Bladder cancer (BC) is one of the most common types of urinary system cancers in men, and with less frequency, in women worldwide [1]. It is a complex disease resulting from both genetic and environmental factors. Aberrations in different genes’ structure and function, molecular derangements, and several environmental factors have been found to play a crucial role in the development of BC. Age, increased body mass index (BMI), occupation, unhealthy diet, and some drugs can increase the risk of BC development [2]. The epigenetic mechanism also involves in BC tumorigenesis [3]. Blood cancers are generally classified as non muscle invasive with low-grade (pTa/T1) and muscle invasive (pT2-4) that are frequently high-grade tumors [4].
Regulation of different processes during cell cycles, including cell growth, differentiation, cell movement, and apoptosis are orchestrated by diverse signaling pathways. When the above mentioned processes are dysregulated secondary to changes in a key genetic element of cellular homeostasis, tumorigenesis can be the outcome. The fibroblast growth factor receptor (FGFR) signaling pathway has been receiving growing attention as one of the major contributors in cell cycle regulation that in turn, introduces this specific molecule as a potential drug target for cancer therapy [5,6,7].
The mammalian fibroblast growth factors (FGFs) are a family of growth factors, consisting of 18-22 members that play an essential role in multiple physiological events such as angiogenesis, wound healing, embryonic development, and various endocrine signaling pathways both in health and disease. Fibroblast growth factors signaling dysregulation is evidently present in a considerable number of BC cases [8]. A subfamily of receptor tyrosine kinases (RTKs) named fibroblast growth factor receptor family comprises of four members (FGFR1-4). They are activated by binding to their ligand FGFs, which results in kinase activation. Different changes exemplified by
Molecular genetic studies on
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
List of primer sets for real-time polymerase chain reaction.Primers Sequences (5′<3′) Amplicon Size FGFR1-F GAA GAC TGC TGG AGT TAA TAC C 159 FGFR1-R TCT TCC AGG GCT TCC AGA AC FGFR3-F CCA CTG TCT GGG TCA AGG AT 180 FGFR3-R AGG ATG GAG CGT CTG TCA C GAPDH-F ATC CTG GGC TAC ACT GAG C 159 GAPDH-R CAC CAC CCT GTT GCT GTA G
Real-time PCR was performed using SYBR Premix EX Taq™II (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:
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
This study comprised 50 BC patients including 43 (86.0%) men and seven (14.0%) women. Analyses of the subjects showed that 36 (72.0%) cases had high-grade and 14 (28.0%) had low-grade tumors. Their median age was 66 years (range 33-84 years). There were 34 patients (68.0%) with a smoking habit, of which 94.0% (32/34) had been cigarette smokers for ≥10 years and 20.0% (10/50) were opium addicts. Of these subjects, 16.0% (8/50) had diabetes and 46.0% (23/50) showed cardiovascular and/or respiratory diseases. The rate of occupational exposure was about 54.0% (27/50). Table 2 shows the demographic and clinicopathological characteristics of all subjects.
Clinicopathological characteristics of the study subjects.Clinicopathological Parameters % Age (years): ≤66 26 52.0 >66 24 48.0 Sex: males 43 86.0 females 7 14.0 Stage: I 16 32.0 II 20 40.0 III 10 20.0 IV 4 8.0 Grade: low 14 28.0 high 36 72.0 Tumor type: non invasive muscle 15 30.0 muscle invasive 35 70.0 Family history: bladder cancer 8 16.0 other cancer 6 12.0 no cancer 36 72.0 Smoker: ≤10 2 4.0 >10 32 64.0 non smoker 16 32.0 Occupational exposure 27 54.0
Increased mRNA expression of
Multivariate linear regression analysis of FGFR1 and FGFR3 expressions with clinicopathological variables.Relative Expression Variable(s) Undersized Coefficients B Standard Error B FGFR1 no significant variable FGFR3 (constant) family history of BC cigarette smoking –28.908 11.031 –2.621 0.021 14.266 4.125 3.458 0.004 11.568 4.968 2.329 0.037
Unexpectedly, FGFR1 mRNA expression in our BC cases was decreased in 60.0% (30/50) of samples. The majority of high grade tumor (75.0%) (27/36) reflected a significant association with decreased level of FGFR1 mRNA expression (
This is the first study that demonstrates FGFR1 and FGFR3 mRNA expression levels in Iranian patients with BCs. The FGF/FGFR pathway is one of the central mechanisms that govern differentiation, proliferation, survival and many other issues of cellular characteristics. Large number of evidence supports the appropriate functionality of FGFs-FGFRs systems is dysregulated at any point within their signaling cascade that could frequently lead to cancer development
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However, it is so complicated to find out which member of the FGFRs system is playing the central role in cancer. Cheng
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To the best of our knowledge, this pilot study is the first report examining the expression of two components of FGFR signaling pathway (
However, under individualized medicine strategy for BC in the future, it seems that FGFRs could provide attractive targets for therapeutic interventions. Because of the heterogeneity between different populations, such objective requires a more comprehensive understanding on the role of FGFRs in BC, coupling with the data on larger sample sizes of the cases and controls. Finally, it is very crucial to remember that the inter-individual phenotypic variation at the cellular and molecular level is not solely dictated by genetic impressions. Accordingly, epigenetic issues, which themselves are influenced by environmental factors, should be taken into account to unravel the reality behind these observations.