Esophageal achalasia is an archetypal esophageal motility disorder, which is characterized by impaired relaxation of the lower esophageal sphincter (LES) and abnormal peristalsis of the esophageal body. Esophageal achalasia can result in an impaired ability to digest food and can thus reduce a patient's quality of life (1-3).

Although this disease was first reported ~300 years ago, its etiology remains unknown (4). Current treatment strategies aim to reduce LES pressure. Methods of treatment include endoscopic balloon dilation, botulinum toxin injection, laparoscopic Heller's myotomy, and in serious advanced cases, surgical resection of the affected esophagus (5). However, in recent years, per-oral endoscopic myotomy (POEM) has been established as an alternative minimally invasive method of treating esophageal achalasia (6). This treatment is effective and safe, even in elderly patients, allowing for short and long-term prognoses (6,7). Sato et al (8) performed per-oral endoscopic biopsies from the muscle layer during POEM, called POEM-b. According to their study, histopathological and immunohistochemical analysis of POEM-b samples showed neurodegenerative signatures rather than inflammatory infiltrates in the muscular layer (8). There was a tendency for preservation of interstitial cells of Cajal in patients with type III achalasia, whereas more severe fibrosis was observed in patients with type I achalasia, based on the Chicago classification criteria high-resolution manometry (HRM) (8,9).

Currently, the proposed causal factors are varied and multifactorial, and are hypothesized to involve complex interactions between the autoimmune and inflammatory response, and this may be initiated by viral infections in patients who are genetically susceptible (2). Causal viral agents include (but not limited to) herpes simplex virus, which is a neurotropic virus that exhibits a predilection for squamous epithelium, as well as varicella-zoster, measles and human papillomavirus (4,5,7,10). Moreover, it has been reported that HSV type 1 (HSV-1) DNA and RNA is detectable in all tissues from patients with achalasia, but not in the control tissues (11). Therefore, HSV-1 infection may be considered particularly relevant in the development and/or progression of achalasia.

microRNAs (miRNAs/miRs) are single-stranded RNAs that regulate gene expression and serve crucial roles in numerous physiological and pathological processes, including viral infections and antiviral response (12-15). Certain viruses, particularly herpes viruses (including HSV-1), express miRNAs, although their pathological roles are not completely understood (12,15). In our previous study, it was shown that expression of HSV1-miR-H1-3p in the esophageal mucosa of patients with achalasia was increased (1). However, the analysis and understanding of the miRNA expression profiles in the muscular layer of the LES are still being elucidated. HSV1-miR-H1 can directly target E3 ubiquitin-protein ligase component n-recognition 1 in vitro (UBR1) (14). This miR-mediated downregulation of the ubiquitin-proteasome system results in the accumulation of neurodegenerative-associated protein fragment β amyloid (15). Subsequently, the autophagy pathway is influenced, which is essential for host defense against viral infection; in particular, autophagy-independent antiviral functions of autophagy-related genes (ATGs) have been reported to be activated (16-18). For example, ATG 16-like 1 (ATG16L1), which is part of the ATG5-ATG12-ATG16L1 complex, is activated following interferon-γ treatment (18). Finally, autophagy also increases interleukin-1β (IL-1β) secretion (19).

The aim of the present study was to analyze the mRNA expression levels of UBR1, ATG16L1 and IL-1B as potential targets for viral miRNAs, and to investigate the mechanisms underlying onset of achalasia.

Esophageal primary achalasia is characterized by aganglionosis or loss of myenteric neurons (2). These features are predominantly caused by the degeneration of inhibitory neurons in the Auerbach's plexus (20). At present, causal factors of idiopathic achalasia are diverse and multifactorial, and they may be involved in complex interactions of autoimmune responses, degenerative neuronal processes, viral infections and the genetic susceptibility of individuals (1,4).

HSV-1 is a member of the Herpesviridae family, and has been proposed as the most likely candidate as a primary target for treatment or management of esophageal primary achalasia, taking into account its neural cell tropism and predilection for squamous epithelium (5,12,15). HSV-1 has a life cycle with two distinct programs consisting of productive and latent phases (13). Certain viral miRNAs can downregulate specific targets or promote viral genome stability, translation and RNA accumulation (13). In our previous study, it was shown that expression of HSV1-miR-H1, an HSV-1 miRNA, was increased in the LES of achalasia patients (1). Hence, the muscular layer of LES may act as a reservoir for HSV-1 and serve as a region for expression of viral miRNAs in patients with achalasia. Additionally, HSV1-miR-H1 is a latency-associated transcript (LAT) that is a non-coding viral miRNA. LAT-derived miRNAs interfere with viral metabolites and regulate the host immune response (13,15). In the present study, it was hypothesized that UBR1 and ATG16L1 were the direct targets of HSV1-miR-H1, as the downregulation of the ubiquitin-proteasome system results in the accumulation of neurodegenerative-associated protein fragment β-amyloid (15), and the downregulation of autophagy-mediated viral clearance is advantageous for the survival of viruses (16-18). In the present study, UBR1 showed decreased expression (although not significant), whereas ATG16L1 was significantly downregulated at the site of LES. Furthermore, there was a weak correlation between hsv-miR-H1-3p and ATG16L1 levels, but not UBR1. These data suggest that ATG16L1 is the target of HSV1-miR-H1. In contrast, IL-1β expression was upregulated in the LES, and the inflammatory pathway may have been influenced by viral miRNAs. A correlation between IL-1B mRNA expression levels and patient age was also observed. However, additional studies with larger cohorts are required to determine if these observations are generalizable. Additionally, the studied mRNAs are only part of a complex of virus-host interactions, and further studies on the involvement of other transcripts are required. The construction of a achalasia mouse model is also important in obtaining a deeper understanding of the pathogenesis in vivo.

A strong correlation was verified between ATG16L1 and IL-1β in the present study. The relationship between HSV1 and autophagy (21-24) and between autophagy and IL-1β production (25-28) have been reported previously. Notably, p62, a selective autophagy receptor, accumulates in ATG16L1 deficient cells, and p62 activates MAPK and Caspase-1, in-turn increasing IL-1β production (29). Based on these previous studies, the mechanism underlying upregulation of IL-1β expression through ATG16L1 via HSV1-miR-H1 hypothesized in the present study is presented in Fig. 4. HSV1-miR-H1 decreases ATG16L1, and the accumulation of p62 is induced by the decrease in ATG16L1. The abundance p62 induces pro-IL-1β via MAPK activation and activates Caspase-1 simultaneously. IL-1β secretion in induced by MAPK and Caspase-1 (Fig. 4).

There are several limitations to this pilot study. As the controls, tissues from non-motility patients with upper gastrointestinal carcinomas not affecting the EGJ, including LES were used. However, the ideal samples would be tissues from healthy individuals, although there are ethical issues related to obtaining such muscular samples. The number of samples was small and each sample in this study was from a patient diagnosed by classical criteria, primarily based on typical findings using barium. Additionally, all samples were used for RNA extraction due to the small sample size; thus protein expression analysis could not be performed. Finally, whether the influence of ATG16L1 was direct or indirect was not determined. To overcome these problems, a prospective study with a larger sample size and detailed pathological analysis is required.

In conclusion, the levels of ATG16L1, a target of HSV1-miR-H1, are reduced in the LES of achalasia patients, and this reduction could be the cause of the esophageal motility disorder.