UJ 35 Summer.pdf 574 | Urological Oncology A Plausible Anti-Apoptotic Role of Up-Regulated OCT4B1 in Bladder Tumors Jamshid Asadzadeh,1 Malek Hossein Asadi,2 Nasser Shakhssalim,3 Mahmoud-Reza Rafiee,4 Hamid Reza Kalhor,5 Mahmoud Tavallaei,6 Seyed Javad Mowla1 Purpose: To investigate and compare the expression of OCT4B1 tumor bladder tissues. Materials and Methods: We investigated the expression of OCT4B1 in 30 tumor and non- tumor surgical specimens of the bladder, using the TaqMan real-time polymerase chain reac- of the variant. Results: OCT4B1 expression, but P < .002). Moreover, the up-regu- lation of OCT4B1 (P < .05). We have also employed the RNA interference strategy to evaluate the functional role of OCT4B1 in a bladder cancer cell line, 5637. Suppression of OCT4B1 caused some Conclusion: OCT4B1 or progression of the bladder cancer. Additionally, OCT4B1 - Keywords: cancer stem cells, urinary bladder neoplasms, apoptosis, neoplasm invasiveness, prognosis Corresponding Author: Seyed Javad Mowla, PhD Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran Tel: +98 21 8288 3464 Fax: +98 21 8288 4717 E-mail: sjmowla@modares.ac.ir Received June 2011 Accepted January 2012 1 Department of Molecular Genet- ics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran 2 Department of Biotechnology, Research Institute of Environmental Sciences, International Center for Science, High Technology & Environ- mental Sciences, Kerman, Iran 3 Urology and Nephrology Research Center , Shahid Labbafinejad Medical Center, Shahid Beheshti University of Medical sciences, Tehran, Iran 4 Nanomedicine and Tissue Engineering Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran 5 Omics Research Center, Golestan University of Medical Sciences, Gorgan, Iran 6 Human Genetics Research Center, Baqiyatallah Medical Sciences University, Tehran, Iran UROLOGICAL ONCOLOGY 575Vol. 9 | No. 3 | Summer 2012 |U R O LO G Y J O U R N A L Up-regulation of OCT4B1 in Bladder Cancer | Asadzadeh et al INTRODUCTION Bladder cancer is the 9 th most common malignancy and the 2nd most common tumor of the genitouri- th most numer- ous cause of cancer death.(1,2) potential, tumorigenesis, resistance to therapy, etc. Similar - Based on this hypothesis, this highly tumorigenic subset of (3-7) Octamer binding protein 4 (OCT4 OCT3, POU5F1, and OTF3 OCT4 ex- respectively).(9) OCT4 cancer stem cells.(10,11) Subsequently, other research groups have reported OCT4 overexpression in the bladder, gastric, prostate, and colorectal cancers.(12-16) Based on its main role OCT4 expression in cancer has been regarded as a possible route (17) ectopic expression of OCT4 has an anti-differentiation effect on the differentiated host cells. Moreover, it has been re- - blasts resulted in complete reprogramming of the host cells OCT4 turned out to be one of the essential players.(19-22) OCT4A), human OCT4 gene variants, designated as OCT4B(10) and OCT4B1.(23) OCT4B - OCT4B cannot maintain the self- (9,10) Our previous reports revealed that OCT4B1 is expressed in pleuripotent and non- pleuripotent cells,(23) (24) and plays a potential role as an anti-apoptotic factor in gastric adenocarcinoma.(15) Although OCT4B1 expression has been studied in gastric and colorectal cancers.(15,16) about its expression and function in other tumors, including bladder cancer. - pression of OCT4B1 in a series of bladder cancer samples and further examined its function in a bladder cancer cell line using RNA interference (RNAi) technology. MATERIALS AND METHODS Clinical Specimens - normal tissues as control. the bladder, and all had a tumoric counterpart in the tumor - - prior to participation. RNA Extraction and Real-Time Polymerase Chain Reac- tion (PCR) - - - - 576 | - negative control. OCT4B1 OCT4 B1 variant: GAPDH: GAPDH mRNA OCT4B1 normalized to GAPDH expression value in each sample. All - using serial dilutions of an embryonic carcinoma cell line, or triplicate. - - state and grade of the tumors. Cell Culture - - 2 2. RNAi OCT4B1 Urological Oncology Clinicopathological characteristics of the patients with blad- der cancer. Characteristic n % Histological type TCC 30 100 Tumor grade High 13 43.34 Low 17 56.66 Tumor stage Ta 4 13.34 pT1 19 63.34 pT2/pT2a 6 20 pT4 1 3.34 Gender Male 28 6.67 Female 2 93.34 577Vol. 9 | No. 3 | Summer 2012 |U R O LO G Y J O U R N A L - Oct4B1 Oct4B1-siRNA2 Target sequence: AAG AGG TGG TAA - quence of OCT4B1, to discriminate it from other variants of OCT4. 4 cells - - - 2 incubator. Cell Cycle Analysis - enediaminetetraacetic acid to yield single cell suspensions. - Optimization of RT-PCR Reactions - and reverse primers and the probe for OCT4B1 other OCT4 variants and pseudogenes. To determine the re- relative expression of OCT4B1 GAPDH tran- probable sampling errors. RESULTS Elevated Expression of OCT4B1 in Bladder Tumors We have detected the expression of OCT4B1 stronger in the tumor samples, compared to their non-tumor OCT4B1 in bladder tumor samples (P the samples (0.39; P < .01). OCT4B1 OCT4B1 high-grade tumors (P - OCT4B1 expression level and the grade of the tumors (0.31; P < .05). Cell Cycle Alterations Following OCT4b1 Knock-Down in 5637 Cells against OCTB1 three days after the transfection. The level of OCT4B1 ex- siRNAs against OCT4B1 Up-regulation of OCT4B1 in Bladder Cancer | Asadzadeh et al 578 | - OCT4B1 - tion (P < .05) in the percentage of the cells in the sub-G1 of cells in G1 phase of OCT4B1 suppressed group declined DISCUSSION initiation and progression is one of the most important aims (3-7) sev- (12-16,25-27) OCT4, is the most notable OCT4 - - OCT4 expression in some cancers, cancer cell lines, and adult stem cells.(11-16) appeared to be controversial, and there is a need to discrimi- Urological Oncology Figure 2. (A) Cells treated with OCT4B1-siRNA, compared to the ones treated with IR-siRNA, demonstrated a dramatic suppression in OCT4B1 expression as determined by real-time PCR. GAPDH was used as an internal control. (B and C) Effect of OCT4B1 knock-down on cell cycle distribution in 5637 cell line. CB Figure 1. (A) Comparison of the relative expression level of the OCT4B1 between tumor and non-tumor samples. Each triangle rep- resents the mean expression level of a single specimen, obtained by averaging values from two to three independent experimental replicates. (B) Comparison of relative expression of OCT4B1 between 13 high-grade and 17 low-grade tumor samples, as determined by real-time PCR. 579Vol. 9 | No. 3 | Summer 2012 |U R O LO G Y J O U R N A L nate the expression of OCT4 variants in different types of cancers. OCT4 variants, OCT4A and OCT4B the OCT4 gene coined OCT4B1.(23) This variant is primar- ily expressed in undifferentiated cells, and might contrib- (15,23,24) expression and function in different cancers. To gain more insight on the role of the OCT4 - of OCT4B1 - desired mRNA transcript for OCT4B1. Our results revealed that OCT4B1 - cantly elevated in tumor samples compared to that of non- tumor samples. OCT4B1 expressed at higher levels in the high-grade tumors compared OCT4B1 expression also existed in most of the non-tumor samples, ie, the expres- - - as OCT4 in adult stem cells,(11,15,27) OCT4B1 is expressed at a basic level in normal tissue of the bladder as a result of existing adult stem cells. Secondly, several lines of evi- dence exist in supporting a clonal expansion of multifocal carcinomas, suggesting derivation of these tumors from a primary transformed progenitor cell. Therefore, it can at other luminal surfaces of the urinary tract could be re- OCT4B1 parts of the bladder. Elevated expression of OCT4B1 in high-grade tumors is quite of differentiation.(23) - vious data on gastric cancer,(15) this novel variant may play an anti-differentiation role as the tumor grade is related to the degree of differentiation. According to the grading system, the degree of differentiation of a given tumor has an inverse relationship to its grade, ie, the higher the grade of tumor, the (31) (15) suppression of OCT4B1 of cells in the sub-G1 fraction, suggesting that OCT4B1 plays CONCLUSION Altogether, our data revealed that OCT4B1 in the bladder cancer and the variant seemed to have a role in tumorigenesis process, probably as an anti-apoptotic fac- tor. Therefore, OCT4B1 can be considered as a novel tumor - tional OCT4B1 of OCT4B1 and its function. ACKNOWLEDGEMENTS CONFLICT OF INTEREST None declared. REFERENCES 1. Siegel R, Ward E, Brawley O, Jemal A. Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA Cancer J Clin. 2011;61:212-36. 2. Parkin DM. The global burden of urinary bladder cancer. Scand J Urol Nephrol Suppl. 2008;12-20. Up-regulation of OCT4B1 in Bladder Cancer | Asadzadeh et al 580 | 18. Lengner CJ, Welstead GG, Jaenisch R. 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