Identification and validation of autophagy-related genes in Hirschsprung’s disease

Introduction

Hirschsprung’s disease (HSCR) is a congenital disorder characterized by the absence of nerve cells in the intermuscular and submucosal nerve networks of the intestines, leading to compromised gastrointestinal function (Amiel & Lyonnet, 2001). HSCR likely occurs due to the premature cessation of vagal neural crest cell migration from the head to the tail during weeks 5 to 12 of gestation. This interruption causes problems in the movement, growth, and specialization of intestinal neural crest cells (Borrego et al., 2013). Autophagy is a tightly regulated biological process that is critical for the degradation and recycling of cellular components, damaged organelles, and protein aggregates within cells (Li et al., 2016). Recent investigations have shown the crucial function of autophagy in preserving cellular stemness and promoting diverse stages of differentiation, such as cell proliferation and migration (Foerster et al., 2021). Autophagy is implicated in a variety of diseases, including intestinal problems. Nevertheless, the autophagy-related genes linked to HSCR have not been extensively studied and require more research.

In this study, we re-analyzed two public microarray datasets, GSE96854 and GSE98502, with a focus on autophagy-related genes. We used multiple bioinformatics approaches for integrated analysis, including functional enrichment and protein–protein interaction (PPI) network analyses. This provided insights into the underlying mechanisms of these genes. Finally, we identified nine hub genes (Fig. 1).

Experimental Procedures and Materials

Autophagy-related genes datasets and microarray data

We acquired a cumulative of 232 genes linked to autophagy from The Human Autophagy Database (http://www.autophagy.lu/index.html). The mRNA expression profiles dataset from GSE96854 was acquired from the GEO database (http://www.ncbi.nlm.nih.gov/geo/). This dataset is part of the GPL18943 platform, specifically the NimbleGen Human Gene Expression 12x135K Array. It includes 30 colon samples, which were pooled into three control and three case specimens, each composed of tissues from 15 HSCR patients and 15 matched controls. The data were preprocessed using background correction and normalization through the RMA method. Quality control included inspecting MA plots and boxplots to ensure consistency across samples.

GO and KEGG Pathway Enrichment Analysis of Genes Associated with Autophagy

In order to confirm the functional importance of prospective targets, a functional enrichment analysis was conducted. Gene Ontology (GO) is a commonly used technique for gene annotation, which includes the categorization of genes into cellular components (CC), molecular functions (MF), and biological processes (BP). The Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database specifically created to facilitate the methodical examination of gene function and genomic information. It focuses on studying the interconnectedness of gene expression within a comprehensive network. GO and KEGG pathway enrichment analyses were performed using the clusterProfiler package (version 3.14.3) with a p-value cutoff of 0.05 and q-value cutoff of 0.05. For KEGG, organism-specific pathway annotations for Homo sapiens were used.

Statistical analysis

The statistical analyses were performed using R software (version 4.2.0; R Core Team, 2022). The gene expression levels of our clinical samples were compared using a two-tailed Student’s t-test, and a significance threshold of P < 0.05 was utilized to determine statistically significant differences.

Results

Retrospective analysis of differential expression of autophagy-related genes in Hirschsprung’s disease

We performed a comprehensive investigation of the expression levels of 232 autophagy-related genes across three pooled samples from HSCR patients and three pooled control samples. PCA was first conducted to assess the variability and repeatability of the data, ensuring robust clustering between HSCR and control samples (Fig. 2A). We identified 20 genes showing significant differential expression, with 15 up-regulated and five down-regulated, based on a significance threshold of P < 0.05 and an absolute fold-change greater than 2.0. These results were visualized using a heatmap to display overall gene expression patterns and a volcano plot to highlight the most significantly up- and down-regulated genes (Figs. 2B and 2C). Further analysis of these expression patterns was conducted through violin plots, which depict the distribution of expression levels for these genes across the HSCR and control groups (Figs. 3A and 3B). The five genes that showed increased expression were NRG1, BAG3, CAPN2, ITGA6, and EGFR. On the other hand, the five genes that showed decreased expression were SERPINA1, BNIP3, NRG2, CDKN2A, and ITPR1. These findings are shown in Figs. 2A and 2B, and summarized in Table 1.

Table 1:

The 20 differentially expressed autophagy-related genes in colon samples from cases and controls.

Gene Symbol	logFC	P-value	Adjusted P-value	T	Changes
NRG1	2.515266384	0.000342269	0.063319673	6.269035768	Up
BAG3	1.290357777	0.002314947	0.107066292	4.555551479	Up
CAPN2	1.248786297	0.000728997	0.067432254	5.548953225	Up
ITGA6	1.19463117	0.006863156	0.251385681	3.717129017	Up
EGFR	1.0902745619	0.024006962	0.370107338	2.834354471	Up
ARSB	0.846961015	0.002203959	0.107066292	4.595465466	Up
NAF1	0.832092075	0.014375713	0.29735965	3.187439413	Up
SIRT1	0.814564303	0.032025328	0.390017682	2.639686981	Up
CHMP2B	0.69288678	0.038279749	0.393430755	2.52017479	Up
EIF2AK3	0.67694849	0.008153049	0.251385681	3.591198144	Up
MAP1LC3B	0.63199153	0.034464467	0.390017682	2.590434178	Up
GOPC	0.559534035	0.02084943	0.356618735	2.930502318	Up
CLN3	0.544895447	0.034039712	0.390017682	2.598747137	Up
ATG3	0.467254333	0.046081265	0.426251697	2.396496742	Up
ATF6	0.430002507	0.042619723	0.414981509	2.448505293	Up
SERPINA1	−1.594610937	0.014466145	0.29735965	−3.183059631	Down
BNIP3	−1.36435527	0.010413534	0.275214823	−3.415036548	Down
NRG2	−1.143834525	0.032292088	0.390017682	−2.634114933	Down
CDKN2A	−1.096129695	0.035839463	0.390017682	−2.564230317	Down
ITPR1	−1.086387215	0.021204357	0.356618735	−2.918958624	Down

DOI: 10.7717/peerj.18376/table-1

Functional and pathway enrichment analysis of the differentially expressed genes associated with autophagy

In order to investigate the possible biological functions of DEGs, we used the R program to perform GO and KEGG enrichment analyses (Table S2). The GO analysis categorized DEGs into 919 biological processes (BPs), 65 molecular functions (MFs), and 112 cellular components (CCs), highlighting key processes such as autophagy, which utilizes macroautophagic mechanisms, and various cellular components including the endoplasmic reticulum and organelle membranes. Additionally, molecular functions such as enzyme binding and cadherin binding were significantly enriched (Figs. 5A–5C). The KEGG pathway analysis further revealed significant enrichment in autophagy-related pathways, including cellular senescence and the FoxO signaling pathway (Figs. 6A and 6B), providing insight into the complex mechanisms underlying HSCR. These enrichments not only highlight the role of autophagy in HSCR but also suggest that pathways related to cellular senescence and FoxO signaling may contribute to the disease’s pathogenesis, emphasizing the intricate interplay between different cellular processes.

Verification of hub gene expression

We selected an HSCR dataset to validate the expression levels of these core genes. The findings revealed an increase in the expression of EGFR, SIRT1, EIF2AK3, and ATG3 in contrast to normal intestinal tissue, although MAP1LC3B showed no change. Nevertheless, ATF6 was suppressed. The bioinformatics study indicated earlier anticipated that these genes will be upregulated. The expression of CDKN2A was decreased, whilst the expressions of ITPR1 and BNIP3 were increased, in contrast to the anticipated outcomes from the bioinformatics analysis (Fig. 8). Out of the genes that were examined, only EGFR, SIRT1, CDKN2A, and ATG3 showed statistical significance and concordance with earlier bioinformatics analysis (P < 0.05), which supports our previous results.

Table 2:

Top nine in network ranked by DC method.

Rank	Gene ID	Gene name	Score	Changes
1	EGFR	Epidermal growth factor receptor	71.33333333	Up
2	MAP1LC3B	Microtubule associated protein 1 light chain 3 beta	63.26666667	Up
3	SIRT1	Sirtuin 1	20.13333333	Up
4	EIF2AK3	Eukaryotic translation initiation factor 2 alpha kinase 3	10.33333333	Up
5	ITPR1	Inositol 1,4,5-trisphosphate receptor type 1	6	Down
6	ATF6	Activating transcription factor 6	2.8	Up
7	CDKN2A	Cyclin dependent kinase inhibitor 2A	2	Down
8	ATG3	Autophagy related 3	1.066666667	Up
9	BNIP3	BCL2 interacting protein 3	1.066666667	Down

DOI: 10.7717/peerj.18376/table-2

In order to confirm the trustworthiness of the GSE96854 dataset, we performed qRT-PCR analysis on four autophagy-related genes that showed varying levels of expression in clinical samples. The aganglionic colon tissue sample showed a significant increase in the expression levels of EGFR, SIRT1, and ATG3 compared to the control sample. This finding aligns with the findings obtained from mRNA microarray analysis of colon tissue samples. Moreover, there was a significant decrease in the levels of CDKN1A expression. However, the levels of SIRT1 expression were the only ones that showed a significant difference between the two groups (P < 0.01) (Fig. 9).

Discussion

This study explored the pathogenic mechanisms of HSCR through comprehensive bioinformatics analysis, leading to the identification of nine hub genes and their potential roles in the disease. The significant upregulation of SIRT1, for example, indicates its pivotal role in modulating autophagy in HSCR. The correlation analysis suggests that SIRT1 may influence key pathways such as cellular senescence and FoxO signaling, both of which are implicated in the regulation of autophagy. This highlights a potential feedback loop where SIRT1 activation could promote autophagy, leading to cellular changes characteristic of HSCR.

Rather than focusing on general autophagy mechanisms, this study specifically identifies how autophagy-related genes, particularly SIRT1, contribute to HSCR pathogenesis. Previous studies have linked SIRT1 to the regulation of autophagy in gastrointestinal diseases, where its role in maintaining cellular homeostasis is critical (Fefelova et al., 2016). Our findings support and extend this research by demonstrating that SIRT1 upregulation in HSCR tissues may reflect an adaptive response to disrupted autophagy processes. The involvement of other hub genes such as ATG3 and EGFR further suggests that these genes are integral to the dysregulation of autophagy in HSCR and could serve as potential therapeutic targets (Wang et al., 2023; Zhang et al., 2017a). For example, Zhang et al. (2017a) demonstrated that SIRT1 is involved in regulating intestinal inflammation through autophagy modulation, providing a possible explanation for its upregulation in HSCR. In addition to SIRT1, other hub genes like EGFR have been implicated in autophagy-related pathways, particularly in cancer biology, where EGFR’s role in autophagy regulation is well established (Wang et al., 2023). These results are consistent with studies showing that EGFR-mediated autophagy can promote cellular survival under stress conditions, which could explain its increased expression in HSCR tissues. Moreover, the unexpected downregulation of CDKN2A despite predictions of upregulation based on bioinformatics models might be attributed to its role in a different cellular context, such as its known tumor-suppressive function in cancer (Morigi, Perico & Benigni, 2018). This discrepancy could reflect distinct regulatory mechanisms in the neural tissues affected by HSCR, where CDKN2A may play a less dominant role. These findings underscore the importance of conducting further experimental validation in specific tissue contexts to refine our understanding of these gene interactions.

Recent research has provided evidence that autophagosome structures are present in the myenteric plexus of individuals with HSCR who have narrow and transitional portions of the intestine. Furthermore, the expression of Beclin1 and LC3 was shown to be most prominent in the narrow segment (Zhang et al., 2017b). Sbrana et al. (2016) have shown the possible role of mir-939 in controlling LRSAM1 in HSCR, and propose that autophagy may possibly play a part in the susceptibility to HSCR. Autophagy has been recognized as a crucial mechanism for maintaining normal gastrointestinal function in recent years. However, when autophagy becomes dysregulated, it may lead to gut dysfunction (Li et al., 2020). There is significant data suggesting that autophagy has a dual effect on gastrointestinal problems. Autophagy is a fundamental mechanism that occurs in all cells and helps maintain energy metabolism and facilitate substance recycling (Qiang, 2016). Nevertheless, its manifestation is swiftly increased in reaction to cellular energy deficiencies resulting from insufficient dietary intake or circumstances such as hunger and hypoxia (Guo et al., 2018). Overstimulation of autophagy may cause excessive breakdown of vital cellular components, eventually resulting in autophagic cell death. Zheng et al. (2021) colleagues discovered that miR-222 was markedly increased in slow transit constipation (STC) mice compared to healthy control rats. Furthermore, they observed that miR-222 may stimulate excessive autophagy and death of isolated ICCs. Furthermore, it has been shown that the increase in miR-222 leads to heightened levels of autophagy-related proteins LC3B and Beclin-1. MiR-222’s regulation of autophagy might result in tissue harm, presumably linked to gut dysfunction. He et al. (2022) found that electroacupuncture (EA) therapy may inhibit the PI3K/AKT/mTOR signaling pathway in the colonic tissues of mice with functional constipation (FC). This leads to the enhancement of autophagy in enteric glial cells (EGCs) and improvement in intestinal motility.

Autophagy is crucial in controlling the activity of different cell types in the intestinal milieu. This process involves both innate and adaptive immune cells, as well as stromal cells. Nevertheless, there is still a great deal of knowledge to be acquired about its precise role in preserving the integrity of the intestinal epithelial barrier. Autophagy is crucial in improving the death of intestinal cells and strengthening the function of the gut barrier. Autophagy has a crucial role in regulating cellular stress and preventing stress-induced apoptosis, which are important processes that cause the death of intestinal epithelial cells (IECs) (Li et al., 2019; Wen et al., 2017). Autophagy and autophagy-related proteins have a vital function in controlling intestinal damage and inflammation by regulating the process of epithelial cell apoptosis and necrosis (Cong et al., 2017; Galluzzi et al., 2017). Tumor necrosis factor inhibits autophagy, resulting in an increase in CLDN2 expression. This, in turn, causes malfunction in epithelial tight junctions and leads to an increased permeability of the barrier (Chen et al., 2021). Furthermore, impairments in Atg9, Atg1, Atg13, and Atg17/FIP200 have been shown to cause a lack of autophagy in Drosophila, which subsequently leads to an elevated permeability of the intestinal barrier (Morigi, Perico & Benigni, 2018). Autophagy-related proteins have a crucial function in the host’s response to infection in the intestinal epithelium. They do this via xenophagy, which involves the removal of foreign pathogens, as well as other processes that help regulate the immune system. This results in both the protection and vulnerability during intestinal infections (Petri et al., 2017). Multiple investigations have shown that the development of Hirschsprung-associated enterocolitis is caused by abnormalities in the structure and function of the intestinal mucosal barrier (Petri et al., 2017; Ravanan, Srikumar & Talwar, 2013). Ultimately, it is reasonable to hypothesize that autophagy may play a role in the development of HSCR. However, the investigation of autophagy-related genes using bioinformatics analysis has not been done in HSCR. Through bioinformatics research, we have discovered 20 putative autophagy-related genes in HSCR for the first time. GO and KEGG enrichment analysis were used to evaluate the probable biological roles of autophagy-related genes that were expressed differently. The findings of our study revealed that certain phrases were significantly linked to autophagy in animals, with a special emphasis on macroautophagy. In addition, by using PPI network and doing key module analysis, we have discovered nine central genes that are linked to HSCR. These genes include EGFR, MAP1LC3B, SIRT1, EIF2AK3, ATF6, ATG3, ITPR1, CDKN2A, and BNIP3.

Out of the nine hub genes anticipated to be associated with diabetic retinopathy, only the expression of SIRT1 was shown to align with the bioinformatics analysis of mRNA chip data. Prior research has shown the crucial function of the sirtuin protein family in controlling cellular metabolism and impacting many biological processes in several organs, such as the central nervous system, liver, pancreas, and intestinal tract (He et al., 2022). SIRT1, widely studied as a target of miR-132/-212, plays a vital role in the development of cancers, inflammatory diseases, neurological disorders, and other illnesses (Doherty & Baehrecke, 2018; Li et al., 2020). Although the exact cause-and-effect relationship between SIRT1 and HSCR is not yet established, current research suggests a potential association between SIRT1 and the development of HAEC. According to the findings of Chen et al. (2021), exosomal miR-18a-5p has the ability to increase inflammatory responses and cause cell death in colonic epithelial cells in HAEC. This is achieved by activating the RORA-dependent SIRT1/NF κB signaling pathway (Chen et al., 2021). Li et al. discovered that lipopolysaccharide (LPS) causes an increase in the expression of miR-132 and miR-212 in human aortic endothelial cells (HAEC), resulting in the inhibition of SIRT1 and promoting the activation of the NLRP3 inflammasome (Li et al., 2020). The results emphasize the important function of the LPS/miR-132/-212/SIRT1/NLRP3 regulatory network in the development of HAEC. Our investigation revealed that SIRT1 was increased in the narrow segments of HSCR, suggesting its involvement in the development of the condition.

Although we have made meticulous efforts, our research is limited by the inclusion of a very small number of clinical samples. Thus, it is crucial to verify our findings in a more extensive group of individuals diagnosed with HSCR. Another limitation of this study is the gender imbalance in the control group, which may introduce bias in the gene expression analysis. Future studies should aim for a more balanced sample to validate these findings. Moreover, while the expression levels of autophagy-related genes that are differently expressed have been validated in clinical specimens, additional research is required to understand the possible processes of these genes in HSCR cells and mice models.

Conclusions

Through bioinformatics research, a total of 20 candidate genes associated with autophagy were discovered in HSCR. Nine hub genes, including EGFR, MAP1LC3B, SIRT1, EIF2AK3, ATF6, ATG3, ITPR1, CDKN2A, and BNIP3, were found by creating a PPI network and identifying important modules. This study found an increase in the expression of SIRT1 in HSCR tissues and in vitro experiments confirmed that it can regulate autophagy, suggesting its potential role in HSCR pathogenesis by regulating autophagy-related pathways. Additional trials are required to investigate the regulatory role of SIRT1 in the development of HSCR.

Supplemental Information