南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (1): 31-38.doi: 10.12122/j.issn.1673-4254.2021.01.04

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长链非编码RNA Kcnq1ot1促进成骨细胞分化和抑制破骨细胞分化

章 坤，时哲敏，任 怡，韩晓辉，王敬朝，洪 伟
  

  • 出版日期:2021-01-26 发布日期:2021-01-26

Kcnq1ot1 promotes osteogenic differentiation and suppresses osteoclast differentiation

  • Online:2021-01-26 Published:2021-01-26

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摘要：

目的 探究长链非编码RNA Kcnq1ot1对成骨细胞分化和破骨细胞分化的调控作用。方法 运用荧光实时定量PCR技术分别在卵巢去势（双侧卵巢切除，n=8）、鼠尾悬吊（后肢悬空，n=14）以及自然衰老（正常饲养至18月龄，n=8）引起的骨质疏松小鼠以及各自对照小鼠（n=6）股骨组织中，小鼠前成骨细胞系MC3T3-E1向成骨细胞分化过程中以及小鼠骨髓源巨噬细胞BMMs和小鼠单核巨噬细胞系RAW264.7向破骨细胞分化过程中检测lnc-Kcnq1ot1、骨钙素（Bglap）、Runt相关转录因子2（Runx2）、碱性磷酸酶基因（Alp）、骨涎蛋白（Bsp）、活化T-细胞核因子c1（Nfatc1）、基质金属蛋白酶9（Mmp9）、组织蛋白酶K（Ctsk）和破骨细胞相关受体（Oscar）的表达水平；运用两对特异性以慢病毒为载体的短发夹RNAs（Lv-shRNAs）或小干扰RNAs（siRNAs）在MC3T3-E1、BMMs和RAW264.7细胞诱导分化过程中沉默lnc-Kcnq1ot1，随后运用荧光实时定量PCR技术检测lnc-Kcnq1ot1、成骨相关基因（Bglap、Alp）和破骨相关基因（Ctsk、Oscar）的表达；运用ALP染色检测碱性磷酸酶活性；运用抗酒石酸磷酸酶染色检测抗酒石酸磷酸酶活性。运用核浆分离联合荧光实时定量PCR技术检测lnc-Kcnq1ot1亚细胞定位。结果 与对照相比，lnc-Kcnq1ot1在卵巢去势、鼠尾悬吊以及自然衰老引起的疏松股骨组织中表达显著降低（P<0.05）；在MC3T3-E1向成骨细胞分化过程中表达增多（P<0.05）；在小鼠BMMs和RAW264.7向破骨细胞分化过程中表达显著降低（P<0.05）。在MC3T3-E1细胞中，沉默lnc-Kcnq1ot1抑制Bglap和Alp的表达（P<0.05），并减弱成骨诱导剂引起的成骨细胞分化；在小鼠BMMs和RAW264.7细胞中，沉默 lnc-Kcnq1ot1 促进 Ctsk 和 Oscar 的表达（P<0.05），并加重 RANKL 诱导的破骨细胞分化。核浆定位显示 lnc-Kcnq1ot1主要定位在MC3T3-E1和RAW264.7细胞核内。结论 lnc-Kcnq1ot1促进成骨细胞分化和抑制破骨细胞分化，可能成为骨质疏松症的一个潜在治疗靶点。

关键词:

Abstract: Objective To investigate the regulatory role of long non-coding RNA Kcnq1ot1 in osteoclast differentiation, osteogenic difffferentiation and osteoporosis. Methods 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, n=8), disuse osteoporosis (induced by tail suspension, n=14) and age-related osteoporosis (18-month-old mice, n=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 lnc-Kcnq1ot1 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. Results The expression of lnc-Kcnq1ot1 was significantly upregulated during osteoblast differentiation but downregulated in the bone tissues of osteoporotic mice and during osteoclast differentiation (P<0.05). Silencing lnc-Kcnq1ot1 obviously decreased the expression of Bglap and Alp (P<0.05) and attenuated osteogenic medium-induced osteoblast differentiation. Knockdown of lnc-Kcnq1ot1 also promoted the expression of Ctsk and Oscar (P<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. Conclusion 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.

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