Identification and experimental validation of autophagy-related genes in abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a fatal condition which threatens public health. It consists of local enlargement of the abdominal aorta with reduced vascular smooth muscle cells (VSMCs) in the vascular middle layer. AAA is a common form of aneurysm, but it is usually asymptomatic and rupture is often accidental and fatal, with a mortality rate of 80% or more [1]. Previous studies have shown that risk factors for AAA include genetic factors, advanced age, male sex, smoking, hypertension, hyperlipidemia, obesity, atherosclerosis and other vascular occlusives [2]. Unfortunately, there is no clinically established effective pharmacological approach to limit the progression of AAA or the risk of rupture in humans, surgical intervention being the only viable treatment [3]. Growing evidence indicates that the pathogenesis of AAA involves multiple biological functions, including chronic inflammation, cell proliferation, apoptosis, and autophagy [4‐6], and that its regulatory mechanism is complex. Therefore, it is important to explore the pathogenesis of AAA and find key targets and markers for its diagnosis and treatment.

Autophagy is a conserved mechanism that transports damaged, denatured, or senescent proteins and organelles to lysosomes for digestion and degradation [7]. Meanwhile, autophagy is a factor in cardiovascular and a range of other diseases. For example, lncRNA CAIF alleviates myocardial infarction and protects cardiac tissue by regulating cardiac autophagy through the p53-cardiomyin axis [8]. In addition, Mir-214-3p directly targets vascular endothelial cell ATG5, reduces ox-LDL-induced autophagy, and regulates the progression of atherosclerosis [9]. Studies have shown that certain signaling pathways affect the biological functions of AAA through autophagy. Autophagy induced by AngII is modulated by JAK2/STAT3 and NF-κB signaling, which is inhibited by BP-1-102, thus affected the progression of AAA [6]. However, the autophagy-related genes involved in AAA remain largely unclear and require further study. The exploration of subclinical ARGs of AAA will offer new latent targets for clinical treatment of AAA.

With rapid development of next-generation sequencing technology, ARG-based signs have been used to evaluate and verify the differential expression of genes in different types of diseases [10, 11]. Recent research has identified the potential roles of characteristic ARGs in the diagnosis of systemic lupus erythematosus (SLE) and revealed the correlation between their expression and DNA methylation [11]. However, the role of ARGs in AAA has not been fully clarified.

In this study, we explored the differentially expressed genes related with autophagy in AAA by analyzing the GSE98278 and GSE57691 datasets in the GEO database. Firstly, we screened 44 candidate genes. Following, PPI analysis and GO enrichment analyses were employed on the candidate genes. Lastly, we further verified the key genes expression levels among the candidate genes in clinical samples with AAA using RT-qPCR. Our results found that IL6, PPARG, SOD1, and MAP1LC3B may influence the process of AAA by regulating autophagy. These will help deepen the understanding of AAA and provide an effective reference for clinical diagnosis and treatment.

AAA is a chronic vascular inflammatory disease and a vital reason of death from aortic rupture in adults, yet no effective clinical treatment is available. Risk factors for AAA include smoking, aging, inflammation, thrombosis, and atherosclerosis. Mounting evidence indicates that autophagy may have a critical role in AAA. For example, AAA risk factors are closely associated with autophagy [16‐19], while Zheng et al. found that ATG is involved in the induce formation of autolysosomes in AAA tissues [20]. In addition, Li et al. showed that rapamycin, a powerful immunosuppressant, can inhibit the pathological process of AAA by inhibiting the mTOR pathway [21]. However, more evidence and further validation is needed to clarify the potential role of autophagy in the pathological mechanism of AAA.

Recently, with the development of next-generation sequencing, bioinformatics analysis has been extensively applied to explore and recognized latent biomarkers of some diseases [22], and many public databases have emerged, such as TGGA and GEO. The latest of a series of studies have explored the pathogenesis of AAA with potential therapeutic value in gene expression. Chen et al. constructed a co-expression network using WGCNA and analyzed gene components in abdominal aortic aneurysm and healthy control states. In their study, hub gene clusters (the most important clusters of the DEG co-expression network chosen by MCODE) including YIPF6, RABGAP1, ANKRD46, GPD1L, and PGRMC2 were identified [23]. These genetic factors have underlying diagnostic implications and may turn into biomarkers for abdominal aortic aneurysm. Moreover, Giusti et al. using expression profiles of microarray data showed that the autophagy gene ATG5 in peripheral venous blood of AAA patients was up-regulated compared with control patients [24]. Despite these advances, the understanding of pathogenesis and genetics of AAA is still not fully clarified. Consequently, it is essential to further discover new targets for the diagnosis and treatment of AAA.

As far as we know, several published articles have explored key genes and the role of autophagy in AAA. For example, a recent study reported on 10 hub genes in AAA, some of which are involved in chronic inflammation in patients [23]. However, bioinformatics analysis of ARGs in AAA remains indistinct. In this survey, we used bioinformatics analysis for the first time to identify 44 potential AAA-related ARGs from two GEO datasets (GSE98278 and GSE57691). One of the previous studies on ARGs of AAA confirmed that mutations in the IL-6-174G/C allele increased the risk of AAA development [25]. In addition, there is evidence that FOXO3a promotes phenotypic transformation of VSMS through the P62/LC3BII autophagy signal channel, accelerating the formation of AAA, and that reducing FOXO3a expression may stop AAA formation [26]. In the future, we plan to explore more subclinical ARGs associated with AAA.

This study used GO and KEGG enrichment analyses to elucidate the biological functions of DEARGs, which were mainly enriched in inflammatory cell activation, cell chemotaxis, the FoxO signaling path, autophagy, cellular senescence, TNF signal channel, longevity controlling pathway, AMPK signaling path, and non-small cell lung cancer. Previous evidence confirms that pathological traits of abdominal aortic aneurysm contain inflammatory disease, AS, and thrombosis [27]. In addition, macrophages, VSMS, and endothelial cells play a role. Autophagy plays a crucial part in all of these processes. For example, in ATG5-deficient ApoE−/− mice, autophagy absence causes hyperactivation of macrophage inflammation complex, accelerating plaque movement [28]. The latest study showed that P2RY12 receptor inhibits autophagy and reduces cholesterol effluence, promoting VSMC-derived foam cytogenic in advanced atherosclerosis. This suggests that P2RY12 receptor plays an important role in the regulation of macro-autophagy/autophagy and the formation of VSMC-derived foam cells in advanced atherosclerosis [29]. In addition, autophagy has been stated to play an essential role in bleeding and thrombotic diseases by regulating the count and function of platelet (PLT), which are the core factors of physiological hemostasia [30]. Therefore, it is necessary to conduct clinical or basic experiments to probe the underlying biological functions of these DEARGs.

This study identified 7 ARGs and found that five of these were distinctively expressed between the two groups. The lack of difference in FOX03 expression levels between groups may be due to large individual differences and small sample sizes and will be further explored and verified in the future. Previous evidence has identified several genes involved in cardiovascular disease. Nishihara et al. demonstrated that suppression of IL-6 can suppress Stat3 activation and expansion of AAA in mouse models [31]. Other studies have found that PPARG polymorphism is weakly associated with the development of AAA [32], and that PPARγ attenuates AAA by inhibiting inflammation and proteolytic degradation [33]. SOD1 has not been reported in AAA, but when treated with hydroxyl ethanol, it inhibits Ang II-induced Alzheimer’s disease, decreases the expressing of NF-κB, P65, TNF-α, and IL-1β, and increases the conveying of SOD1, MMP9, and GCLC in mice [34]. However, the precise mechanisms of these genes in AAA remain largely unknown and require further exploration.

However, this study has some limitations. First, we obtained bioinformatics results from public chip data and did not obtain adequate clinical information. Second, the clinical sample size was small, and the results must be verified in a larger cohort. Third, this study validated the DEARGs in clinical specimens only and did not research the latent mechanisms of these genetic factors in AAA cells and animal models. Consequently, advanced exploration and investigation are needed in the future.

In conclusion, hub genes IL6, PPARG, SOD1, and MAP1LC3B may influence the onset and development of AAA by controlling autophagy. This study proposes a new characteristic of ARGs, enhances understanding of AAA, and may advance its diagnosis and therapy.