元宇^1,2, 张玲莉¹
1. 华南师范大学 体育科学学院，广东 广州 510006;
2. 广州体育学院 运动与健康学院，广东 广州 510500
收稿日期：2020-07-24；接收日期：2020-10-10；网络出版时间：2020-11-18
基金项目：国家自然科学基金(No. 81901430)，中国博士后科学基金(Nos. 2018M640792，2019T120739) 资助

摘要：骨代谢的平衡取决于骨形成及骨吸收之间的动态平衡，Wnt/β-catenin信号通路能够广泛参与骨吸收及骨形成的调控，在维持骨代谢平衡中发挥着重要作用。近年来有研究表明，长链非编码RNA (Long non-coding RNA, lncRNA) 也广泛参与骨代谢各阶段的调节，还能通过Wnt/β-catenin信号通路参与骨代谢平衡的调控。目前关于lncRNA介导Wnt/β-catenin信号通路调控骨代谢的综述报道较为鲜见。鉴于此，文中主要以Wnt/β-catenin信号通路为切入点，概述lncRNA在骨代谢中的调控作用，发现lncRNA能够通过靶向作用于miRNA间接调控Wnt/β-catenin信号通路，也能通过Wnt/β-catenin信号通路上的关键因子直接激活或抑制Wnt/β-catenin信号通路，进而发挥其对骨代谢的调控作用，这些发现为lncRNA调控骨代谢作用机制的研究提供了新的思路和方向。
关键词：长链非编码RNAWntβ-连环蛋白骨代谢
Long non-coding RNA regulates bone metabolism via Wnt/β-catenin signaling pathway
Yu Yuan^1,2, Lingli Zhang¹
1. School of Physical Education, South China Normal University, Guangzhou 510006, Guangdong, China;
2. School of Exercise and Health, Guangzhou Sport University, Guangzhou 510500, Guangdong, China
Received: July 24, 2020; Accepted: October 10, 2020; Published: November 18, 2020
Supported by: National Natural Science Foundation of China (No. 81901430), China Postdoctoral Science Foundation Funded Project (Nos. 2018M640792, 2019T120739)
Corresponding author: Lingli Zhang. Tel: +86-20-39310260; E-mail: lingliwdc@163.com.

Abstract: The balance of bone metabolism depends on the dynamic balance between bone formation and bone resorption. Wnt/β-catenin signaling pathway is involved in the regulation of bone resorption and bone formation, and plays an important role in maintaining the balance of bone metabolism. Recently, long non-coding RNA (lncRNA) is shown to play an essential role in different process of bone metabolism. LncRNA can also regulate the balance of bone metabolism via Wnt/β-catenin signaling pathway. Few studies report that lncRNA regulates bone metabolism via Wnt/β-catenin signaling pathway. Therefore, we summarize here the role of lncRNA in bone metabolism from the perspective of Wnt/β-catenin signaling pathway. LncRNA indirectly regulates Wnt/β-catenin signaling pathway by targeting miRNAs as well as activating or inhibiting Wnt/β-catenin signaling pathway via targeting the key molecules of Wnt/β-catenin signaling pathway, thus to regulate bone metabolism. These findings provide new ideas and directions for the study of the mechanism whereby lncRNA regulates bone metabolism.
Keywords: lncRNAWntβ-cateninbone metabolism
成骨细胞主导的骨形成与破骨细胞主导的骨吸收之间的动态平衡是维持骨代谢平衡的关键，骨代谢失衡将诱发骨质疏松症、骨硬化症等骨疾病的发生^[1]。大量研究表明，Wnt/β-catenin信号通路能够通过调控骨髓间充质干细胞(Bone marrow mesenchymal stem cells，BMSCs)、成骨细胞及破骨细胞的增殖与分化参与骨代谢的调节^[2-6]。近年的研究发现，长链非编码RNA (Long non-coding RNA，lncRNA) 广泛参与骨代谢的调控，对微小RNA (microRNAs，miRNAs) 发挥海绵效应，作为竞争性内源RNA (Competing endogenous RNA，ceRNA) 吸附miRNAs，或与miRNAs竞争性结合mRNA参与骨形成及骨吸收的调控^[7-8]。此外，lncRNA还能通过Wnt信号通路参与骨代谢的调控。目前关于lncRNA介导Wnt/β-catenin信号通路调控骨代谢的综述较为鲜见，因此，本文主要综述Wnt/β-catenin信号通路与lncRNA在骨代谢中的调控作用以及lncRNA介导Wnt/β-catenin信号通路调控骨代谢的相关报道，为lncRNA在骨疾病中的诊断及治疗提供理论基础。
1 Wnt/β-catenin信号通路在骨代谢中的调控作用Wnts是一类高度保守的分泌型糖蛋白，在细胞增殖、分化及凋亡等生物学过程中发挥着重要的调控作用^[9]。Wnts家族成员众多，目前在人类基因组中共发现19种Wnt蛋白^[10]。Wnt/β-catenin信号通路又称为经典Wnt信号通路，在Wnt/β-catenin信号通路中，β-catenin是最关键的信号转导因子。当细胞外的Wnt蛋白与细胞膜上的卷曲蛋白受体(Frizzled) 和低密度脂蛋白受体相关蛋白5/6 (Low density lipoprotein receptor related proteins，LRP5/6) 结合时，Wnt/β-catenin信号通路被激活，导致腺瘤息肉蛋白(Adenomatous polyposis coli，APC)、Axin及糖原合酶激酶3β (Glycogen synthase kinase 3β，GSK3β)组成的复合体解体，抑制β-catenin磷酸化，细胞内β-catenin的水平升高并进入细胞核与转录因子TCF结合，进而启动下游靶基因的转录。当Wnt未能与Frizzled、LRP 5/6结合时，Wnt/β-catenin信号通路被抑制，APC、Axin及GSK3β组成的复合体促使β-catenin磷酸化，磷酸化的β-catenin被泛素化，然后被蛋白酶体降解^[11-12]。
Wnt/β-catenin信号通路通过干预BMSCs、成骨细胞以及破骨细胞等骨组织细胞的增殖及分化广泛参与骨代谢的调控。在一定的诱导条件下，BMSCs能够分化为成骨细胞、脂肪细胞及软骨细胞。BMSCs向成骨细胞分化能力的衰退可能是骨质疏松的病因之一。Wnt/β-catenin信号通路能够调控BMSCs的分化，激活Wnt/β-catenin信号通路能够通过上调Runt相关转录因子2 (Runt related transcription factor 2，Runx2) 及骨钙素(Osteocalcin，OCN) 等成骨因子的表达促进BMSCs向成骨细胞分化，通过抑制CCAAT/增强子结合蛋白α (CCAAT/enhancer-binding protein alpha，C/EBPα) 等相关成脂因子的表达，抑制BMSCs向脂肪细胞分化^[13-15]。
在成骨细胞中，Wnt/β-catenin信号通路也发挥着重要作用。激活Wnt/β-catenin信号通路能够促进小鼠成骨细胞前体细胞系MC3T3E1中Runx2、碱性磷酸酶(Alkaline phosphatase，ALP)、Ⅰ型胶原α1 (Collagen TypeⅠAlpha 1，Col1α1)、OCN以及骨形态发生蛋白4 (Bone morphogenetic protein4，BMP4) 等成骨因子的表达，进而促进成骨细胞的增殖及分化^{[2, 16]}。我们前期的研究也发现，适宜的机械应力能够上调成骨细胞中ALP、OCN、Runx2及Osterix的mRNA水平，增强Wnt1和β-catenin的蛋白表达，通过激活Wnt/β-catenin信号通路促进成骨细胞的分化^[17]。与野生型小鼠比较，成骨细胞条件性Wnt10b基因敲入小鼠股骨干骺端有更高的骨密度、骨体积分数及骨小梁数目。而Wnt10b基因敲除小鼠则表现为骨量及骨小梁数量下降，骨形成率降低^[18]。成骨细胞条件性Wnt1基因敲除小鼠出现骨丢失的症状，与之相反，成骨细胞条件性Wnt1基因敲入小鼠则出现骨量增加的症状^[19]。而人体中Wnt1基因功能缺失突变则会导致早发性骨质疏松及成骨不全症^[20]。LRP5基因敲除小鼠也表现出成骨细胞功能缺失以及骨量降低的症状^[21]。但在条件性LRP5基因敲除的小鼠模型中，只有骨细胞条件性LRP5基因敲除小鼠出现骨量下降，而肠LRP5基因条件性敲除小鼠则未出现类似症状，提示LRP5能够在骨组织中特异性调控骨量增长^[22]。成骨细胞条件性LRP5基因敲除小鼠在16周龄时开始出现骨丢失的症状，成骨细胞条件性LRP6基因敲除小鼠则在4周龄时开始出现骨丢失。成骨细胞条件性LRP5/6双敲除小鼠则表现出更加严重的骨量流失症状^[23]。人类遗传学的相关研究也表明，人体中LRP5基因功能缺失突变将导致骨形成能力低下，诱发骨质疏松-假性神经胶质瘤综合征。而人体LRP5基因功能获得性突变则与高骨量及骨硬化症有关^{[10, 24]}。在骨细胞中，Wnt/β-catenin信号通路能够通过LRP5介导骨细胞对机械刺激信号的应答，对于骨稳态的维持有着必不可少的作用^[25-26]。骨细胞能特异性分泌骨硬化蛋白(Sclerostin)，Sclerostin是Wnt/β-catenin信号的拮抗剂，由SOST基因编码，人体SOST基因功能缺失突变将导致硬化性骨化病以及弥漫性骨皮质增生症(Van Buchem病)^[26-27]。此外，β-catenin在骨细胞中也发挥着重要作用，骨细胞条件性β-catenin基因敲除小鼠出现早发性的骨质流失、轻度生长迟缓以及过早死亡等症状，其松质骨质量及皮质骨厚度均明显减少，这种骨量丢失的表型与骨细胞骨保护素(Osteoprotegerin，OPG) 分泌减少所导致的破骨细胞数量增加及骨吸收能力增强有关^[28]。
Wnt/β-catenin信号通路也参与破骨细胞生成的调控，激活Wnt/β-catenin信号通路能通过下调活化T细胞核因子c1 (Nuclear factor of activated T cells cl，NFATc1) 的表达，抑制破骨细胞前体细胞向破骨细胞分化。有研究报道，破骨细胞前体细胞条件性β-catenin基因敲除小鼠具有破骨细胞分化能力增强且骨量减少的症状^[29]。破骨细胞前体细胞条件性LRP5/6基因敲除小鼠则表现出骨吸收及骨形成能力同时衰退的现象，且同样存在骨量减少的症状^[30]。上述研究从细胞实验、动物实验及人类遗传学等多个维度充分证实了Wnt/β-catenin信号通路在骨代谢平衡的调控中有着不可或缺的作用。
2 LncRNA在骨代谢中的调控作用LncRNA广泛参与机体的生理和病理过程，是一类长度大于200个核苷酸(Nucleotide，nt) 的非编码RNA，在表观遗传、细胞周期及细胞分化的调控中发挥着重要作用。近年的研究表明，lncRNA能够通过调节BMSCs、成骨细胞以及破骨细胞等骨组织细胞的增殖及分化广泛参与骨代谢相关的生物学过程的调控^[31-33]。在BMP2诱导人骨髓间充质干细胞(hBMSCs) 向成骨分化的过程中，lncRNA LOC100506178表达上调，过表达lncRNA LOC100506178能够提高BMSCs的ALP活性以及Runx2、Osterix与ALP的mRNA水平，此外，还能增强BMP2的促成骨作用^[34]。国内也有研究报道，在诱导hBMSCs细胞株向成骨分化的过程中，lncRNA-1708表达下调，过表达lncRNA-1708能够降低Runx2及ALP等相关成骨因子的表达，而低表达则能上调相关成骨因子的表达，提示lncRNA-1708可能抑制hBMSCs的成骨分化^[35]。Zhang等研究发现，骨质疏松病人血浆中lncRNA UCA1的水平是普通人的11.54倍。在成骨细胞系中低表达lncRNA UCA1能够上调Col1α1、Runx2以及骨桥蛋白(Osteopontin，OPN) 等相关成骨因子的表达，激活BMP-2/(Smad1/5/8) 信号通路，进而促进成骨细胞的增殖及分化^[33]。在破骨细胞中，lncRNA也发挥着重要的调控作用，如lncRNA AK077216在破骨细胞生成的过程中表达上调，过表达lncRNA AK077216能够上调c-Fos、NFATc1及CTSK等相关骨吸收因子的表达，下调NIP45的表达，促进破骨细胞分化，而低表达lncRNA AK077216则与之相反，当在破骨细胞中同时过表达lncRNA AK077216及NIP45时，破骨细胞生成能力减弱，提示lncRNA AK077216能够通过抑制NIP45来上调NFATc1的表达，进而增强破骨细胞的生成及功能^[36]。LncRNA Bmncr在破骨细胞中的调控作用则与此不同，其在骨质疏松小鼠的骨髓及脾脏中低表达，且在破骨细胞分化的过程中表达逐渐下调，过表达lncRNA Bmncr能够减少破骨细胞数量并抑制其骨吸收能力，低表达lncRNA Bmncr则与之相反。这些结果表明，lncRNA Bmncr能够抑制破骨细胞的分化，进而缓解骨质疏松的进程^[37]。
在骨代谢中，lncRNA还能够作为miRNAs的诱饵分子，对miRNAs发挥海绵吸附作用，抑制miRNA对相关成骨因子或骨吸收因子的调控^{[36, 38]}。如lncRNA AK131850通过抑制破骨细胞中的miR-93-5p增强VEGFA的分泌，促进内皮祖细胞血管生成^[38]。LncRNA LOC100506178能够通过海绵吸附miR-214-5p上调BMP2的表达，进而促进BMSCs向成骨分化^[34]。除此之外，lncRNA也能够与miRNAs竞争性结合调控骨代谢的mRNA，减少miRNA与相关mRNA的结合位点，进而实现对骨代谢的调控。如在软骨细胞中，lncRNA MSR能够通过和miR-152竞争性结合胸腺素β4 (Tymosin β4，TMSB4)，进而参与软骨退化的调控。上述研究表明，lncRNA在骨代谢中发挥着重要的调控作用，能够通过调节相关成骨因子或骨吸收因子的表达调控骨形成及骨吸收，也能够通过与miRNAs的相互作用实现对骨代谢的间接调控。
3 LncRNA通过Wnt/β-catenin信号通路调控骨代谢如前文所述，lncRNA和Wnt/β-catenin信号通路均在骨代谢中发挥着重要的调控作用。近年的研究发现，lncRNA能够通过Wnt/β-catenin信号通路调控BMSCs及成骨细胞的增殖及分化，进而发挥其对骨代谢的调控作用。如lncRNA H19通过吸附miR-141及miR-22激活Wnt/β-catenin信号通路，促进间充质干细胞向成骨分化^[39]。LncRNA H19还能通过靶向作用于Dickkopf-4 (DKK4)，抑制DKK4的表达，促进Wnt/β-catenin信号通路的激活。低表达lncRNA H19则上调DKK4的表达，抑制Wnt/β-catenin信号通路，抑制大鼠成骨细胞系UMR106的成骨分化^[40]。LncRNA LINC00707能够通过海绵吸附miR-145上调LRP5的表达，激活Wnt/β-catenin信号通路，进而促进BMSCs的成骨分化^[41]。LncRNA HOTTIP能够与WD重复域蛋白5 (WD repeat domain 5，WDR5) 相互作用，促进β-catenin的转录并激活Wnt/β-catenin信号通路，促进人BMSCs的成骨分化^[42]。
一些临床的证据也表明，骨折延迟愈合患者的骨组织中lncRNA KCNQ1OT1表达显著下调，在hFOB1.19人成骨细胞系中过表达lncRNA KCNQ1OT1能够激活Wnt/β-catenin信号通路，促进成骨细胞增殖并抑制细胞凋亡，而低表达lncRNA KCNQ1OT1则与之相反^[43]。此外，lncRNA KCNQ1OT1还能通过靶向作用于β-catenin促进间充质干细胞向成骨分化，激活Wnt/β-catenin信号通路，进而促进骨形成^[44]。在骨质疏松患者的血清及BMSCs中，lncRNA HOTAIR表达上调。在大鼠BMSCs中低表达lncRNA HOTAIR能够上调ALP、OCN、OPN及β-catenin等成骨因子的表达，下调DKK1的表达，促进BMSCs向成骨分化，而高表达lncRNA HOTAIR则与之相反。此外，在低表达lncRNA HOTAIR的过程中使用DKK1干预能够削弱其促成骨作用，DKK1是Wnt/β-catenin信号通路的拮抗蛋白，提示lncRNA HOTAIR能够通过Wnt/β-catenin信号通路抑制大鼠BMSCs的成骨分化^[45]。在骨关节炎中，lncRNA HOTAIR则能够靶向作用于miR-17-5p间接调控α-(1, 2)岩藻糖基转移酶2 (Alpha-1, 2 fucosyl transferase 2，FUT2)的表达，进而通过Wnt/β-catenin信号通路抑制软骨细胞增殖，促进软骨细胞凋亡，加速关节炎的发生^[46]。在关节炎大鼠的滑膜组织中，低表达lncRNA HOTAIR能够通过抑制Wnt/β-catenin信号通路缓解滑膜炎症及滑膜细胞增殖，促进滑膜细胞凋亡，进而缓解关节炎^[45]。这些研究都表明，lncRNA能够通过Wnt/β-catenin信号通路发挥对骨代谢的调控作用(表 1)。其主要调控路径可能是通过直接作用于Wnt/β-catenin信号通路上的关键因子，或者与miRNAs相互作用间接调节Wnt/β-catenin信号通路，但这两种调控途径之间的关系是相互独立还是相互作用仍未可知。此外，由于目前相关研究数量较少且主要集中在骨形成的研究，相关机制研究也主要以细胞实验为主，因此，lncRNA介导Wnt/β-catenin信号通路调控骨代谢平衡的整体效应及其重要性还有待更深入的研究论证。
表 1 LncRNA介导Wnt/β-catenin信号通路调控骨代谢Table 1 LncRNA regulates bone metabolism via Wnt/β-catenin signaling pathway

LncRNA	Sample resources	Targets	Function	References
LncRNA H19	hMSCs	miR-141↓ miR-22↓	Osteogenesis↑	[39]
LncRNA H19	Femur from SD rats rat osteoblast/osteocyte- like cell line (UMR106)	DKK4↓	Osteogenesis↑	[40]
LncRNA KCNQ1OT1	mMSCs	β-catenin↑	Osteogenesis↑	[44]
LncRNA KCNQ1OT1	Tibia(human/rabbit) hFOB1.19 cell line;	-	Cell proliferation↑ Cell apoptosis↓	[43]
LncRNA DANCR	serum from fracture patients MC3T3-E1 osteoblast cell line	-	Osteoblast proliferation↓ Osteogenesis↓	[47]
LncRNA TUG1	Osteoblasts (SD rat)	-	osteoblast Proliferation↑ Osteogenesis↑	[48]
LncRNA LINC00707	hBMSCs	miR-145↓	Osteogenesis↑	[41]
LncRNA HOTTIP	hBMSCs	WDR5↑	Osteogenesis↑	[42]
LncRNA HOTAIR	Serum from osteoporotic patients BMSCs (human/rat)	-	Osteogenesis↓	[49]
LncRNA HOTAIR	Synovial tissue from osteoarthritis rats synoviocytes (SD rat)	-	Synovial inflammation↑ Synoviocyte proliferation ↑ Synoviocyte apoptosis↓	[45]
LncRNA HOTAIR	Cartilage tissues from osteoarthritis patients and rats	miR-17-5p↓	Chondrocyte apoptosis↑ Chondrocyte proliferation↓ Osteoarthritis↑	[46]
hMSCs: human mesenchymal stem cells; mMSCs: mouse mesenchymal stem cells; ↑: promotion or up-regulation; ↓: inhibition or down-regulation; -: no mention.

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4 总结与展望在骨代谢中，Wnt/β-catenin信号通路及lncRNA均发挥着重要的调控作用。它们能够通过调控BMSCs、成骨细胞以及破骨细胞等骨组织细胞的增殖及分化广泛参与骨代谢相关的各个生物学过程的调控。LncRNA能够直接作用于相关的成骨因子或破骨因子参与对骨组织细胞的调控，也能够通过靶向作用于miRNA间接调控相关成骨因子或骨吸收因子的表达。如lncRNA能够通过miRNA或者Wnt/β-catenin信号通路上的关键因子，激活或抑制Wnt/β-catenin信号通路，进而实现对骨代谢的调控(图 1)。

图 1 LncRNA介导Wnt/β-catenin信号通路调控成骨分化示意图 Fig. 1 The model for regulating effects of lncRNA on osteogenesis via Wnt/β-catenin signaling pathway.

图选项

目前lncRNA介导Wnt/β-catenin信号通路调控骨代谢的相关研究较少，且主要以研究BMSCs及成骨细胞的成骨分化为主，破骨细胞及骨细胞的相关研究较为鲜见，其在骨代谢调控中的重要性还有待进一步验证。随着相关研究的逐渐深入，相信lncRNA与Wnt/β-catenin信号通路在骨代谢中的相互作用及关系将逐渐成为相关领域的研究热点，其对骨细胞和破骨细胞的作用及调控机制也将会被一一揭晓，这为进一步揭示lncRNA调控骨代谢的作用机制提供了新的研究方向。
此外，相关机制研究主要以细胞实验为主，在体实验部分仅检测分析临床骨疾病患者或动物骨组织及血清的lncRNA水平，仅极少数研究使用shRNA或慢病毒载体构建lncRNA低表达或高表达动物模型，应用lncRNA条件性基因敲除小鼠的研究较为鲜见。在后续的研究中，为了进一步验证lncRNA介导Wnt/β-catenin信号通路调控骨代谢的整体效应及相关机制，lncRNA低表达及高表达动物模型将普遍应用在相关研究中。随着基因编辑技术的日趋完善及普及，lncRNA条件性敲除小鼠在相关研究的应用也将进一步普及化。
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