DIFFERENTIAL EXPRESSION OF FGFRs SIGNALING PATHWAY COMPONENTS IN BLADDER CANCER: A STEP TOWARD PERSONALIZED MEDICINE
Ousati Ashtiani Z, Tavakkoly-Bazzaz J, Salami SA, Pourmand MR, Mansouri F, Mashahdi, 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: 7

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.



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