Freitas Jaime, Moura Sara Reis, Barbosa Mário Adolfo, Santos Susana G, 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.
Front Bioeng Biotechnol. 2023 Jul 4;11:1155596. doi: 10.3389/fbioe.2023.1155596. eCollection 2023.
Long non-coding RNAs (lncRNAs) are master regulators of gene expression and have recently emerged as potential innovative therapeutic targets. The deregulation of lncRNA expression patterns has been associated with age-related and noncommunicable diseases in the bone tissue, including osteoporosis and tumors. However, the specific role of lncRNAs in physiological or pathological conditions in the bone tissue still needs to be further clarified, for their exploitation as therapeutic tools. In the present study, we evaluate the potential of the lncRNA CASC2 as a regulator of osteogenic differentiation and mineralization. Results show that CASC2 expression is decreased during osteogenic differentiation of human bone marrow-derived Mesenchymal Stem/Stromal cells (hMSCs). CASC2 knockdown, using small interfering RNA against CASC2 (siCASC2), increases the expression of the late osteogenic marker Bone Sialoprotein (BSP), but does not impact ALP staining level nor the expression of early osteogenic transcripts, including RUNX2 and OPG. Although siCASC2 does not impact hMSC proliferation nor apoptosis, it promotes the mineralization of hMSC cultured under osteogenic-inducing conditions, as shown by the increase of calcium deposits. Mass spectrometry-based proteomic analysis revealed that 89 proteins are regulated by CASC2 at late osteogenic stages, including proteins associated with bone diseases or anthropometric and musculoskeletal traits. Specifically, the Cartilage Oligomeric Matrix Protein (COMP) is highly enhanced by CASC2 knockdown at late stages of osteogenic differentiation, at both transcriptional and protein level. On the other hand, inhibition of COMP impairs osteoblasts mineralization as well as the expression of BSP. The results indicate that lncRNA CASC2 regulates late osteogenic differentiation and mineralization in hMSC via COMP and BSP. In conclusion, this study suggests that targeting lncRNA CASC2 could be a potential approach for modulating bone mineralization.
长链非编码RNA（lncRNAs）是基因表达的主要调节因子，最近已成为潜在的创新治疗靶点。lncRNA表达模式的失调与骨组织中与年龄相关的非传染性疾病有关，包括骨质疏松症和肿瘤。然而，lncRNAs在骨组织生理或病理条件下的具体作用仍需进一步阐明，以便将其开发为治疗工具。在本研究中，我们评估了lncRNA CASC2作为成骨分化和矿化调节因子的潜力。结果表明，在人骨髓间充质干/基质细胞（hMSCs）的成骨分化过程中，CASC2表达降低。使用针对CASC2的小干扰RNA（siCASC2）敲低CASC2，可增加晚期成骨标志物骨唾液酸蛋白（BSP）的表达，但不影响碱性磷酸酶（ALP）染色水平，也不影响早期成骨转录本（包括RUNX2和骨保护素（OPG））的表达。虽然siCASC2不影响hMSC的增殖和凋亡，但它促进了在成骨诱导条件下培养的hMSC的矿化，钙沉积增加表明了这一点。基于质谱的蛋白质组学分析表明，在成骨晚期有89种蛋白质受CASC2调节，包括与骨疾病或人体测量及肌肉骨骼特征相关的蛋白质。具体而言，在成骨分化后期，软骨寡聚基质蛋白（COMP）在转录和蛋白质水平上均因CASC2敲低而高度上调。另一方面，抑制COMP会损害成骨细胞的矿化以及BSP的表达。结果表明，lncRNA CASC2通过COMP和BSP调节hMSC的晚期成骨分化和矿化。总之，本研究表明靶向lncRNA CASC2可能是调节骨矿化的一种潜在方法。
