Balkan Journal of Medical Genetics

KDM3A, A NOVEL BLOOD-BASED BIOMARKER IN COLORECTAL CARCINOGENESIS
Polat D.1, Onur E.2,*, Yılmaz N.3, Sökücü M.4, Gerçeker O.F.1
*Corresponding Author: Assoc. Prof. Elif Onur, Department of Medical Biology, Faculty of Medicine, SANKO University, 27090 Gaziantep, Turkey; Phone:+90 342 211 6562; Fax: +90 342 211 6566; Email: elif.onur@sanko.edu.tr
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MATERIALS AND METHODS

Study design The study consisted of two groups; 50 healthy controls without CRC, inflammatory bowel disease, and positive family history, and 50 pathologically confirmed CRC patients in different stages. All blood samples collected from the patient and control groups were taken early in the morning. Furthermore, blood samples were collected from the patient groups at the time of diagnosis before treatment was started. Usage of drugs, hormones, immune suppressors, cytotoxins, or free radical scavengers were exclusion criteria for all groups. Depending on the severity of the CRC, patients were divided into four groups (stage I-IV). The study was approved by the Ethics Committee of SANKO University (2018/05-03). Written informed consent was acquired from all individuals who agreed to participate in the study. Total RNA Isolation and cDNA Synthesis PureLink® (Thermo Fisher) RNA Mini Kit was used to extract the total RNA from peripheral blood leukocytes, according to the protocol recommended by the manufacturer. The RNA concentration of each sample was measured, and purities were evaluated by the NanoDrop spectrophotometer. Then, cDNA transcriptions from RNA samples (1μg) were done using High Capacity cDNA Reverse Transcription Kit (Thermo Fisher). Quantitative Real Time-PCR The gene expression levels of HIF-1α, KDM3A, Ecadherin, Claudin-1, Slug, and ZEB-1 were determined using quantitative real-time polymerase chain reaction (qRT-PCR) from total leukocyte RNA of peripheral blood samples. StepOnePlus QRT-PCR (Qiagen, Germany) was used for cDNA amplifications. The PCR mixture was composed of SYBR Green PCR Master Mix (Qiagen), 20 pmol of forward/reverse primers, RNase-free water. The cDNAs were constructed in a total volume of 20 μL. β-actin (ACTB), the housekeeping gene, expression level was used as an internal control to evaluate the integrity of each sample. The PCR primers of each gene are described in table 1. Cycling conditions were 95°C for 15 min, 40 cycles; at 95°C for 15 sec; at 60°C for 1 min, and at 72°C for 30 sec. Data were examined by Rotor-Gene Q Series software v2.1.0 (Qiagen), and expression levels were calculated by using the standard curve method. Each gene was analyzed separately and ran by duplicate. The mean CT threshold values for each sample were used to calculate the relative gene expression levels using the 2−ΔΔCT method expressed as Fold-Change (FC) (15). Statistical analysis Statistical analysis was performed using SPSS software (standard version 22.0; SPSS). The normal distribu-tions of expressions in groups were analyzed using the Shapiro-Wilk method. As descriptive statistics, median values were used when expression levels were not normally distributed, and mean values were used when expression levels were normally distributed. A Non-parametric Mann-Whitney U test was used for non-normally distributed genes and the student-t test was used for normally distributed Claudin-1 gene. Differences between patient subgroups were tested using Kruskal-Wallis with Dunn’s multiple comparison test. The KDM3A diagnostic value was assessed by the ROC curve. P <0.05 was considered statistically significant.

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