A Systematic Analysis on DNA Methylation and the Expression of Both mRNA and microRNA in Bladder Cancer

Zhu, Jialou; Jiang, Ziyu; Gao, Fei; Hu, Xueda; Zhou, Liang; Chen, Jiahao; Luo, Huijuan; Sun, Jihua; Wu, Song; Han, Yonghua; Yin, Guangliang; Chen, Maoshan; Han, Zujing; Li, Xianxin; Huang, Yi; Zhang, Weixing; Zhou, Fangjian; Chen, Tong; Fa, Pingping; Wang, Yong; Sun, Liang; Leng, Huimin; Sun, Fenghao; Liu, Yuchen; Ye, Mingzhi; Yang, Huanming; Cai, Zhiming; Gui, Yaoting; Zhang, Xiuqing

doi:10.1371/journal.pone.0028223

Cited by 36 publications

(41 citation statements)

References 78 publications

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“…The other genes are mostly tumor suppressor genes that should have a low degree of promoter methylation (0–10%) in normal urothelium [48]–[54]. Methylation levels of 10–15% for C1QTNF6 in UROtsa were reported before, being in good agreement with our value of 12% in UROtsa-4 [29].…”

Section: Discussionsupporting

confidence: 90%

Cross-Contamination of a UROtsa Stock with T24 Cells – Molecular Comparison of Different Cell Lines and Stocks

et al. 2013

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BackgroundUROtsa is an authentic, immortalized human urothelial cell line that is used to study the effects of metals and other toxic substances, mostly in the context of bladder cancer carcinogenesis. Unusual properties on the molecular level of a provided UROtsa cell line stock prompted us to verify its identity.MethodsUROtsa cell line stocks from different sources were tested on several molecular levels and compared with other cell lines. MicroRNA and mRNA expression was determined by Real-Time PCR. Chromosome numbers were checked and PCR of different regions of the large T-antigen was performed. DNA methylation of RARB, PGR, RASSF1, CDH1, FHIT, ESR1, C1QTNF6, PTGS2, SOCS3, MGMT, and LINE1 was analyzed by pyrosequencing and compared with results from the cell lines RT4, T24, HeLa, BEAS-2B, and HepG2. Finally, short tandem repeat (STR) profiling was applied.ResultsAll tested UROtsa cell line stocks lacked large T-antigen. STR analysis unequivocally identified our main UROtsa stock as the bladder cancer cell line T24, which was different from two authentic UROtsa stocks that served as controls. Analysis of DNA methylation patterns and RNA expression confirmed their differences. Methylation pattern and mRNA expression of the contaminating T24 cell line showed moderate changes even after long-term culture of up to 56 weeks, whereas miRNAs and chromosome numbers varied markedly.ConclusionsIt is important to check the identity of cell lines, especially those that are not distributed by major cell banks. However, for some cell lines STR profiles are not available. Therefore, new cell lines should either be submitted to cell banks or at least their STR profile determined and published as part of their initial characterization. Our results should help to improve the identification of UROtsa and other cells on different molecular levels and provide information on the use of urothelial cells for long-term experiments.

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Section: Discussionsupporting

confidence: 90%

Cross-Contamination of a UROtsa Stock with T24 Cells – Molecular Comparison of Different Cell Lines and Stocks

et al. 2013

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“…Epigenetic silencing of miRs via hypermethylation of CpG shore regions has been documented in BC [7], though some have reported that the genome-wide methylation patterns between BC and normal adjacent tissue are similar [46]. Saito showed that tumor suppressor miR-126, which was down-regulated in cell lines and BC, could be up-regulated in cell lines by DNA methylation and histone deacetylation inhibitors [47].…”

Section: Resultsmentioning

confidence: 99%

The evolving understanding of microRNA in bladder cancer

Guancial

Bellmunt

Yeh

et al. 2014

Urologic Oncology: Seminars and Original Investigations

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Purpose MicroRNA (miR) expression is altered in urologic malignancies, including bladder cancer (BC). Individual miRs have been shown to modulate multiple signaling pathways that contribute to BC. We reviewed the primary literature on the role of miRs in BC and provide a general introduction to the processing, regulation, and function of miRs as tumor suppressors and oncogenes; and critically evaluate the literature on the implications of altered miR expression in BC. Materials and Methods We searched the English language literature for original and review articles in PubMed® from 1993 to March 2013, using the terms “microRNA” and “bladder cancer,” “transitional cell carcinoma,” or “urothelial carcinoma.” This search yielded 133 unique articles with greater than 85% published within the last three years. Results To date, the majority of miR studies in BC use profiling to describe dynamic changes in miR expression across stage and grade. Generalized down regulation of miRs, including those that target the fibroblast growth factor 3 (FGFR3) pathway such as miR-145, miR-101, miR-100 and miR-99a, has been observed in low grade, non-muscle invasive bladder cancer (NMIBC). In contrast, generalized increased expression of miRs is observed in high grade, muscle invasive bladder cancer (MIBC) compared with adjacent normal bladder urothelium, including miRs predicted to target p53, such as miR-21 and miR-373. Furthermore, p53 suppresses transcriptional factors which promote mesenchymal differentiation, ZEB-1 and ZEB-2, through regulation of the miR200 family. Conclusions Aberrations in miR expression identified between NMIBC and MIBC support and provide insight into molecular alterations known to distinguish the two parallel pathways of bladder carcinogenesis. The heterogeneity of tumor specimens and research methods limits the reproducibility of changes in miR expression profiles between studies and underscores the importance of in vivo validation in a field that utilizes in silico miR target prediction models.

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“…[126][127][128][129][130] Although the mechanisms underlying miRNA dysregulation in human bladder cancer are not fully determined, proof-of-principle experiments have established a tight link between epigenetic changes, such as aberrant DNA methylation and histone modifications, and altered miRNA expression in bladder carcinogenesis. [131][132][133] For instance, Saito et al 134 have shown that simultaneous treatment of T24 human bladder cancer cells with 5-aza-2'-deoxycytidine (5-Aza-CdR) and 4-phenylbutyric acid, which inhibit DNA methylation and histone deacetylase, respectively, resulted in upregulation (>3-fold) of 17 out of 313 (5.4%) human miRNAs examined. Epigenomic studies in various types of human cancer, including bladder cancer, have shown that the number of dysregulated miRNA genes with aberrant DNA methylation at their promoter CpG islands and/ or the flanking CpG "shores" (regions with less CpG density) is rapidly rising.…”

Section: Concluding Remarks: Potential Challenges and Future Directionsmentioning

confidence: 99%

Alterations of DNA methylome in human bladder cancer

Besaratinia¹,

Cockburn

Tommasi

2013

Epigenetics

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A Systematic Analysis on DNA Methylation and the Expression of Both mRNA and microRNA in Bladder Cancer

Cited by 36 publications

References 78 publications

Cross-Contamination of a UROtsa Stock with T24 Cells – Molecular Comparison of Different Cell Lines and Stocks

Cross-Contamination of a UROtsa Stock with T24 Cells – Molecular Comparison of Different Cell Lines and Stocks

The evolving understanding of microRNA in bladder cancer

Alterations of DNA methylome in human bladder cancer

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