Moura Sara Reis, Olesen Jacob Bastholm, Lindberg-Larsen Martin, Barbosa Mário Adolfo, Søe Kent, Almeida Maria Inês
i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.
INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal.
Cell Commun Signal. 2025 Aug 12;23(1):370. doi: 10.1186/s12964-025-02324-7.
The interplay between osteoblasts and osteoclasts (OCs) is a highly regulated and coordinated process essential for maintaining bone skeletal integrity and health. Disruption of this balance marks the onset of various bone disorders, such as osteoporosis. In our previous study, we demonstrated that non-coding RNAs (ncRNAs) were able to regulate OC behaviour. Specifically, the long ncRNA DLEU1 and the small ncRNA miR-16 independently control OCs multinuclearity, fusion and resorption. Herein, we investigate whether OCs modulated with ncRNAs impact the behaviour of osteoblast lineage cells (OBs) in a biomimetic co-culture system. Communication between OB and OC comprises numerous molecules and processes, including the secretion of coupling factors, release of matrix-derived signals during bone resorption, and exposure of previously occluded proteins within the bone resorption cavities. Firstly, we explored how ncRNAs influence the composition of the secretome and the remodelling of resorption cavities left by the ncRNA-engineered-OCs on OBs responses. In our OC/OB co-culture system, conducted in the absence of FBS and exogenous RANKL, we observed changes in the levels of secreted factors 72 h after DLEU1 levels were decreased in mature OCs (siDLEU1-OC condition), specifically in RANKL, leptin, osteocalcin, MMP7, MMP8, TIMP1 and TIMP2. Meanwhile, leptin and MMP8 levels were increased in the OC/OB co-culture when miR-16 was overexpressed in OCs (miR-16-OC condition). The results also show that OBs co-cultured with siDLEU1-OCs exhibited impaired migratory capacity and increased ALP levels, while OBs co-cultured with miR-16-OCs displayed a tendency towards decreased elongation. On bone slices, OBs persisted for a longer duration in the trenches created by siDLEU1-OCs. Besides altering OBs behaviour, silencing of DLEU1 reduced the area of each individual trench cavity, while miR-16 overexpression significantly hindered the total eroded surface and inhibited trench formation in the OC/OB co-culture system. Overall, these findings identify DLEU1 and miR-16 as key regulators in the interplay between human primary OBs and OCs, highlighting the potential of targeting ncRNAs in OCs to modulate their paracrine and matrix-mediated effects on OBs.
成骨细胞与破骨细胞（OCs）之间的相互作用是一个高度调控且协调的过程，对于维持骨骼完整性和健康至关重要。这种平衡的破坏标志着各种骨疾病的开始，如骨质疏松症。在我们之前的研究中，我们证明了非编码RNA（ncRNAs）能够调节破骨细胞的行为。具体而言，长链非编码RNA DLEU1和小非编码RNA miR-16分别独立控制破骨细胞的多核化、融合和吸收。在此，我们研究了在仿生共培养系统中，经ncRNAs调节的破骨细胞是否会影响成骨细胞系细胞（OBs）的行为。成骨细胞与破骨细胞之间的通讯包括众多分子和过程，包括偶联因子的分泌、骨吸收过程中基质衍生信号的释放，以及骨吸收腔内先前被封闭蛋白质的暴露。首先，我们探讨了ncRNAs如何影响分泌组的组成以及由经ncRNAs工程改造的破骨细胞留下的吸收腔的重塑对成骨细胞反应的影响。在我们的破骨细胞/成骨细胞共培养系统中，在无胎牛血清（FBS）和外源性核因子κB受体活化因子配体（RANKL）的条件下进行实验，我们观察到在成熟破骨细胞中降低DLEU1水平（siDLEU1-破骨细胞条件）72小时后，分泌因子水平发生了变化，特别是在RANKL、瘦素、骨钙素、基质金属蛋白酶7（MMP7）、基质金属蛋白酶8（MMP8）、金属蛋白酶组织抑制剂1（TIMP1）和金属蛋白酶组织抑制剂2（TIMP2）方面。同时，当在破骨细胞中过表达miR-16（miR-16-破骨细胞条件）时，破骨细胞/成骨细胞共培养体系中瘦素和MMP8水平升高。结果还表明，与siDLEU1-破骨细胞共培养的成骨细胞迁移能力受损且碱性磷酸酶（ALP）水平升高，而与miR-16-破骨细胞共培养的成骨细胞显示出伸长减少的趋势。在骨切片上，成骨细胞在由siDLEU化破骨细胞形成的沟槽中持续存在的时间更长。除了改变成骨细胞的行为外，DLEU1的沉默减少了每个单独沟槽腔的面积，而miR-16的过表达显著阻碍了总侵蚀表面并抑制了破骨细胞/成骨细胞共培养系统中的沟槽形成。总体而言，这些发现确定DLEU1和miR-16是人类原代成骨细胞与破骨细胞之间相互作用的关键调节因子，突出了靶向破骨细胞中的ncRNAs以调节其对成骨细胞的旁分泌和基质介导作用的潜力。
