After cardiovascular diseases and accidents, cancer is the most common cause of death worldwide (
1)
. Esophageal cancer (EC) is the sixth main cause of cancer-related mortality and the eighth most common malignancy in the world (
2,
3). In spite of many treatments like surgery and chemotherapy, EC has a poor prognosis with a five-year survival rate (
4). Histologically, EC is classified into two general types: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (
4,
5). ESCC is the most widespread type of EC mainly in low- or medium-income countries, such as Iran (
4). In the Golestan Province located in the northeastern part of Iran, EC has an age-standardized incidence rate of 43.4 per 100,000 men and 36.3 per 100,000 women (
6)
. It has been suggested that EC development may be dependent on several risk factors, including genetic susceptibility, poor oral health, hot tea consumption and poor economic and social status (
7). In addition, smoking increases the risk of ESCC development by 1.4 fold (
8, 9).
Long non-coding RNAs (lncRNAs) are molecules of longer than 200 nucleotides that do not encode proteins. These molecules are associated with some cellular progression processes such as parental imprinting, cell proliferation, apoptosis and metastasis via epigenetic modification, chromatin remodeling and sponging miRNAs (10,
11). Evidence suggest that lncRNAs are involved in the onset and progression of cancer and can act as a tumor suppressor or an oncogene (
12). Recent investigations reveal that there may be a relationship between upregulation of some lncRNAs such as HOTAIR, POU3F3, FOXCUT and MALAT1 and development of ESCC (13,
14). Taurine up-regulated 1 (
TUG1), a newly discovered lncRNA, with the length of 7.1 kb, was first identified in a genomic screening study for the treatment of taurine in retinal cells (
15). TUG1 often associates with polycomb repressive complex 2 (PRC2) to silence target genes.
Based on the reports from LncRNADisease v2.0 database (http://www.rnanut.net/lncrnadisease/), TUG1 has a role in the pathogenesis of diseases such as B-cell lymphoma, malignant glioma, lung squamous cell carcinoma, renal cell carcinoma, ESCC, intrahepatic cholangiocarcinoma and oral squamous cell carcinoma. Moreover, lncRNA TUG1 has been recently found to contribute to tumor progression via regulation of different cellular processes, including migration, invasion, proliferation, differentiation and apoptosis (
16). Considering the unknown factors affecting ESCC progression, identifying biomarkers that detect ESCC is of great importance for clinical surveillance and clinical decision making (
13).
In a previous study by Yang et al., it has been reported that alcohol drinking and tobacco smoking together significantly enhance the risk of ESCC among Chinese men (
9). Pakzad et al. also reported the high incidence rate of EC in less developed countries because of lifestyle habits, smoking, alcohol use and poor nutrition (
17). In ESCC, heavy smokers have been proved to have a two-times higher odds of p53 mutation than non-smokers (
18)
. Dysregulation of TUG1 is probably associated with p53 mutation during ESCC development (
5); therefore, TUG1 may be a therapeutic option for ESCC.
In this study, we evaluate overexpression of lncRNA TUG1 in the tumor tissue and adjacent non-cancerous tissue of ESCC patients in the Golestan Province, northeastern Iran. Moreover, we investigated the relationship between the regulation of lncRNA TUG1 and ESCC clinical parameters.
MATERIALS AND METHODS
Tissue samples
A total of 31 pairs of ESCC and adjacent normal tissues were collected from ESCC patients (15 men and 16 women). The mean age of cases was 60.70 ± 13 years. Written informed consent was obtained from all patients prior to participation in the study.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from the tissues using Trizol reagent (Invitrogen, CA, USA). The quantity and quality of the extracted RNA were evaluated using a spectrophotometer (Picodrop, UK). Complementary DNA (cDNA) was synthesised using PrimeScript™ RT Reagent Kit (Takara, Shiga, Japan) and DNase Ι, RNase-free Kit (SinaClon, Iran) to remove additional DNA. Next, qRT-PCR was performed using the SYBR RealQ Plus Master Mix Green (Ampliqon, Denmark) and 2 μl of cDNA in an ABI 7300 thermocycler (Applied Biosystems, CA, USA). All the above-mentioned procedures were conducted based on the instructions provided by the manufacturers. The forward and reverse primers used in this study were TUG1 F: 5ˊ-CTGAAGAAAGGCAACATC-3ˊ, TUG1 R: 5ˊ-GTAGGCTACTACAGGATTTG-3, GAPDH F: 5ˊ-GAAGGTGAAGGTCGGAGT-3ˊ, and GAPDH R: 5ˊ-GAAGATGGTGATGGGATTTC-3ˊ. The relative expression of TUG1 was calculated and normalised using the 2−ΔΔCt method relative to GAPDH, as the internal control. The cycling conditions for the qRT-PCR experiment are listed in table 1.
TUG1 may bind to PRC2 and epigenetically regulate gene expression. HOXB7 regulates cell growth, mainly by activating the MAPK and PI3K/AKT pathways, and the destruction of TUG1 can elevate p-ERK, p-AKT and p-GSK3β levels. Regulation of cell growth under TUG1 occurs partly through HOXB7 regulation (
30). Chen et al. have also indicated that HOXB7 is one of the HOX genes expressed only in the ESCC tissue and not in normal esophageal mucosa (
31)
. On the other hand, Huang et al. reported that lncRNA expression can be regulated by some transcription factors such as p53 and SP1 (
32). Based on RAID v2.0 database (
www.rna-society.org/raid/), TUG1 may interact with EZH2 and SUZ12 proteins. Hence, evaluating of the function and interaction of these proteins with TUG1 in ESCC in future studies can be beneficial for finding novel strategies to control ESCC progression.
In our study, the qRT-PCR analysis showed lncRNA TUG1 overexpression in an Iranian population of ESCC patients. However, further investigations with a larger study population are needed to clarify the exact role of TUG1 in ESCC. In this survey, due to the high cost of testing, the clinical function of TUG1 was evaluated only with qRT-PCR and bioinformatics analysis. More studies are required to understand the exact function of TUG1 in EC and its association with the mentioned proteins.
CONCLUSION
Our findings reveal that TUG1 is overexpressed in ESCC tissues, which may be associated with history of smoking. Our results also illustrate the importance of lifestyle and epigenetic factors in the development and progression of ESCC. Therefore, TUG1 can be considered as a potential therapeutic target in ESCC. The bioinformatics analysis also revealed a strong association between the proteins EZH2, SUZ12, and HOXB7 and TUG1, which may be associated with EC development
.
ACKNOWLEDGMENTS
We would like to thank Massoud Bazori and Naeme Javid for their helpful comments.
DECLARATIONS
Funding
This study received financial support from the Gonbad Kavous University, Iran.
Ethics approvals and consent to participate
Written informed consent was obtained from all patients prior to participation in the study.
Conflict of interest
The author declares that there is no conflict of interest regarding publication of this article.
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