APE1 is an important DNA repair enzyme that plays a key role in maintaining genome integrity and stability[18] Previous studies have shown that APE1 also plays an important role in biological processes such as cell proliferation, apoptosis, oxidative stress, and inflammation [19]. In order to gain a deeper understanding of the function and regulatory mechanism of APE1, this study applied RNA-seq technology to analyze the esophageal cancer TE-1 cell line with APE1 knockdown, and explored the differential genes and their functions related to APE1 knockdown.
Through RNA-seq analysis, we identified differential genes whose expression levels were significantly changed after knocking down the APE1 gene. A total of 2060 differential genes were screened out, including 1063 up-regulated genes and 997 down-regulated genes. Through GO analysis, we found that differentially expressed genes were mainly enriched in functions such as multicellular organism processes, phylogeny, regulation of multicellular organism processes, and cell edges. The enrichment of multicellular organismal processes suggests that APE1 may be involved in cell-cell interactions and tissue structure regulation in esophageal cancer cells[20]. During the development of esophageal cancer, abnormal proliferation and differentiation of cells are important features, and the regulation of multicellular organismal processes may be closely related to these processes[21] Furthermore, phylogenetic enrichment suggested that APE1 may play an important role in the formation and development of esophageal cancer cells. The cell edge is the contact area between cells or between cells and the extracellular matrix, and its abnormal changes may promote the invasion and metastasis capabilities of esophageal cancer cells[22]. The enrichment of cell edge functions suggests that APE1 may be related to the invasion and metastasis process of esophageal cancer cells.
Through KEGG analysis, we found that differential genes were mainly enriched in cancer pathways, calcium signaling pathways, PI3K-Akt signaling pathways, and complement and coagulation cascade reaction pathways. The enrichment results of cancer pathways further verified the important role of APE1 in esophageal cancer cells. Studies have shown that genetic alterations in calcium signaling pathway gene modification in colorectal cancer can be identified through the tumor microenvironment, and changing the rs12538364 T allele in PDE1C to the rs12538364 C allele significantly increases the risk of colorectal cancer [23]; In addition, KDM5a plays a crucial role in the occurrence of hepatocellular carcinoma, promoting the formation of hepatocellular carcinoma lesions by regulating miR-433-FXYD3-PI3K-Akt signaling [24]. The enrichment of complement and coagulation cascade reaction pathways indicates the importance of immune regulation and tumor microenvironment [25, 26]. Studies by Zheng Gong et al. have shown that complement and coagulation pathways such as C2, C1QB, SERPING1, CFB, MASP1, F8, and F2R The expression of cascade reaction pathway genes is related to ICIs response and overall survival in patients with urothelial cancer. Patients with low expression usually have better ICIs response rate and longer overall survival [27]. The above studies show that APE1 may mainly affect cell proliferation, survival, metastasis, immune regulation and tumor immune microenvironment through the above pathways in esophageal cancer cells.
In order to further understand the interactions and regulatory networks between target genes, we constructed a PPI protein interaction network. Through PPI network analysis, we found that there are multiple interactions between target genes. Finally, among these differential genes, we selected FN1, TNF and IL6 as target genes. Among them, TNF is an up-regulated gene; FN1 and IL6 are down-regulated genes.
FN1 (fibronectin 1) is an extracellular matrix protein, involved in cell adhesion and migration, and closely related to the invasion and metastasis of cancer [28, 29]. The APE1 gene may cause the expression of these genes to be up-regulated, which may promote the invasion ability of esophageal cancer cells. FN1 is mainly derived from tumor-associated fibroblasts (CAFs), and FN1 in the tumor microenvironment (TME) causes tumor invasion by forming physical scaffolds or regulating signaling pathways [30], promoting Cell proliferation provides an environment for angiogenesis. Studies have found that in transgenic mice knocking out the FN1 gene, the density of blood vessels in the mice is significantly reduced and there are morphological abnormalities [31], and in the environment of tumors or chronic inflammation, FN1 can also promote pathological blood vessel formation [32]. In addition, studies by Yuwei Liu and others have shown that FN1 present in breast cancer tumor cells regulates changes in cell phenotype at both the protein and mRNA levels[33].
There is a close relationship between APE1 and FN1[34]. On the one hand, APE1 can affect the production and function of FN1 by regulating gene expression [35]. Studies have shown that overexpression of APE1 can enhance the transcriptional activity of FN1, leading to the overproduction of FN1 [36]. FN1 plays an important role in the assembly and stability of tumor extracellular matrix, and overexpression of APE1 can increase the production of FN1, thereby promoting the formation of tumor extracellular matrix and increasing the invasion and metastasis ability of tumor cells [37]. On the other hand, FN1 can also regulate the expression and function of APE1. Studies have found that FN1 can regulate the expression and activation of APE1 in cervical cancer cells through signal transduction pathways, such as the pyrophosphorylated tyrosine kinase (FAK) signaling pathway. Overexpression of FN1 can activate the FAK signaling pathway and increase the expression level of APE1 in cervical cancer cells[38]. Studies by Yuri Sakai and others have shown that in cervical cancer HeLa cell lines and lung cancer A549 cell lines, APE1 plays an important role in DNA repair and oxidative stress response, and the regulation of FN1 may affect the function of APE1, thereby affecting the cell's function. DNA repair capacity and oxidative stress response APE1 downregulation promotes actin rearrangement, induces actin accumulation in HeLa cells and the dissipation of stress fibers in A549 cells, and APE1 knockdown enhances cell motility of A549 cells, and this Motility is inhibited by inhibiting transforming growth factor (TGF)-β1 signaling, and therefore, APE1 controls the composition of the actin cytoskeleton by regulating TGF-β1 expression [34].
TNF is a pleiotropic factor that induces a variety of cellular responses by binding to the TNF receptor (TNFR), including the production of inflammatory cytokines, cell survival, cell proliferation, and apoptosis[39]. Initially, TNF was discovered to be produced by immune cells (such as macrophages, lymphocytes, and natural killer cells) in immune responses, and its name was derived from its direct killing effect on tumor cells. With the deepening of research, TNF-α has now been discovered to play a broader role in the immune system. When the body is infected, injured or otherwise stimulated, immune cells will release TNF-α, triggering an inflammatory response in response to the outside world. Invasion. The effects of TNF-α are dual in nature, offering both benefits and potential risks. In normal immune responses, TNF-α activates the immune system for immune regulation; however, excessive production or abnormal release of TNF-α may lead to immune diseases such as ankylosing spondylitis and psoriasis [40].
Kelly Verhelst et al. used TNF to act on human liver cell lines and found that TNF can activate the NF-κB pathway and accelerate the transition of liver cells from the G0/G1 phase to the S phase, resulting in accelerated and disordered liver cell cycles. It is speculated that TNF can pass through It is related to regulating the NF-kB pathway and thus affecting the occurrence and development of hepatocellular carcinoma[41]. Starska et al. found through immunohistochemical staining and RT-PCR technology that STAT3 is closely related to the expression of EGFR and IL-8 in laryngeal cancer tissues and is synergistic, but TNF and STAT3 are negatively correlated[42]; therefore, the expression of TNF is closely related to STAT3 Pathway related.
APE1 and TNF play different roles in tumor occurrence and development. Overexpression of APE1 is related to the proliferation, metastasis and treatment resistance of various tumor cells, and TNF, as a molecule that can induce tumor cell apoptosis, has potential anti-tumor effects in tumor treatment[43]. However, overexpression of APE1 may limit the anti-tumor effect of TNF, thus weakening its therapeutic effect [44, 45]. Studies have shown that in liver cancer cells, the APE1 inhibitor E3330 prevents the functional activation of the NF-κB pathway by altering APE1 subcellular trafficking and reduces TNF and fatty acid accumulation-induced IL by blocking redox-mediated NF-κB activation. -6 and IL-8 expression, thus affecting the chemotherapy resistance and inflammatory response of liver cancer cells [44]. Other studies have shown that APE1/ref-1 in endothelial cells can alleviate TNF-induced monocyte adhesion and the expression of vascular cell adhesion molecules, and this anti-adhesion property of APE1/ref-1 is mainly caused by NOS. mediated by a dependent mechanism, in addition, APE1/ref-1 may inhibit VCAM-1 expression by inhibiting superoxide production and p38 MAPK activation [45].
Interleukin is a lymphokine that interacts between white blood cells and immune cells. It plays an important role in mediating T and B cell activation, proliferation and differentiation, and inflammatory responses. IL6 (interleukin 6) has the most extensive functions. One of the cytokines involved in inflammatory response and immune regulation [46]. Studies have found that IL6 can activate members of the tyrosine kinase family after binding to its receptor. The activation of tyrosine kinase family members can activate multiple downstream pathways, such as MAPKs, PI3Ks, and STAT, and these pathways are involved in regulating cell proliferation, survival, metabolic regulation, and interstitial fibrosis and other biological behaviors [47, 48]. IL6 has been found to be overexpressed in a variety of tumors. Studies have confirmed that chronic inflammation plays a key role in the process of tumor formation, and IL6 is a tumor-related inflammatory factor that is highly expressed in the malignant tumor microenvironment and has the ability to increase tumor invasion, promote tumor distant metastasis, and Angiogenesis, participation in the formation of tumor drug resistance, and regulation of interstitial fibrosis[49, 50].
There is a complex interaction between APE1 and IL-6. On the one hand, APE1 can affect IL-6 production and signaling pathways by regulating gene expression. Jedinak et al. demonstrated that APX3330 inhibits the secretion of inflammatory cytokines in lipopolysaccharide-stimulated macrophages, including TNF, IL-6, IL-12 and the inflammatory mediator nitric oxide (NO), by inhibiting TFs NF-κB and AP-1. and prostaglandin E2 (PGE2) [51, 52]. Studies have found that overexpression of APE1 can enhance the transcriptional activity of IL-6, leading to overproduction of IL-6. IL-6 is an autocrine factor, and its overproduction can form a self-amplifying loop to further promote the proliferation and survival of tumor cells[52]. On the other hand, IL-6 can also regulate the expression and function of APE1. IL-6 can increase the expression level of APE1 by activating signal transduction pathways, such as the JAK/STAT3 signaling pathway [53, 54]. In addition, IL-6 can also affect the function of APE1 by regulating oxidative stress response and other molecules in the DNA repair pathway. High-level expression of IL-6 may lead to an increase in intracellular oxidative stress, thereby enhancing APE1 activity and DNA repair capacity [55]. The interaction between APE1 and IL-6 has an important impact on the occurrence and development of tumors. High levels of APE1 and IL-6 expression may work synergistically to promote the proliferation, invasion and metastasis of tumor cells. In addition, they may also promote tumor immune evasion and resistance to treatment by interfering with immune surveillance and inhibiting apoptosis [51].
Our research still has some limitations. Further functional studies can experimentally verify the role of these core genes and deeply explore their regulatory mechanisms by sh down-regulating APE1. For example, gene knockout or overexpression methods can be used again to study these core gene methods to explore their impact on the biological behaviors of esophageal cancer cells, such as proliferation, adhesion, migration, and apoptosis, as well as on esophageal cancer genomics and Metabolomics implications.. In addition, the interaction between shAPE1 and these genes can be further explored to find potential regulatory mechanisms. Although this study found through KEGG analysis that the differential genes were mainly enriched in pathways such as "Pathways in cancer", "Calcium signaling pathway", "PI3K-Akt signaling pathway" and "Complement and coagulation cascades", the discussion section did not analyze these pathways. The specific regulatory mechanisms are discussed. Further research can experimentally verify the functions of these pathways in esophageal cancer cells through in vitro or in vivo experiments, and explore the regulatory mechanism of shAPE1 on these pathways.
In summary, through RNA-Seq analysis and functional studies, this study revealed that knockdown of the APE1 gene triggered the differential expression of multiple genes in esophageal cancer cells and discussed the potential functions of these genes in esophageal cancer cells. These findings provide important clues for further studying the mechanism of shAPE1 in the development of esophageal cancer and developing related treatment strategies. Future studies can further verify the functions of these differentially expressed genes and explore the regulatory network of APE1 and its interactions with other pathways.