PURPOSE: To investigate and compare the expression of OCT4B1 between tumor and non-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 reaction approach and by carefully designing primers and probes specific for the amplification of the variant. RESULTS: Most tumor and non-tumor samples of the bladder showed OCT4B1 expression, but its expression level was significantly higher in the tumors (P < .002). Moreover, the up-regulation of OCT4B1 was more significant in high-grade tumors compared to the low-grade ones (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 changes in cell cycle distribution, and significantly elevated the rate of apoptosis in the cells. CONCLUSION: Our findings suggest that OCT4B1 plays a potential role in tumor initiation and/or progression of the bladder cancer. Additionally, OCT4B1 can be regarded as a new tumor marker for detection, classification, and treatment of the bladder cancer. However, more experimental studies are needed to replicate our findings

Hamid Reza Kalhor

Jamshid Asadzadeh

Mahmoud Tavallaei

Mahmoud-Reza Rafiee

Malek Hossein Asadi

Nasser Shakhssalim

Seyed Javad Mowla

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574 | Urological OncologyA Plausible Anti-Apoptotic Role of Up-Regulated OCT4B1 in Bladder Tumors Jamshid Asadzadeh,1 Malek Hossein Asadi,2 Nasser Shakhssalim,3 Mahmoud-Reza Rafiee,4Hamid Reza Kalhor,5 Mahmoud Tavallaei,6 Seyed Javad Mowla1Purpose: To investigate and compare the expression of OCT4B1tumor 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: OCT4B1or progression of the bladder cancer. Additionally, OCT4B1-Keywords: cancer stem cells, urinary bladder neoplasms, apoptosis, neoplasm invasiveness, prognosisCorresponding Author:Seyed Javad Mowla, PhDDepartment of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, IranTel: +98 21 8288 3464Fax: +98 21 8288 4717E-mail: sjmowla@modares.ac.ir Received June 2011Accepted January 20121Department of Molecular Genet-ics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran2Department of Biotechnology, Research Institute of Environmental Sciences, International Center for Science, High Technology & Environ-mental Sciences, Kerman, Iran3Urology and Nephrology Research Center , Shahid Labbafinejad Medical Center, Shahid Beheshti University of Medical sciences, Tehran, Iran4Nanomedicine and Tissue Engineering Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran5Omics Research Center, Golestan University of Medical Sciences, Gorgan, Iran6Human Genetics Research Center, Baqiyatallah Medical Sciences University, Tehran, Iran UROLOGICAL ONCOLOGY575Vol. 9   |   No. 3   |   Summer 2012   |UROLOGY  JOURNALUp-regulation of OCT4B1 in Bladder Cancer   |  Asadzadeh et alINTRODUCTIONBladder cancer is the 9th 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 (OCT4OCT3, POU5F1, and OTF3OCT4 ex-respectively).(9)OCT4cancer 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 OCT4expression 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 METHODSClinical 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. OCT4B1OCT4 B1 variant:GAPDH:GAPDH mRNA OCT4B1normalized 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--22.RNAiOCT4B1Urological OncologyClinicopathological characteristics of the patients with blad-der cancer.Characteristic     n    %Histological typeTCC                                           30                            100Tumor gradeHigh                                          13 43.34Low                                           17 56.66Tumor stageTa                                               4 13.34pT1                                           19 63.34pT2/pT2a                                  6 20pT4                                            1 3.34Gender  Male                                          28 6.67Female                                        2 93.34577Vol. 9   |   No. 3   |   Summer 2012   |UROLOGY  JOURNAL-Oct4B1Oct4B1-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 OCT4B1other OCT4 variants and pseudogenes. To determine the re-relative expression of OCT4B1GAPDH tran-probable sampling errors. RESULTSElevated Expression of OCT4B1 in Bladder TumorsWe have detected the expression of OCT4B1stronger in the tumor samples, compared to their non-tumor OCT4B1 in bladder tumor samples (Pthe samples (0.39; P < .01).OCT4B1OCT4B1high-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 Cellsagainst OCTB1three days after the transfection. The level of OCT4B1 ex-siRNAs against OCT4B1Up-regulation of OCT4B1 in Bladder Cancer   |  Asadzadeh et al578 |-OCT4B1 -tion (P < .05) in the percentage of the cells in the sub-G1 of cells in G1 phase of OCT4B1 suppressed group declined DISCUSSIONinitiation 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 OncologyFigure 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. CBFigure 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   |UROLOGY  JOURNALnate the expression of OCT4 variants in different types of cancers.OCT4 variants, OCT4Aand OCT4Bthe 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. OCT4B1expressed 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-OCT4B1parts 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 OCT4B1of cells in the sub-G1 fraction, suggesting that OCT4B1 plays CONCLUSIONAltogether, our data revealed that OCT4B1in 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 OCT4B1of OCT4B1and its function. ACKNOWLEDGEMENTSCONFLICT OF INTERESTNone declared.REFERENCES1. 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 al580 |18. Lengner CJ, Welstead GG, Jaenisch R. 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A Plausible Anti-Apoptotic Role of Up-Regulated OCT4B1 in Bladder Tumors

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A Plausible Anti-Apoptotic Role of Up-Regulated OCT4B1 in Bladder Tumors

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