Background
Ulcerative colitis (UC) is a chronic, recurrent, and intestinal inflammatory disease; however, its pathogenesis remains unclear. It is generally considered that the intestinal mucosal immune system of patients with UC produces abnormal amplification of the immune response to intestinal microbial antigens in a specific environment, which can cause intestinal mucosal inflammatory damage [
1]. The imbalance between inflammation and mucosal immunity is an important feature of colorectal carcinogenesis [
2]. Therefore, patients with UC have an increased risk of UC-associated colorectal cancer (CAC).
Although an increasing number of studies have investigated coding gene-related biomarkers in CAC, protein-coding genes only account for 2% of the human genome. The competitive regulatory crosstalk of different molecular species, especially the competition between protein-coding and non-coding RNA transcripts, is a key link in the occurrence of disease [
3,
4]. MicroRNAs (miRNAs), as an abundant class of small, non-coding, single-stranded oligoribonucleotides, act as regulators in various cellular processes and function as sequence-specific silencers of target gene transcription after binding, thereby affecting the expression levels of more than half of all protein-coding genes [
5,
6]. Therefore, Salmena et al. [
7] proposed the competing endogenous RNA (ceRNA) hypothesis, which posits that most RNA molecules act in a “many-to-many” manner. As each miRNA molecule may potentially target miRNA response elements on multiple messenger RNA (mRNA) molecules, each mRNA molecule can be targeted by multiple miRNA molecules [
8]. This suggests that miRNA molecules have a pivotal role in competitively regulating crosstalk and lead to gene silencing by binding to mRNAs, and that ceRNA regulates gene expression by competitively binding to miRNAs.
Numerous ceRNA studies in different diseases (ie tumors, inflammatory bowel diseases, liver fibrosis, and cardiovascular diseases) have suggested that ceRNA can influence dysregulation of the immune microenvironment by regulating the interaction of different types of RNAs, thereby promoting the occurrence and development of diseases [
9‐
15]. However, the specific immunoregulatory mechanisms in the CAC process remain unclear. We hypothesized that the ceRNA regulatory axis can alter the immune microenvironment during the development of CAC.
To verify this hypothesis, we integrated immune-related genes (IRGs), weighted gene co-expression network analysis (WGCNA), and differential expression analysis results, and then constructed a ceRNA immunoregulatory network based on the principle of reverse prediction. Subsequently, small-molecule medicines with possible applications in therapy for CAC were investigated. Pathway enrichment analysis and immune cell infiltration analysis were performed to identify the key pathways and immune cells. Correlation analysis was conducted to identify the key ceRNA regulatory axis from the network. This study provides a foundation for improving the understanding of the pathophysiological processes of CAC and insights into the variations in the immunological microenvironment of the disease.
Discussion
UC is a chronic inflammatory disease of unknown etiology, and its cumulative incidence of progressing to CAC has markedly increased over the years [
44]. Because of chronic inflammation caused by UC, various layers of the intestinal wall are infiltrated by immune cells, forming an immune microenvironment and participating in the induction of CAC through the production of cytokines and chemokines [
45]. However, the underlying mechanisms remain unclear. Here, we explored the role of the ceRNA-immunoregulatory axis in the immune regulation of CAC at the transcriptional level.
We integrated three bioinformatics methods, including immune gene list, WGCNA, and differential gene expression analysis, to identify 130 HIGs. We found that metronidazole, 3-acetamidocoumarin, heptaminol, and isometheteptene reversed the expression levels of HIGs and play a potential role in the treatment of CAC. Compared with the other three drugs not reported in CAC, metronidazole has been shown to inhibit the occurrence of tumors and reduce the degree of inflammation in CAC animal models [
46]. Based on the ceRNA hypothesis, we predicted miRNAs and lncRNAs using HIGs and constructed a ceRNA-immunoregulatory network to better understand the CAC-related molecular mechanisms and biological phenomena at the transcriptional level.
To determine the underlying mechanisms affecting the occurrence of CAC, we analyzed the differences in pathways between the CAC and NC groups using two major pathway enrichment methods. The results showed that the largest number of genes (93 genes) was enriched in the NF-κB pathway, revealing that this pathway is closely related to CAC. The NF-κB pathway is involved in the immune response in vivo through classical and non-classical pathways, allowing the massive release of pro-inflammatory cytokines that cause tissue damage and participate in tumor invasion and metastasis by regulating the expression of angiogenesis-related genes [
47‐
50]. A previous study showed that the NF-κB pathway exerts a tumor-promoting effect in a CAC mouse model [
51].
We further evaluated alterations in the immune microenvironment of CAC and found that only neutrophils had the same expression trend (high in the CAC group and low in the NC group) across the three methods used to calculate the degree of immune cell infiltration. These results were consistent with those reported in previous studies showing that neutrophil infiltration is significantly higher in the colonic mucosal layer of a CAC mouse model, promoting CAC occurrence by secreting chemokines and, consequently, recruiting chemotactic receptors [
52,
53]. Correlation analysis showed that the NF-κB pathway was positively correlated with neutrophils. Further analysis of 93 core genes enriched in the NF-κB pathway revealed that IL6ST was positively associated with neutrophils. We subsequently screened the miRNAs regulating IL6ST (miR-1-3p) and its upstream lncRNA from the ceRNA network (NEAT1). Previous studies suggested that NEAT1 promotes tumor development by downregulating target miRNAs. Zhou et al. [
54] reported that NEAT1 targets and downregulates miR-500a-3p, promoting gastric cancer cell proliferation and invasion; Huang et al. [
55] suggested that NEAT1 promotes pancreatic cancer progression by negatively regulating miR-506-3p; whereas Zhang et al. [
56] showed that upregulation of NEAT1 is involved in the proliferation of glioma cells by negatively regulating miR-324-5p. However, the role of NEAT1 in CAC has rarely been reported. MiR-1-3p, a downstream target of NEAT1, has been shown to play a facilitative role in tumors. For instance, Peng et al. [
57] reported that miR-1-3p affects gastric cancer cell proliferation by promoting the oxygen glycolytic pathway, and Liu et al. [
58] indicated that downregulation of miR-1-3p expression is involved in the growth and motility of lung cancer cells. Our results suggest that IL6ST is a target gene of miR-1-3p in CAC. IL6ST is a subunit of the IL-6 receptor, and the IL-6/IL-6 receptor complex only functions by binding to IL6ST [
59]. Previous studies showed that IL-6 promotes the occurrence of CAC by interacting with IL6ST [
60]. Our data further supported that NEAT1 can competitively bind to miR-1-3p and upregulate IL6ST at the transcriptional level, affecting the NF-κB pathway and neutrophil infiltration as well as promoting the occurrence and development of CAC.
We constructed a ceRNA-immunoregulatory network by integrating multiple bioinformatics tools and deeply analyzed the alterations of the immune microenvironment and pathways in the process of CAC. Ultimately, we identified a ceRNA immunoregulatory axis closely related to CAC. However, our study had some limitations; (1) we only used public datasets for the analysis, which may be biased because of the limited sample size. (2) Although we conducted a bioinformatic analysis and database prediction, the direct relationship between the ceRNA immunoregulatory axes requires experimental verification. (3) In vivo and in vitro functional experiments are needed to determine the biological role of the ceRNA regulatory axis in CAC. (4) Currently, bioinformatic analysis for the prediction of drug utility is limited; however, we believe that this study may provide valuable insights in designing drugs to treat CAC.
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