1. Introduction
sophageal cancer, as a lethal malignancy, is the seventh most frequent cancer and the sixth cause of cancer death worldwide; it occurs at high incidence in the countries located in the Asian esophageal cancer belt region. It has low survival, poor prognosis, and the highest incidence rate in Iran [1, 2, 3]. Radiotherapy, chemotherapy, and esophagostomy are the most common treatment approaches for esophageal cancer. Despite numerous improvements in diagnosis and multimodality therapy, the overall 5-years survival rate remains low. Moreover, >85% of esophageal cancer patients die within 2 years of diagnosis [4, 5]. Several genetic and epigenetic alterations, as well as environmental factors, are involved in the carcinogenesis of esophageal cancer, however; its precise underlying mechanism remains undiscovered. Therefore, investigating the molecular alterations of esophagus cancer is essential for improved early detection and effective treatment. Alteration in microRNAs (miRNA) expression represents a main epigenetic change in the carcinogenesis process. Additionally, miRNAs, are a class of small-regulatory non-coding RNA, i.e., critical in basic physiological processes. It can post-transcriptionally regulate gene expression through transcript degradation or translation inhibition. Aberrant miRNA expression is involved in the etiology of human cancers, including esophageal cancer and their expression pattern can be applied as useful biomarkers for cancer diagnosis [6, 7]. In this regard, researchers focused to identify new cancer miRNA biomarkers. Otherwise, miRNA expression varies according to cancer type, disease stage, and tumor histological characteristics [8]. Thus, identifying miRNA involved in esophageal cancer can provide great insight into its tumorigenesis and facilitate improved therapies. In the present study, the expression level of miRNA-138 was evaluated in fresh tumors and normal esophageal tissues, and its association was assessed with the clinicopathological characteristics of patients.
2. Methods
In total, 35 samples of tumor tissues along with adjacent normal tissues of the esophagus were collected from patients referring to Imam Khomeini Hospital following esophagectomy. All obtained samples were confirmed for origination from the esophagus and the status of differentiation was identified by pathological examination. The study was performed at Payame Noor University in 2020. This study was approved by the Ethics Committee of Payame Noor University (Code: IR.PNU.REC.1399.078). Following sample collection, total RNA was isolated applying TriPure Isolation Reagent (Roche, Germany) according to the manufacturer’s procedure. After removing the genomic DNA with RNase-free DNase I (Fermentas, INC, MD, USA), cDNA synthesis was performed by miScript II RT Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Subsequently, real-time PCR was applied to evaluate the expression level of miRNA-138 using miScript SYBR Green kit (Qiagen, Hilden, Germany) on Light Cycler 4 system (Roach, Basel, Switzerland). The reaction mixture was prepared in the total volume of 25 μL containing 10 μL of SYBER Green Master Mix, 2 μL specific forward primer, 2 μL reverse primer (miScript Universal Primer), 2 μL of cDNA, and an adequate quantity of PCR grade water. RNU6B (MiScript PCR Control, Qiagen, Hilden, Germany) gene was used as an internal control. All reactions were performed in triplicate. Statistical analysis was performed using SPSS. The Pearson’s correlation coefficient and Chi-squared test were used to analyze the associations between miRNA-138 expression and the clinicopathological characteristics. P<0.05 was considered statistically significant.
3. Results
The expression level of miRNA-138 was assessed in tumor and adjacent normal esophageal tissues per patient by real-time PCR. Totally, 35 patients (18 males & 17 females) were explored in this study. The age range of the patients at the time of diagnosis was 47-88 years with a mean of 64 years. According to the obtained results, the expression of miR-138 was considerably decreased in tumor tissues, compared with normal adjacent tissues (n=35; P<0.05). The relative expression of miRNA-138 in tumor tissues was 69% lower than that in the normal tissues. The patients were grouped into a<69% downregulation group and a>69% downregulation group according to the mean of downregulation level (69%), i.e., used as the cutoff point. The correlation between miRNA-138 expression and clinicopathological characteristics was assessed by statistical analysis (Table 1). 


Accordingly, there was no significant correlation between miRNA-138 downregulation level and gender (P=0.064) or the age (P=0.163) of patients. However, the level of miRNA-138 reduction was significantly associated with the differentiation degree of the tumors (P=0.036) and lower levels of miRNA-138 expression were observed in the poorly differentiated samples. In other words, patients at the late stages of disease had a higher possibility of miRNA-138 downregulation, compared to early stages patients. Moreover, a significant correlation was observed between the metastasis occurrence and miRNA-138 decreased level (P=0.016); patients with a lower level of miRNA-138 expression had a higher chance of metastasis and aggressive malignancy. 
4. Discussion and Conclusion
Esophageal cancer is a complicated disease with a poor prognosis, low survival rate, and high incidence in Iran. However, its exact molecular mechanism is less known and demands further investigation. According to the present study, miRNA-138 was downregulated in esophageal tumor tissues; however, it was expressed at a considerably higher level in the adjacent normal tissues. Moreover, the level of downregulation was significantly related to the differentiation degree and metastatic behavior of tumors. The significant downregulation of miRNA-138 in esophageal cancer indicated the role of this epigenetic alteration in esophageal carcinogenesis. Thus, miRNA-138 could be potentially considered as a molecular biomarker for esophageal cancer.

Ethical Considerations
Compliance with ethical guidelines
This study was approved by the Ethics Committee of Payame Noor University of Tehran (Code: IR.PNU.REC.1399.078).

Funding
This article is taken from the research project of the responsible author, approved by Payame Noor University and sponsored by Payame Noor University.

Conflicts of interest
The author declared no conflict of interest.


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