The development and premature fusion of cranial sutures are elaborately orchestrated by mechanical and molecular signals. Therefore, the unexplored etiology of single suture craniosynostosis has aroused widespread hypotheses, varying from genetics to epigenetics(Clarke et al., 2018, Miller et al., 2017, Lattanzi et al., 2017, Dudakovic et al., 2015). Our data showed a significant differentially expression of lncRNAs in closed sutures and patent sutures from single suture craniosynostosis patients. Furthermore, gene enrichment and pathway analyses implied that those lncRNAs involve multiple biological processes and pathways to participate in the development of craniosynostosis.
A total of 223 lncRNAs have differentially expressed (P < 0.05, fold change > 1.5), of which 98 are up-regulated, and 125 are down-regulated. Such differentiation implied their potential roles in the premature fusion of cranial sutures. In the differentially expressed lncRNAs, we found lncRNA ENSG00000264727 associated gene SHISA6 down-regulated in closed sutures. SHISA6 was previously proved to be a cell-autonomous Wnt inhibitor and resistant to the Wnt/β-catenin signaling(Tokue et al., 2017). The canonical Wnt pathway is involved in maintaining patent sutures, and its inhibition is responsible for ossification and suture fusion(Behr et al., 2010, Behr et al., 2011).
lncRNA MEG33 was found significantly down-regulated in the closed sutures group. lncRNA MEG3 has been clarified to be a key regulator during mesenchymal stem cells (MSCs) osteogenic differentiation in bone remodeling in multiple pathways. However, the underlying mechanism remains controversial. In a recent study, Liu et al. revealed that down-regulation of MEG3 could promote osteogenic differentiation of bone marrow MSCs in skull repairing through activating the Wnt/β-catenin signaling pathway. In addition, their results showed a remarkable upregulation in bone mineral density, bone volume, and new bone generation(Liu et al., 2022). Considering that the Wnt signaling pathway is the canonical contributor to osteogenesis and tissue regeneration of the neural crests in frontal bones, whether lncRNA MEG3 can regulate osteogenic differentiation of suture MSCs via the Wnt signaling pathway may be explored in the future(Quarto et al., 2010). Furthermore, lncRNA T010926 associated gene RNF11 and BIG-lncRNA-524.1 associated gene TLL1 were proved to regulate the bone formation, respectively. mRNA RNF11 was found to be highly expressed in osteoblasts of multiple skeletal elements during embryonic bone formation in mice(Gao et al., 2005). TLL1 is closed related to the osteogenesis gene BMP1, interacting together to promote osteoblast differentiation(Muir et al., 2014).
GO analysis showed that the top differentially expressed genes were rich included in “extracellular matrix organization,” “extracellular matrix,” and “extracellular matrix structural constituent.” Extracellular matrix protein was found to be related to skull development and craniosynostosis. Bai et al. reported that Periostin, which is expressed by suture mesenchymal cells, can reduce suture cell proliferation and differentiation through the upregulation of Wnt/β-catenin signaling, thus mitigating coronal craniosynostosis in Twist1+/− mice(Bai et al., 2018). Additionally, differentially expressed genes were rich in “skeletal system development,” a paramount process of craniosynostosis. The GO analysis results also indicated that differentially expressed genes were rich in “regulation of osteoblast differentiation.” Based on KEGG analysis, the most enriched pathways included the mTOR signaling pathway, Hematopoietic cell lineage, signaling pathways regulating pluripotency of stem cells, and Osteoclast differentiation. The results demonstrated the essential regulatory mechanism of mesenchymal stem cell-osteoblast differentiation and hematopoietic stem cell-macrophage progenitor cells-osteoclast differentiation in cranial sutures(Byron, 2006, De Pollack et al., 1996).
Although so far, limited studies have been reported on the direct regulation of lncRNAs in craniosynostosis or suture biology. Emerging research has elucidated that lncRNAs play a pivotal part in bone biology, especially in regulating osteogenic differentiation and maintaining bone homeostasis. HOTAIR, a star lncRNA molecule, although not found differentially expressed in the current study, has been confirmed to participate extensively in osteoblast differentiation and further facilitate the disease progress. In the skeletal development field, it has been investigated that the knock-out HOTAIR homozygous mice exhibited lumbosacral transformation and fusion of the metacarpal and carpal bones(Li et al., 2013). Interestingly, unlike most studies focusing on osteoblast, our previous research showed that HOTAIR in craniosynostosis could promote osteoclast differentiation via the HOTAIR-miR-152-CAMKIIα axis(Dong et al., 2022).
The current study is a novel study to investigate differentially expressed lncRNAs and mRNAs profiles from craniosynostosis patients. The study has several limitations. Firstly, the sample size was relatively small. Due to RNA degradation, a certain amount of cranial sutures samples were not able to perform microarray assay after RNA quality control. Secondly, the expression of differently expressed lncRNAs was not validated in a larger sample of single suture craniosynostosis patients. Lastly, the sophisticated mechanisms of lncRNAs in the underlying pathogenesis should be further investigated.
In summary, we have identified multiple novel lncRNAs differentially expressed in closed and patent sutures from single suture craniosynostosis patients. Our findings indicate the potential roles of lncRNAs in abnormal cranial suture development and their underlying functions and pathways. Future studies will concentrate on the verifications of identified lncRNA signatures and functional elucidation.