Dong Chenbin, Liu Xiangqi, Li Jun, Lan Dongyi, Zheng Shan
Department of Plastic Surgery, Children's Hospital of Fudan University, Shanghai, China.
Front Genet. 2022 Mar 10;13:787734. doi: 10.3389/fgene.2022.787734. eCollection 2022.
Craniosynostosis is one of the most common craniofacial deformities demanding surgical treatment in infancy. LncRNA HOTAIR has verified its important role in osteogenesis and osteoarthritis. However, whether HOTAIR plays an essential role in the development of craniosynostosis is still unclear. In this study, we aimed to investigate the molecular role of HOTAIR in the osteoclast function and development of craniosynostosis.For osteoclast differentiation, RAW264.7 cells were induced by 50 ng/ml of RANKL and 10 ng/mL M-CSF, followed by TRAP staining. Cell proliferation and apoptosis were assayed by the CCK-8 kit and Annexin V-FITC apoptosis detection kit, respectively. The expression of HOTAIR was determined in PBMCs by qRT-PCR. Protein levels of all those involved genes were measured by Western blot assay. A luciferase reporter assay was used to determine the miRNA target validation. The HOTAIR expression in PBMCs from children with craniosynostosis was significantly downregulated. The results of cell proliferation and apoptosis assays indicated that silencing of HOTAIR could inhibit osteoclast differentiation and increase cell apoptosis. Moreover, the luciferase reporter assay revealed that the regulatory axis and HOTAIR-miR-152-CAMKIIα were the regulatory mechanisms of HOTAIR in the osteoclast function and development of craniosynostosis.In this study, our data showed that HOTAIR could promote osteoclast differentiation by binding miR-152. Furthermore, the HOTAIR/HOTAIR-miR-152-CAMKIIα axis was found to regulate osteoclast differentiation. These results indicate that the HOTAIR plays a crucial role in the development of osteoclasts.
颅缝早闭是婴儿期最常见的需要手术治疗的颅面畸形之一。长链非编码RNA HOTAIR已证实其在骨生成和骨关节炎中发挥重要作用。然而，HOTAIR在颅缝早闭发展过程中是否发挥关键作用仍不清楚。在本研究中，我们旨在探讨HOTAIR在破骨细胞功能及颅缝早闭发展中的分子作用。对于破骨细胞分化，用50 ng/ml的RANKL和10 ng/mL的M-CSF诱导RAW264.7细胞，随后进行抗酒石酸酸性磷酸酶（TRAP）染色。分别用CCK-8试剂盒和膜联蛋白V-FITC凋亡检测试剂盒检测细胞增殖和凋亡情况。通过qRT-PCR测定外周血单核细胞（PBMCs）中HOTAIR的表达。所有相关基因的蛋白水平通过蛋白质免疫印迹法检测。采用荧光素酶报告基因检测法确定miRNA靶点验证。颅缝早闭患儿PBMCs中HOTAIR的表达显著下调。细胞增殖和凋亡检测结果表明，沉默HOTAIR可抑制破骨细胞分化并增加细胞凋亡。此外，荧光素酶报告基因检测显示，调控轴HOTAIR-miR-152-CAMKIIα是HOTAIR在破骨细胞功能及颅缝早闭发展中的调控机制。在本研究中，我们的数据表明HOTAIR可通过结合miR-152促进破骨细胞分化。此外，发现HOTAIR/HOTAIR-miR-152-CAMKIIα轴调节破骨细胞分化。这些结果表明HOTAIR在破骨细胞发育中起关键作用。
