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RESEARCH ARTICLE ISSN: 2211-5366 eISSN: 2211-5374
Chronic Exposure to Cigarette Smoke and Chewing Tobacco Alters Expression of microRNAs in Esophageal Epithelial Cells BENTHAM SCIENCE
Aafaque Ahmad Khan1,2,‡, Jayshree Advani1,3,‡, Krishna Patel1,4, Vishalakshi Nanjappa1, Keshava K. Datta1, Hitendra Singh Solanki1,2, Prashant Kumar1, Premendu Prakash Mathur1,2, Bipin Nair4, Thottethodi Subrahmanya Keshava Prasad1,5, Aditi Chatterjee1,* and Harsha Gowda1,* 1
Institute of Bioinformatics, International Technology Park, Bangalore 560066, India; 2School of Biotechnology, KIIT University, Bhubaneswar 751024, India; 3Manipal Academy of Higher Education, Manipal 576104, India; 4School of Biotechnology, Amrita Vishwa Vidyapeetham, Kollam, India; 5YU-IOB Center for Systems Biology and Molecular Medicine, Yenepoya University, Mangalore 575018, India Abstract: Background: Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers with high mortality rate. Cigarette smoke and chewing tobacco are well known risk factors associated with ESCC. However, molecular mechanisms associated with development of ESCC among smokers and chewers are poorly understood. MicroRNAs play an important role in regulating physiological and disease processes including esophageal cancer. ARTICLE HISTORY Received: August 24, 2017 Revised: October 17, 2017 Accepted: December 01, 2017 DOI: 10.2174/2211536607666171213123907
Objective and Methods: In this study, we developed an in vitro model by treating non-neoplastic Het1A esophageal cell line with cigarette smoke and chewing tobacco. We carried out miRNA sequencing on Illumina HiSeq 2500 platform and compared miRNA expression pattern across cigarette smoke and chewing tobacco treated Het-1A cells with untreated cells. Results: We identified and quantified 433 miRNAs in both smoke exposed and chewing tobacco treated cells, of which 13 miRNAs showed significantly altered expression in cigarette smoke exposed cells while 25 miRNAs showed significantly altered expression in chewing tobacco treated cells. In addition, we predicted novel miRNAs from these data-sets. We evaluated miRNAs that showed selective or context dependent expression pattern in cigarette smoke exposed or chewing tobacco treated cells. Conclusion: In this study, we have comprehensively mapped miRNA expression pattern in response to cigarette smoke and chewing tobacco in Het-1A cells. We identified miRNAs that show altered expression in these cell models.
Keywords: Chewing tobacco extract, cigarette smoke condensate, esophageal cancer, miRNA sequencing, nicotine, novel miRNAs.
MicroRNA
1. INTRODUCTION Esophageal squamous cell carcinoma (ESCC) is the sixth leading cause of cancer associated deaths [1]. Five-year survival rate of ESCC patients remains less than 20% (American cancer society: Cancer facts and figures 2017. Atlanta: American cancer society 2017). Cigarette smoke and chewing tobacco are well-known risk factors associated with the development of ESCC [2, 3]. The International Agency for Research on Cancer (IARC) has reported more than 60 compounds from smoke to be carcinogenic. The carcinogens *Address correspondence to these authors at the Institute of Bioinformatics, 7th floor, Discoverer building, International Tech Park, Bangalore 560 066, India; Tel: 91-80-28416140; E-mails:
[email protected]; and
[email protected] ‡These authors contributed equally 2211-5374/18 $58.00+.00
belong to an array of chemicals including polycyclic aromatic hydrocarbons, N-nitrosamines, aromatic amines, volatile hydrocarbons and several aldehydes. Tobacco-specific N-nitrosamines, including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosonornicotine (NNN), N′nitrosoanabasine (NAB) and N′-nitrosoanatabine(NAT), occur widely in tobacco and tobacco smoke [3]. Globally, tobacco is consumed mostly in the form of cigarette smoke, however, in India chewing tobacco products which include chewing tobacco, betel quid with tobacco, moist snuff, tobacco tooth powder and variety of new products account for over one-third of all tobacco used [4]. The use of chewing tobacco or cigarettes is associated with nicotine delivery and addiction and development of multiple cancers including ESCC. However, molecular alterations associated with development of ESCC among tobacco smokers and chewers are poorly understood. © 2018 Bentham Science Publishers
Chronic Exposure of Cigarette Smoke Alters Expression of miRNA
miRNAs are small noncoding RNAs approximately 1925 nucleotides long and are known as key regulators of gene expression [5]. miRNAs are known to be involved in regulating several cellular processes including differentiation, proliferation and apoptosis [6]. Several miRNAs are reported as potential therapeutic targets for various cancers as strong correlation has been observed between altered miRNA expression and development and progression of several cancers [7]. Depending on the context, particular miRNA may function as tumor suppressor or tumor promoter [8]. Therefore, cellular context is important in understanding the role of miRNAs in carcinogenesis. In addition, circulating miRNAs that have greater stability in body fluids as compared to mRNAs [9] are also reported as non-invasive tumor specific biomarkers in various cancers including gastric cancer [10], pancreatic cancer [11], ovarian cancer [12], and esophageal cancer [13, 14]. In ESCC, several studies have reported altered miRNA expression in tissues and body fluids and suggested their diagnostic and prognostic potential for early detection of ESCC [15, 16]. Expressions of hsa-miR-31-5p and hsa-miR142-3p were found to be correlated with histological differentiation, and high hsa-miR-142-3p expression was found to be associated with poor prognosis [17]. It is reported that downregulation of hsa-miR-138-5p contributes to constitutive NF-kB activation and ESCC progression [18]. NF-kB can directly bind to the promoter of hsa-miR-34a-5p and elevate its expression [19]. The role of miRNAs in tumor invasion and metastasis is also explored in ESCC. For example, hsa-miR-10b-5p post-transcriptionally regulates expression of KLF4 and has a positive role in invasion and migration of ESCC cell lines [17, 20]. Similarly, decreased expression of hsa-miR-28-5p and hsa-hsa-miR-98-5p in ESCC promotes metastasis [21]. Another well-known miRNA, hsamiR-21-5p enhances cell growth [22] and its increased expression was found to be associated with metastasis and invasion of ESCC [23]. In this study, we have carried out miRNA expression profiling of non-neoplastic esophageal Het-1A cells chronically exposed to cigarette smoke and chewing tobacco using next generation sequencing platform. 2. MATERIALS AND METHODS 2.1. Preparation and Treatment of Cigarette Smoke Condensate and Chewing Tobacco Extract Cigarette smoke condensate (CSC), which was prepared from 3R4F research grade cigarettes, was purchased from Murty Pharmaceuticals, Inc, Lexington, KY. Human esophageal cell line Het-1A was purchased from American Type Culture Collection (ATCC, Manassas, VA). Het-1A cells were grown in keratinocyte serum free media (K-SFM) supplemented with growth factors. Het-1A cells were chronically treated with 0.1% CSC for six months. To control for the vapor effect of the CSC, cells were grown in a smoke dedicated incubator [24]. The untreated parental cells were grown in a separate incubator, where no cells were treated or exposed to CSC or its vapor phase. Cells were treated with chewing/smokeless tobacco extract (STE) as described previously [25]. Briefly, 10 grams of STE was purchased from local vendor finely ground into powder followed by homogenization. It was dissolved in
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100ml of 1X phosphate buffer saline (PBS). The mixture was stirred for 24 hours, followed by a centrifugation at 2000 g for 15 minutes. The supernatant was collected and sterile filtered using 0.22 µm filter. This was considered as 100% extract of STE. Het-1A cells were treated with 1% STE for six months. Henceforth, CSC treated cells will be referred to as Het1A-Smoke or smoke exposed cells and chewing tobacco treated cells will be referred to as Het-1A-tobacco or tobacco treated cells. 2.2. RNA Isolation and miRNA Enrichment RNA was isolated using the Qiagen RNA easy isolation kit from Het-1A-parental, Het-1A-Smoke and Het-1ATobacco cells. RNA quality was checked using agarose gel electrophoresis. Integrity number and quality was determined using Bioanalyzer RNA 6000 Pico chip. RNA isolated from Het-1A-parental, Het-1A-Smoke and Het-1A-Tobacco cells were used to construct sequencing libraries with the Illumina TruSeq Small RNA Sample Prep Kit (Illumina). 3′ and 5′ adapters were ligated to small RNA and then reverse transcribed and amplified by polymerase chain reaction (PCR) to create cDNA. To develop fragments with adapter molecules on both ends, cDNA was amplified with 50 PCR cycles using a common primer and a primer containing an index tag to allow sample multiplexing. Amplified cDNA constructs were purified using agarose gel electrophoresis, and validated by checking size, purity, and concentration of amplicons on the Agilent Bioanalyzer High Sensitivity DNA chip (#5067-4626, Genomics Agilent, Santa Clara, CA). Generated libraries were pooled in equimolar amounts, and sequenced on an Illumina HiSeq 2500 instrument to generate 50-base pair reads. 2.3. miRNA Sequencing and Data Analysis Sequencing data was acquired in FASTQ format from Illumina HiSeq 2500 was assessed for quality using FASTQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Bases with Phred score
