In this study, 37 common differentially expressed genes were screened using bioinformatics analysis form GSE54720 and GSE78061 datasets, and the 15 hub genes were finally identified. Then, TAGLN3, KIF5C, and SNAP91 were identified by alignment in PubMed, OMIM, DisGeNET, and GeneCards databases, which have never been reported in the literature or experimentally verified. Additionally, TAGLN3, KIF5C, and SNAP91 were all upregulated in 2 datasets (GSE54720 & GSE78061). Subsequently, TAGLN3, KIF5C and SNAP91 were high expression in the neuroblastoma SH-SY5Y cells by qPCR verified, consistent with our bioinformatics analysis.
In our study, KEGG pathway analysis showed that 37 common differentially expressed genes were mainly related to dopaminergic synapses, phenylalanine metabolism and Tyrosine metabolism. Most human neuroblastoma cell lines are dopaminergic neuroblastoma cells and exhibit characteristics of dopaminergic neurons (Kovalevich & Langford, 2013). Researchers have been studying the relationship between neuroblastoma and dopamine for more than 20 years. Approximately 80% of patients with neuroblastoma exhibit increased expression levels of catecholamines and their metabolites, including dopamine, vanillylmandelic acid (VMA), and homovanillic acid (HVA), making these molecules promising tumor markers for the diagnosis of neuroblastoma (Candito et al., 1992; Nakagawara et al., 1988; LaBrosse et al., 1976). Therefore, the alteration of dopamine expression level mediated by its related signaling pathways may play an important role in the molecular mechanism of neuroblastoma.
Additionally, both phenylalanine and tyrosine are essential amino acids in our diet. Many nutrients, such as amino acids, are required for the rapid proliferation of tumor cells and are also considered potential biomarkers for malignant diseases (Vettore et al., 2020). Tyrosine, phenylalanine, and tryptophan are reduced in the plasma of patients with esophageal cancer (Lai et al., 2005). In contrast, tyrosine, phenylalanine, and tryptophan are increased in the urine, gastric contents, and tissues of gastric cancer patients (Wiggins et al., 2015). Notably, phenylalanine is required for the production of the nonessential amino acid tyrosine (Womack & Rose, 1934). This conversion is catalyzed by phenylalanine hydroxylase, and it has been shown that the activity of phenylalanine hydroxylase can be altered in inflammation or malignancy (Deng et al., 2011; Tang et al., 2023). Consequently, Therefore, changes in phenylalanine and tyrosine metabolism mediated by phenylalanine-related and tyrosine-related signaling pathways may play a role in the initiation and development of neuroblastoma.
GO term enrichment analysis showed that TAGLN3, KIF5C and SNAP91 expression in BP, CC, and MF are mainly associated with axon guidance, neuron projection guidance, axon genesis, axon development, distal axon, site of polarized growth, growth cone, neuronal cell body, and synaptic vesicle transport, indicating that may be involved in protein binding, plasma membrane, membrane composition, nucleus, and other biological functions.
Followed qPCR, we found that TAGLN3, KIF5C, and SNAP91 were significantly high expression in human neuroblastoma SH-SY5Y cells. Current studies have reported that TAGLN3 belongs to the actin-binding protein family, also known as neuron protein 22, NP22, or NP25, and is only found in highly differentiated neural cells and involved in central nervous system development (Ren et al., 1994). The amino acid sequence of TAGLN3 shared homology (from 67–42%) with four other proteins, SM22alpha, calponin, myophilin, and mp20, suggesting a potential interaction of TAGLN3 with cytoskeletal elements and possible mediating regulatory signal transduction pathways in neurons (Ren et al., 1994; Fan et al., 2001). TAGLN3 is significantly downregulated in the brains of SAD patients and in glioma tissues (Arnaud et al., 2022; Su et al., 2022). In contrast, TAGLN3 is specifically expressed in brain tissue and upregulated in the frontal cortex and hippocampus of chronic alcoholics and rats (Kim et al., 2018; Mori et al., 2004; Ren et al., 1994). Upregulated TAGLN3 inhibits Notch signaling during hypothalamic development (Ratié et al., 2013). Studies by Zage et al. found that neuroblastoma tumor cell lines and patient tumors have essentially inactivated Notch signaling, and Notch pathway activation leads to decreased proliferation of neuroblastoma cells (Zage eta l., 2012). Taken together with our study that TAGLN3 expression level was significantly higher in the human neuroblastoma SH-SY5Y cell line than in the ARPE-19 cell line, we hypothesized that upregulated TAGLN3 affects the development and progression of neuroblastoma by blocking Notch activity.
KIF5C, a member of the kinin-1 heavy chain family, which helps transport specific cargoes required for neurite maturation along microtubules, selectively transports molecules from the cell body, and is essential for neuronal development (Kanai et al., 2000; Poirier et al., 2013; Schäfer et al., 2008). Previous studies have shown that KIF5C is downregulated in colon and adenocarcinoma (Tong and Fan, 2023; Aquino et al., 2023), and highly expressed in mouse and human brain neurons during early developmental stage, and esophageal cancer (Kanai et al., 2000; Li et al., 2022; He et al., 2019). Mutations in the KIF5C gene can cause common neurodevelopmental disorders in children, including cortical dysplasia, microcephaly, epilepsy, developmental delay/mental retardation, and autism-like features (Schäfer et al., 2009; Willemsen et al., 2014). In addition, A miRNA has variety of target genes, and a gene can be regulated by multiple miRNAs. He et al. 's study found that KIF5C is a direct target of miR203, and KIF5C overexpression partially counteracted the tumor suppressive effect of miR-203 on esophageal carcinoma cells (He et al., 2019). Similarly, Zhao et al. showed that the expression of Sam68 expression was significantly upregulated in neuroblastoma tissues, and that miR-203 has an inhibitory effect on the malignant progression of neuroblastoma by targeting Sam68 (Zhao et al., 2015). Indeed, miR-203 deregulation has been shown to be associated with various cancer types, including glioma, hepatocellular carcinoma, esophageal carcinoma, and prostate carcinoma, among others (He et al., 2013; Furuta et al., 2010; He et al., 2019; Boll et al., 2013). Our study found that KIF5C is upregulated in neuroblastoma cell line SH-SY5Y, and we speculate that miR-203 target KIF5C is also involved in the development of neuroblastoma progression, but further studies are needed.
SNAP91 (KIAA0656/AP180) encodes a synapse-associated protein with the highest expression in the brain (Ishikawa et al., 1998), which is mainly distributed in the polar part of the synapse and is involved in the vesicular transport of neurotransmitters (Schwartz et al., 2010). Previous studies have shown that SNAP91 is associated with neurological diseases, such as Parkinson's disease, schizophrenia, epilepsy, and Alzheimer's disease (Yemni et al., 2019; Kooet al., 2015; Takata et al., 2017; Cao et al., 2010). Previous studies have shown that SNAP91 is significantly reduced in the hippocampus of patients with Alzheimer's disease and low expression in glioma and acute lymphoblastic leukemia (Cao et al., 2010; Yu et al., 2022; Qi et al., 2021). In contrast, SNAP91 was overexpressed in prostate cancer (PCa), correlated with the metastatic phenotype of PCa, and promoted PCa tumor metastasis (Sun et al., 2021). In our study, the bioinformatics analysis and qPCR results are shown KIF5C is upregulated in neuroblastoma cell line SH-SY5Y. Thus, SNAP91 may be involved in the development of neuroblastoma.
So far, no study has mentioned the role of TAGLN3, KIF5C, and SNAP91 in neuroblastoma. In this study, we analyzed data from two datasets from the GEO database and verified by qPCR, and found that Tagln3, KIF5C and Snap91 were upregulated in neuroblastoma. It also provides new ideas and theoretical basis for screening diagnostic, prognostic markers or therapeutic targets of neuroblastoma. However, the novel biomarkers of neuroblastoma screened by bioinformatics methods in this study still need further study.