Balkan Journal of Medical Genetics

DIFFERENTIAL EXPRESSION OF FGFRs SIGNALING PATHWAY COMPONENTS IN BLADDER CANCER: A STEP TOWARD PERSONALIZED MEDICINE
Ousati Ashtiani Z1,2, Tavakkoly-Bazzaz J2,*, Salami SA3, Pourmand MR4, Mansouri F5,6, Mashahdi R1, Pourmand G1,*
*Corresponding Author: Professor Gholamreza Pourmand, Urology Research Center, Sina Hospital, Tehran University Medical Sciences, Hasan Abad Square, Tehran, 113746911, Iran. Tel: +98-216-634-8560. Fax: +98-216-634-8561. Email: pourmand@tums.ac.ir and/ or Associate Professor Javad Tavakkoly-Bazzaz, Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Poursina Street, Tehran, 1417613151, Iran. Tel: +98-218-895-3005. Fax: +98-218-895-3005. Email: tavakkolybazzazj@tums.ac.ir
page: 75

MATERIALS AND METHODS

Patients and Tissue Samples. Paired samples, both bladder tumor and adjacent normal tissue were obtained from 50 Iranian individuals who underwent transurethral bladder tumor resection or radical cystectomy at two university teaching hospitals (Sina and Imam Khomeini Hospitals) in Tehran, Iran. Bladder tumor and non tumor samples from a standard distance were rapidly frozen in liquid nitrogen following collection and stored at –80 °C until subsequent RNA extraction. Of the 50 patients, 43 were males and seven were females. The median age was 66 years, ranging from 33 to 84 years. None of the patients received any treatments, such as Bacillus Calmette-Guerin (BCG) therapy, chemotherapy, which might alter the situation of the FGFR signaling pathway in terms of its status and activity. Clinicopathological information including grade, stage, lymph node metastasis, age, gender, smoking, alcohol use, family history of cancer, was provided for all subjects. In this research, written informed consent was signed by all participants, after being informed about the goals of the study. This study was approved by the Research Review Board and also the Ethics Committee of Tehran University of Medical Sciences (TUMS), Tehran, Iran. Total RNA from both tumor and adjacent non tumor tissues were isolated using TriPure Isolation Reagent (Roche Life Science, Mannheim, Germany) according to the manufacturer’s protocol. The quality and quantity of extracted RNAs were measured by the absorbance ratio at 280/260 nm using NanoDrop-2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). In order to remove possible DNA contamination from RNA, DNase I treatment was performed. The cDNA was synthesized from 1 μg RNA by oligo dT, Random 6-mer and reverse transcription Enzyme using PrimeScript™ RT reagent kit (Takara, Kusatsu, Shiga, Japan) according to the manufacturer’s instructions. It was designed to perform optimized reverse transcription-polymerase chain reaction (RT-PCR). Thermal Cycler (Senso Quest GmbH, Göttingen, Germany) was used for the incubation reaction mixture at 37 °C for 15 min. and 85 °C for 5 seconds. The cDNAs were stored at –20 °C until further use. For real-time PCR, specific sets of primers were designed for FGFR1, FGFR3 and GAPDH as housekeeping genes. All amplicon lengths for real-time PCR were less than 200 bp long. Primer sets were checked by primer- BLAST and Oligoanalyzer software (https://eu.idtdna. com/ calc/analyzer). Table 1 shows the 5’>3’ sequence of the primers and amplicon lengths. Real-time PCR was performed using SYBR Premix EX TaqTMII (Takara). The reaction mixture was prepared according to the manufacturer’s protocol. The cycling conditions were: 10 seconds at 95 °C (Takara Master does not need longer hold), followed by 40 cycles at 95 °C for 10 seconds and 60 °C for the 20 seconds. Amplification reactions were performed in triplicate for each sample. A melting curve was obtained following amplification. No template control (NTC; nuclease-free water) was included in each run. The quantitative PCR (qPCR) analysis was completed using Rotor-Gene Q (Brisbane, Queensland, Australia). Cycle threshold (ct) values were collected for the genes of interest and glyceraldehyde 3-phosphate dehydrogenase as housekeeping gene during the log phase of the cycle. Results were normalized to the GAPDH as a reference gene. Agarose gel electrophoresis was used to determine the specificity of the RT-PCR reaction products. Gene expression data analysis was carried out using the 2–ΔΔCT method according to the following formula: Δct1 = cttarget-cthousekeeping, Δct2 = ctnormal-cthousekeeping, ΔΔct = Δct1-Δct2. Statistical Analysis. Statistical analysis was performed by the the Statistical Package for Social Sciences (SPSS) version 21 (IBM SPSS Inc., Chicago, IL, USA). The Kolmogorov-Smirnov test was used to assess the normality of quantitative data. Comparison of normalized expression between tumor and non tumor tissues was done using the parametric t-test for FGFR3 and nonparametric Mann-Whitney test for FGFR1. A multivariate linear regression analysis was performed to find the relationship between expression and clinicopathological parameters as independent variables by a stepwise method in the model. In these tests, a p value of ≤0.05 was considered to indicate a significant difference.

Number 27 VOL. 27 (1), 2024	Number 26 Number 26 VOL. 26(2), 2023 All in one
Number 26 VOL. 26(2), 2023	Number 26 VOL. 26, 2023 Supplement
Number 26 VOL. 26(1), 2023	Number 25 VOL. 25(2), 2022
Number 25 VOL. 25 (1), 2022	Number 24 VOL. 24(2), 2021
Number 24 VOL. 24(1), 2021	Number 23 VOL. 23(2), 2020
Number 22 VOL. 22(2), 2019	Number 22 VOL. 22(1), 2019
Number 22 VOL. 22, 2019 Supplement	Number 21 VOL. 21(2), 2018
Number 21 VOL. 21 (1), 2018	Number 21 VOL. 21, 2018 Supplement
Number 20 VOL. 20 (2), 2017	Number 20 VOL. 20 (1), 2017
Number 19 VOL. 19 (2), 2016	Number 19 VOL. 19 (1), 2016
Number 18 VOL. 18 (2), 2015	Number 18 VOL. 18 (1), 2015
Number 17 VOL. 17 (2), 2014	Number 17 VOL. 17 (1), 2014
Number 16 VOL. 16 (2), 2013	Number 16 VOL. 16 (1), 2013
Number 15 VOL. 15 (2), 2012	Number 15 VOL. 15, 2012 Supplement
Number 15 Vol. 15 (1), 2012	Number 14 14 - Vol. 14 (2), 2011
Number 14 The 9th Balkan Congress of Medical Genetics	Number 14 14 - Vol. 14 (1), 2011
Number 13 Vol. 13 (2), 2010	Number 13 Vol.13 (1), 2010
Number 12 Vol.12 (2), 2009	Number 12 Vol.12 (1), 2009
Number 11 Vol.11 (2),2008	Number 11 Vol.11 (1),2008
Number 10 Vol.10 (2), 2007	Number 10 10 (1),2007
Number 9 1&2, 2006	Number 9 3&4, 2006
Number 8 1&2, 2005	Number 8 3&4, 2004
Number 7 1&2, 2004	Number 6 3&4, 2003
Number 6 1&2, 2003	Number 5 3&4, 2002
Number 5 1&2, 2002	Number 4 Vol.3 (4), 2000
Number 4 Vol.2 (4), 1999	Number 4 Vol.1 (4), 1998
Number 4 3&4, 2001	Number 4 1&2, 2001
Number 3 Vol.3 (3), 2000	Number 3 Vol.2 (3), 1999
Number 3 Vol.1 (3), 1998	Number 2 Vol.3(2), 2000
Number 2 Vol.1 (2), 1998	Number 2 Vol.2 (2), 1999
Number 1 Vol.3 (1), 2000	Number 1 Vol.2 (1), 1999
Number 1 Vol.1 (1), 1998

About the journal ::: Editorial ::: Subscription ::: Information for authors ::: Contact