; OD, optical density.

Fig. 4. In vivo experiments show the regulatory effect of long non-coding RNA anti-differentiation noncoding RNA (lncRNA-ANCR) on osteoid formation. (A) Hematoxylin-eosin (HE) staining of osteoid formation in mice injected with siRNA-ANCR (si-ANCR)-transfected osteoblast cells (bar=200 µm). Black and gray arrows indicate hydroxyapatite-tricalcium phosphate and osteoblast cells, respectively. (B) Osteoid/Total area (%). ^*p<0.01 vs. NC-ANCR group (negative control group, cell line was transfected with lncRNA-ANCR negative control) and BLANK group (blank control group, cells without treatment).

Fig. 5. Expression of enhancer of zeste homolog 2 (EZH2), runt related transcription factor 2 (RUNX2), and OSTERIX in osteoblast cells transfected with siRNA-anti-differentiation noncoding RNA (si-ANCR). (A) Quantitative real-time polymerase chain reaction (qRT-PCR) results for the mRNA and protein expression of EZH2, Runx2, and Osterix. (B and C) Western blot results for mRNA and protein expression of EZH2, RUNX2, and OSTERIX. ^*p<0.001 vs. NC-ANCR group (negative control group, cell line was transfected with lncRNA-ANCR negative control) and BLANK group (blank control group, cells without treatment). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 6. The interaction between long non-coding RNA anti-differentiation noncoding RNA (lncRNA-ANCR) and enhancer of zeste homolog 2 (EZH2). (A) Expression of EZH2 in ANCR and ANCR-Mut detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. (B) The expression of ANCR in EZH2 and EZH2-control detected by qRT-PCR and Western blot. ^*p<0.001, lncRNA-ANCR group vs. lncRNA-ANCR-Mut group, EZH2 group vs. EZH2-control group.