Department of Respiratory, Qingdao Eighth People's Hospital, Qingdao, Shandong, China.
Department of Orthopedic Surgery, Qingdao University, Qingdao, Shandong, China.
Yonsei Med J. 2019 Aug;60(8):751-759. doi: 10.3349/ymj.2019.60.8.751.
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).
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.
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.
Silencing ANCR promotes the osteogenesis of PMOP osteoblast cells. The specific binding of ANCR with EZH2 suppressed RUNX2, thereby inhibiting osteogenesis.
本研究旨在探讨长链非编码 RNA（lncRNA）反分化非编码 RNA（ANCR）对绝经后骨质疏松症（PMOP）成骨细胞成骨的作用及其机制。
建立 PMOP 小鼠模型。通过实时荧光定量聚合酶链反应（qRT-PCR）和激光共聚焦显微镜分别检测 ANCR 表达和细胞内钙离子。通过 siRNA-ANCR（si-ANCR）转染沉默 PMOP 小鼠成骨细胞中的 ANCR。分别通过 MTT 和流式细胞术分析成骨细胞的增殖和凋亡。通过碱性磷酸酶（ALP）和茜素红染色试验分别检测 ALP 活性和钙结节。通过 qRT-PCR 和 Western blot 检测增强子的锌指蛋白 2（EZH2）、 runt 相关转录因子 2（RUNX2）和 OSTERIX 的表达。此外，构建了小鼠成骨模型，通过苏木精-伊红（HE）染色观察类骨质形成。通过 RNA 下拉试验进一步鉴定 lncRNA-ANCR 与 EZH2 之间的相互作用。
PMOP 小鼠中 ANCR 表达和细胞内钙离子增加。Si-ANCR 显著增加成骨细胞的增殖、ALP 活性、钙沉积，减少凋亡。Si-ANCR 下调 ANCR 和 EZH2，而上调 RUNX2 和 OSTERIX。Si-ANCR 还促进了羟磷灰石-三钙磷酸盐处理的小鼠类骨质形成。此外，ANCR 特异性结合 EZH2。
沉默 ANCR 促进 PMOP 成骨细胞的成骨作用。ANCR 与 EZH2 的特异性结合抑制 RUNX2，从而抑制成骨作用。
