LINC02381, a sponge of miR-21, weakens osteogenic differentiation of hUC-MSCs through KLF12-mediated Wnt4 transcriptional repression

• Published in: Journal of bone and mineral metabolism Vol. 40; no. 1; pp. 66 - 80
• Main Authors: Zhao, Gang, Luo, Wen-Dong, Yuan, Yong, Lin, Feng, Guo, Li-Min, Ma, Jing-Jing, Chen, Han-Bo, Tang, Huang, Shu, Jun
• Format: Journal Article
• Language: English
• Published: Singapore Springer Singapore 2022; Springer Nature B.V
• Subjects: Alkaline phosphatase; Animals; Bone growth; Cell Differentiation; Cells, Cultured; Chromatin; Collagen; Cord blood; Gene regulation; Gene silencing; Humans; Immunofluorescence; Immunohistochemistry; Immunoprecipitation; Kruppel-Like Transcription Factors - genetics; Medicine; Medicine & Public Health; Mesenchymal Stem Cells; Metabolic Diseases; Mice; Mice, Nude; MicroRNAs - genetics; Nuclear transport; Original Article; Orthopedics; Osteogenesis; Osteogenesis - genetics; Osteoporosis; RNA, Long Noncoding - genetics; Signal transduction; Therapeutic targets; Umbilical cord; Wnt protein; Wnt Signaling Pathway - genetics; Wnt4 Protein; β-Catenin; Osteoporosis; hUS-MSCs; KLF12; miR-21; LINC02381
• ISSN: 0914-8779; 1435-5604
• DOI: 10.1007/s00774-021-01277-4

Introduction
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.
Materials and methods
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.
Results
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.
Conclusion
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.