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Notum, which belongs to the α/β hydrolase family, is a deacylated extracellular protein that regulates the Wnt signaling pathway. Studies have found that Notum participates in the progression of colorectal cancer and hepatocellular carcinoma, but its role in oral squamous cell carcinoma (OSCC) is currently unclear. This study aimed to explore the role of Notum in regulating OSCC and further reveal the underlying mechanisms. Various approaches including bioinformatics analysis, enzyme-linked immunosorbent assay and immunohistochemical staining were used to detect the expression of Notum in OSCC cells and tissues. Cell counting kit-8 assay, clone formation assay, wound healing assay, transwell assay and in-gel zymography assay were explored to evaluate the regulation of Notum in OSCC proliferation and migration. Hoechst 33342/PI assay, cell immunofluorescence, flow cytometry and in vivo tumorigenesis experiment were applied for OSCC apoptosis. Real-time quantitative polymerase chain reaction analysis was performed for mRNA level while western blotting was conducted to detect protein expression. The results showed that Notum was highly expressed in OSCC tissues and cells, and Notum promoted the proliferation and migration of OSCC cells while it inhibited their apoptosis. Furthermore, signaling pathway analysis showed that Notum led to potential pro-survival of OSCC through crosstalk between sonic hedgehog (Shh) and Wnt/β-catenin signaling via phosphorylation of glycogen synthase kinase-3 beta. These results will help to elucidate the mechanism and also provide new ideas for targeted treatment of OSCC. [ABSTRACT FROM AUTHOR]

• Notum expression increased in gingival tissues. • Notum expression increased in periodontal ligament. • Notum inhibited human periodontal ligament stem cell (hPDLSC) osteogenesis. • Notum regulated hPDLSC osteogenesis through Wnt/beta-catentin signaling. The aims of this study were to explore: (ⅰ) the effect of Notum on periodontitis in vivo ; (ⅱ) the effect of Notum on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in vitro ; and (ⅲ) the potential mechanism of Notum in inhibiting the osteogenic differentiation of hPDLSCs. C57BL/6J mice were randomly assigned into two groups: control group (n = 4) and periodontitis group (n = 4). Immunohistochemical staining was used to evaluate the expression of Notum. In in vitro experiments, Western blot, qRT- PCR and ELISA were used to examine the expression of Notum in a lipopolysaccharide-induced inflammation model. Alkaline phosphatase staining was used to evaluate alkaline phosphatase activity. Western blot and qRT - PCR were used to measure the expression of osteogenic-related markers after adding human recombinant Notum and Notum inhibitor ABC99. In addition, LiCl, an agonist of the Wnt/Beta-catenin signaling pathway, was added to explore using Western blot whether Notum was involved in regulating the osteogenic differentiation of human periodontal ligament stem cells through the Wnt/Beta-catenin signaling pathway. Notum was highly expressed in periodontal tissues of mice and lipopolysaccharide-induced inflammation cell model. The protein and messenger ribonucleic acid levels of hPDLSCs osteogenic markers were reduced after adding human recombinant Notum. However, the inhibitory effect of Notum on the osteogenic differentiation of hPDLSCs could be significantly reversed by adding LiCl. These results demonstrated that Notum inhibited the osteogenic differentiation of hPDLSCs probably via the Wnt/Beta-catenin the downstream signaling pathway. [ABSTRACT FROM AUTHOR]