SPI1 Regulates Neural Stem Cell Differentiation by Inhibiting FTO-Regulated m6A Modification of IL6R

Abstract

Background: The comprehensive role of salmonella pathogenicity island 1 (SPI1) in neural stem cells (NSCs) remains unclear. Therefore, this study aimed to explore the effect of SPI1 on the differentiation of NSCs. Methods: NSCs were administered with lipopolysaccharides (LPS, 100 ng/mL) to construct an NSC model in vitro. Relative mRNA expression levels of SPI1 and fat mass and obesity-associated protein (FTO) were assessed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Protein levels of phosphor (p)-janus kinase 1 (JAK1), p-signal transducer and activator of transcription 3 (STAT3), and interleukin-6 receptor (IL6R) were determined using Western blot analysis. Furthermore, the viability of NSCs was assessed using a cell counting kit-8 assay. NSC identification and neurite outgrowth were observed using immunofluorescence. The N6-methyladenosine (m6A) modification sites of IL6R were determined using the RNA Modification Base Database (RMBase). Additionally, the correlation between FTO and IL6R was confirmed through RNA-binding protein immunoprecipitation-qRT-PCR. Results: NSCs exhibited an increase in mRNA level of SPI1 over time, reaching to peak at day 12 (p < 0.001). Moreover, LPS treatment significantly reduced the expression of SPI1 and neurite growth, and inhibited the JAK1/STAT3 pathway, while enhancing the expression of FTO (p < 0.01). The inhibitory impact of LPS was enhanced by silencing SPI1, while SPI1 overexpression reversed its inhibitory effects on NSCs (p < 0.05). Furthermore, IL6R was predicted to contain m6A modification sites. The increased FTO expression in LPS-treated NSCs inhibited the growth of neurite, the expression levels of the JAK1/STAT3 pathway, and IL6R (p < 0.01). Moreover, silencing SPI1 enhanced the effect of FTO, while overexpression of SPI1 reversed this effect (p < 0.05). Conclusions: SPI1 negatively regulates FTO expression, thereby elevating m6A modification of IL6R, hence promoting NSC differentiation. This finding presents a novel strategy for propelling the clinical application of NSCs.