NAT10 promotes osteogenic differentiation of periodontal ligament stem cells by regulating VEGFA-mediated PI3K/AKT signaling pathway through ac4C modification.

Periodontal tissue regeneration engineering based on human periodontal ligament stem cells (hPDLSCs) provides a broad prospect for the treatment of periodontal disease. N-Acetyltransferase 10 (NAT10)-catalyzed non-histone acetylation is widely involved in physiological or pathophysiological processes. However, its function in hPDLSCs is still missing. hPDLSCs were isolated, purified, and cultured from extracted teeth. Surface markers were detected by flow cytometry. Osteogenic, adipogenic, and chondrogenic differentiation potential was detected by alizarin red staining (ARS), oil red O staining, and Alcian blue staining. Alkaline phosphatase (ALP) activity was assessed by ALP assay. Quantitative real-time PCR (qRT-PCR) and western blot were used to detect the expression of key molecules, such as NAT10, Vascular endothelial growth factor A (VEGFA), PI3K/AKT pathway, as well as bone markers (RUNX2, OCN, OPN). RNA-Binding Protein Immunoprecipitation PCR (RIP-PCR) was used to detect the N4-acetylcytidine (ac4C) mRNA level. Genes related to VEGFA were identified by bioinformatics analysis. NAT10 was highly expressed in the osteogenic differentiation process with enhanced ALP activity and osteogenic capability, and elevated expression of osteogenesis-related markers. The ac4C level and expression of VEGFA were obviously regulated by NAT10 and overexpression of VEGFA also had similar effects to NAT10. The phosphorylation level of PI3K and AKT was also elevated by overexpression of VEGFA. VEGFA could reverse the effects of NAT10 in hPDLSCs. NAT10 enhances the osteogenic development of hPDLSCs via regulation of the VEGFA-mediated PI3K/AKT signaling pathway by ac4C alteration.

Solute carrier family 12 member 8 impacts the biological behaviors of breast carcinoma cells by activating TLR/NLR signaling pathway.

This study aimed to understand the expression of solute carrier family 12 member 8 (SLC12A8) in breast carcinoma and its biological functions, as well as its effect on the Toll-like receptor /NOD-like receptor (TLR/NLR) signaling pathway. The expression of SLC12A8 was analyzed using the public RNA sequencing dataset from TCGA database and the two datasets from Oncomine database. The former dataset was also used to evaluate the prognostic value of SLC12A8 in breast carcinoma. Real-time qPCR and western blot were applied to measure relative expression of SLC12A8. Functionally, the effect of SLC12A8 on the cells proliferation and motion was studied using cell counting kit 8 and Transwell assays respectively. Mechanistic studies were conducted using Gene Set Enrichment Analysis (GSEA) and confirmed by western blot. As a result, SLC12A8 was upregulated in breast carcinoma, and high levels of SLC12A8 led to a poorer prognosis and can be regarded as an independent prognosticator for patients with breast carcinoma. Functional experiments demonstrated that SLC12A8-knockdown suppressed while SLC12A8-overexpression elevated the viability, invasiveness and motility of breast carcinoma cells. Furthermore, GSEA indicated that high SLC12A8 was positively correlated with TLR/NLR signaling pathway. Silencing SLC12A8 significantly reduced the protein expression of TLR/NLR-related markers, whereas overexpression of SLC12A8 caused an elevation on the protein expression of these markers. All these data suggested that SLC12A8 plays a promoting effect on the cells viability, invasiveness and motility in breast carcinoma by activating TLR/NLR signaling pathway.