Zhang Kun, Shi Zhemin, Ren Yi, Han Xiaohui, Wang Jingzhao, Hong Wei
Department of Histology and Embryology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2021 Jan 30;41(1):31-38. doi: 10.12122/j.issn.1673-4254.2021.01.04.
To investigate the regulatory role of long non-coding RNA Kcnq1ot1 in osteoclast differentiation, osteogenic differentiation and osteoporosis.
The expression of lnc-Kcnq1ot1, Bglap, Runx2, Alp, Bsp, Nfatc1, Mmp9, Ctsk and Oscar were detected by real-time quantitative PCR (qRT-PCR) in the femoral bones from mouse models of postmenopausal osteoporosis (ovariectomized mice, =8), disuse osteoporosis (induced by tail suspension, =14) and agerelated osteoporosis (18-month-old mice, =8), and also in MC3T3-E1 cells during osteoblast differentiation and in murine bone marrow-derived macrophages (BMMs) and RAW264.7 cells during osteoclast differentiation. MC3T3-E1 cells with lncKcnq1ot1 knockdown by lentivirus infection were induced to differentiate into osteoblasts using osteogenic induction medium, and the expression of lnc-Kcnq1ot1, Alp and Bglap was detected with qRT-PCR and ALP activity was assessed with ALP staining. BMMs and RAW264.7 cells were transfected with siRNAs targeting lnc-Kcnq1ot1 and stimulated with RANKL and/or M-CSF, and the expression of lnc-Kcnq1ot1, Ctsk and Oscar was detected by qRT-PCR, and TRAP activity was assessed by TRAP staining. The subcellular localization of lnc-Kcnq1ot1 in MC3T3-E1 and RAW264.7 cells was determined using cell fractionation followed by qRT-PCR.
The expression of lnc-Kcnq1ot1 was significantly upregulated during osteoblast differentiation but downregulated in the bone tissues of osteoporotic mice and during osteoclast differentiation ( < 0.05). Silencing lnc-Kcnq1ot1 obviously decreased the expression of Bglap and Alp ( < 0.05) and attenuated osteogenic medium-induced osteoblast differentiation. Knockdown of lnc-Kcnq1ot1 also promoted the expression of Ctsk and Oscar ( < 0.05) and aggravated RANKL-induced osteoclast differentiation. The results of cell fractionation and qRT-PCR demonstrated that lnc-Kcnq1ot1 was located mainly in the nuclei of MC3T3-E1 and RAW264.7 cells.
Our data demonstrate that lnc-Kcnq1ot1 promotes osteogenic differentiation and alleviates osteoclast differentiation, suggesting the potential of lnc-Kcnq1ot1 as a therapeutic target against osteoporosis.
探讨长链非编码RNA Kcnq1ot1在破骨细胞分化、成骨细胞分化及骨质疏松症中的调控作用。
采用实时定量PCR（qRT-PCR）检测绝经后骨质疏松小鼠模型（去卵巢小鼠，n = 8）、废用性骨质疏松小鼠模型（尾部悬吊诱导，n = 14）及年龄相关性骨质疏松小鼠模型（18月龄小鼠，n = 8）股骨中lnc-Kcnq1ot1、Bglap、Runx2、Alp、Bsp、Nfatc1、Mmp9、Ctsk和Oscar的表达，同时检测成骨细胞分化过程中MC3T3-E1细胞以及破骨细胞分化过程中鼠骨髓来源巨噬细胞（BMMs）和RAW264.7细胞中上述基因的表达。通过慢病毒感染敲低lncKcnq1ot1的MC3T3-E1细胞，使用成骨诱导培养基诱导其分化为成骨细胞，采用qRT-PCR检测lnc-Kcnq1ot1、Alp和Bglap的表达，通过碱性磷酸酶（ALP）染色评估ALP活性。用靶向lnc-Kcnq1ot1的小干扰RNA（siRNAs）转染BMMs和RAW264.7细胞，并用核因子κB受体活化因子配体（RANKL）和/或巨噬细胞集落刺激因子（M-CSF）刺激，采用qRT-PCR检测lnc-Kcnq1ot1、Ctsk和Oscar的表达，通过抗酒石酸酸性磷酸酶（TRAP）染色评估TRAP活性。采用细胞分级分离结合qRT-PCR确定lnc-Kcnq1ot1在MC3T3-E1和RAW264.7细胞中的亚细胞定位。
lnc-Kcnq1ot1在成骨细胞分化过程中表达显著上调，但在骨质疏松小鼠骨组织及破骨细胞分化过程中表达下调（P < 0.05）。沉默lnc-Kcnq1ot1明显降低Bglap和Alp的表达（P < 0.05），并减弱成骨培养基诱导的成骨细胞分化。敲低lnc-Kcnq1ot1还促进Ctsk和Oscar的表达（P < 0.05），并加重RANKL诱导的破骨细胞分化。细胞分级分离和qRT-PCR结果表明，lnc-Kcnq1ot1主要位于MC3T3-E1和RAW264.7细胞的细胞核中。
我们的数据表明，lnc-Kcnq1ot1促进成骨细胞分化并减轻破骨细胞分化，提示lnc-Kcnq1ot1作为骨质疏松症治疗靶点的潜力。
