Department of Emergency Surgery, The Second Affiliated Hospital of Kunming Medical University, Yunnan Osteoporosis Research Center, Yunnan Trauma Surgery Research Center, Kunming, 650101, Yunnan, People's Republic of China.
J Bone Miner Metab. 2022 Jan;40(1):66-80. doi: 10.1007/s00774-021-01277-4. Epub 2021 Nov 15.
Human umbilical cord blood-derived MSCs (hUC-MSCs) have the potential to differentiate into osteoblasts. This study investigated the function and potential mechanisms of a novel lncRNA LINC02381 in hUC-MSC osteogenic differentiation.
hUC-MSCs were maintained in osteogenic differentiation medium. RT-qPCR assay was performed to assess LINC02381 expression. Alizarin Red S (ARS) and alkaline phosphatase (ALP) staining were performed to evaluate osteogenic differentiation. The interaction between miR-21 and LINC0238/KLF12 was determined by luciferase reporter and RNA immunoprecipitation (RIP) assays. Chromatin immunoprecipitation (ChIP) assay was used to confirm the transcriptional regulation of KLF12 on Wnt4 promoter. The nuclear translocation of β-catenin was evaluated using immunofluorescence. hUC-MSCs seeded on Bio-Oss Collagen scaffolds were transplanted into nude mice to assess in vivo osteogenesis. Bone formation was observed by H&E and Masson's trichrome staining. OSX and OPN levels were assessed by immunohistochemistry.
LINC02381 was up-regulated in the clinical samples of osteoporotic patients. However, LINC02381 expression was reduced during osteogenic differentiation of hUC-MSCs. Enforced expression of LINC02381 suppressed the osteogenic differentiation of hUC-MSCs. Mechanistically, LINC02381 sponged miR-21 to enhance KLF12 expression, which led to the inactivation of Wnt/β-catenin signaling pathway. Furthermore, miR-21 mimics or KLF12 silencing counteracted LINC02381-induced inhibition of osteogenic differentiation, whereas IWP-4 (an inhibitor of Wnt pathway) abolished this effect.
In summary, LINC02381 repressed osteogenic differentiation of hUS-MSCs through sponging miR-21 to enhance KLF12-mediated inactivation of Wnt/β-catenin pathway, indicating that LINC02381 might be a therapeutic target for osteoporosis.
人脐带血间充质干细胞（hUC-MSCs）具有分化为成骨细胞的潜力。本研究探讨了新型长链非编码 RNA LINC02381 在 hUC-MSC 成骨分化中的功能和潜在机制。
将 hUC-MSCs 维持在成骨分化培养基中。通过 RT-qPCR 检测 LINC02381 的表达。茜素红 S（ARS）和碱性磷酸酶（ALP）染色评估成骨分化。通过荧光素酶报告和 RNA 免疫沉淀（RIP）测定确定 miR-21 与 LINC0238/KLF12 的相互作用。染色质免疫沉淀（ChIP）测定用于证实 KLF12 对 Wnt4 启动子的转录调控。使用免疫荧光评估β-catenin 的核转位。将接种在 Bio-Oss 胶原支架上的 hUC-MSCs 移植到裸鼠体内以评估体内成骨作用。通过 H&E 和 Masson 三色染色观察骨形成。通过免疫组织化学评估 OSX 和 OPN 水平。
在骨质疏松症患者的临床样本中上调了 LINC02381。然而，在 hUC-MSCs 的成骨分化过程中 LINC02381 的表达减少。LINC02381 的过表达抑制了 hUC-MSCs 的成骨分化。机制上，LINC02381 海绵 miR-21 以增强 KLF12 的表达，从而导致 Wnt/β-catenin 信号通路失活。此外，miR-21 模拟物或 KLF12 沉默逆转了 LINC02381 诱导的成骨分化抑制，而 IWP-4（Wnt 通路抑制剂）消除了这种作用。
总之，LINC02381 通过海绵 miR-21 抑制 hUS-MSCs 的成骨分化来增强 KLF12 介导的 Wnt/β-catenin 通路失活，表明 LINC02381 可能是骨质疏松症的治疗靶点。
