Balkan Journal of Medical Genetics

TARGETED microRNA PROFILING IN GASTRIC CANCER WITH CLINICAL ASSESSEMENT
Pehlevan Ozel H, Dinç T, Tiryaki RS2, Keşkus AG, Konu O, Kayilioglu SI, Coşkun F
*Corresponding Author: Tolga Dinç, M.D., Associate Professor, Department of General Surgery, Health Sciences University, Ankara City Hospital, Üniversiteler Mahallesi 1604. Cadde No: 9 Çankaya/ Ankara/Turkey. Tel./Fax: +90-312-552-60-00. Intercom: 121514. Mobile: +90-532-481-22-75. Email: tolga_dr@hotmail.com
page: 55

MATERIALS AND METHODS

Ethics Approval and Informed Consent. Ethics committee approval was obtained from the Ethics Committee for the study with the decision numbered E-16-1152. The study protocol was explained to all the patients who participated in the study and a signed informed consent was obtained from all the subjects. Inclusion Criteria. A total of 20 patients aged >18 years were included in this study. They were pathologically diagnosed with gastric adenocarcinomas (two were diagnosed as signet ring cell carcinoma, a subtype of adenocarcinoma), surgically treated and did not receive neoadjuvant chemotherapy or radiotherapy, and were not diagnosed with linitis plastica at the General Surgery Department, Ankara Numune Training and Research Hospital, Ankara, Turkey, between March 2018 and April 2019. Collection of Tissue Samples. Samples collected from the cancerous stomach tissue of patients within the first half hour after the surgical removal of gastric tissue from the abdomen in the operating room along with those collected from normal stomach tissue of the same patients were rapidly frozen in liquid nitrogen. These samples were stored in a storage area at –80 °C until examination. Selection of microRNAs. The miRCancer database (accessed February 18, 2019) [7], citing 5723 articles as references, were reviewed to identify relevant articles for the miRNAs we included in this study. According to the MiRCancer database, 323 miRNAs were associated with gastric cancer [7]. In addition, The National Center for Biotechnology Information [8] and the miRCancer databases were reviewed, and in total, eight miRNAs were identified, including seven that have previously been associated with gastric cancer in one or more articles, and one novel miRNA, not investigated in gastric cancer literature in detail. RNU6-1, recognized as the universal reference, was used as the reference miRNA in the study [9]. microRNAs used herein include hsa-mir-375-3p, hsa-mir-148a-3p, hsamir- 196a-5p, hsa-mir-376c-3p, hsa-mir-129-5p, hsa-mir- 34c-5p, the newly identified hsa-mir-767-5p, and the novel hsa-mir-662 (Table 1) [10-22]. Isolation of microRNAs and cDNA Synthesis. RNA isolation of the tissues to be used in the study was performed using miRNeasy mini kit (Cat. #217004; Qiagen Sciences Inc., Germantown, MD, USA) as described in the catalog. cDNA synthesis was performed using miScript kit II (Cat. #218161; Qiagen Sciences Inc.) according to the manufacturer’s instructions. microRNA Expression Detection and Analysis. In order to evaluate the miRNA expressions, cDNA samples were analyzed with qPCR. Qiagen miScript primer assays with miScript SYBR Green kit (Cat. #218073; Qiagen) and Roche Multiwell Plate 96 (Cat. #4729692001; Hoffman La-Roche Ltd., Indianapolis, IN, USA) were used for this study. LightCycler 480 Instrument II (Roche Diagnostics International AG, Basel, Switzerland was used to determine the miRNA levels. Quantification of the miRNA expression values, and the threshold cycle (Ct) and melting temperature (Tm) determinations were performed using Light Cycler Software. In this study, eight miRNAs (Table 1), whose expression levels were normalized to those of RNU6-1 and their corresponding normal tissues using ΔCt and ΔΔCt methods [23]. Statistical Analyses. Microsoft Excel 2010 (Microsoft, Albuuerque, NM, USA) was used to collect and group the patient data and to perform basic mathematical operations on these data. The Ct values of the miRNAs were evaluated using parametric paired t-tests in comparison of cancerous and normal tissues. Parametric t-test was also used to assess the relationship of demographic, clinical, and pathological results with miRNA expressions with categorical variables, and Pearson correlation score was used for continuous variables. The one-tailed F-test was used to test the difference in the variance of miRNA expression between tumor and normal samples in Microsoft Excel 2010, and those with p values of <0.05 were considered significant. Hierarchical cluster analysis was performed using the “Euclidean” distance and the “complete” linkage methods, while a heatmap was created using the log fold changes in miRNAs with age, smoking and the number of metastatic lymph nodes in the R platform with various R programing tools for plotting data (ggplots) package [24]. All the statistical analyses and graphic drawings for miRNAs and patient data were conducted using GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA) and Matlab 2017 (MathWorks Inc., Natick, MA, USA).

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