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Promoter hypomethylation as potential confounder of Ras gene overexpression and their clinical significance in subsets of urothelial carcinoma of bladder

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Abstract

Overexpression of normal Ras and its aberrant CpG island methylation in the promoter regions have been shown to direct cells for uncontrolled abnormal growth and bladder tumor formation and therefore, fetched recent attention as a marker of diagnosis and prognosis to predict the biological behavior of urothelial carcinoma of bladder (UCB). Methylation pattern at CpG islands of the promoter regions of rat sarcoma (Ras) gene homologues namely Kristen-Ras (K-Ras), Harvey (H-Ras), and Neuroblastoma (N-Ras) were examined by methylation specific polymerase chain reaction (MSP). Real time-quantitative polymerase chain reaction (RT-qPCR) was done to determine transcriptomic expressions of these Ras isoforms in the prospective series of 42 NMIBC (non-muscle invasive bladder cancer) and 45 MIBC (muscle invasive bladder cancer) biopsies. CpG loci in H-Ras and K-Ras were observed to be more hypomethylated in MIBC, whereas more hypomethylation in N-Ras was noted in NMIBC. Strong association of hypomethylation index with tumor stage, grade, type and size validate them it as marker of diagnosis in UCB patients. Differential overexpression of H-Ras, N-Ras and K-Ras genes in NMIBC and MIBC and their association with patients’ demographics identify them as important diagnostic markers in pathogenesis of UCB. Given the reported ability of promoter hypomethylation to activate Ras expression, correlation studies examined positive significant association between hypomethylation index and expression. Study concludes that promoter hypomethylation of N-Ras and K-Ras could be a potential confounder of their increased expression in NMIBC. Biological significance of simultaneous presence of higher expression and promoter hypomethylation of Ras gene isoforms in MIBC is difficult to resolve in a given cohort of patients.

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Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

Authors thank Department of Science and Technology, Ministry of Science and Technology, Govt. of India [Research Grant No. SR/SO/HS-0113/2010] for providing financial support to carry out research work. Authors, KT is thankful to University Grants Commission (UGC), Govt. of India (22/12/2013(ii)EU-V) for providing research fellowship.

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Authors and Affiliations

  1. Department of Biochemistry, University of Lucknow, Lucknow, 226007, India

    Kiran Tripathi & Minal Garg

  2. Department of Urology, King George Medical University, Lucknow, 226003, India

    Apul Goel

  3. Department of Pathology, King George Medical University, Lucknow, 226003, India

    Atin Singhai

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  1. Kiran Tripathi
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  2. Apul Goel
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Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by KT and MG. The first draft of the manuscript was written by KT and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Minal Garg.

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Ethical clearance was obtained from Bioethics Cell, Institutional Ethics Committee (IEC), King George’s Medical University (Reference no.89th ECM II A/P8), Lucknow, India. Studies were performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.

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Tripathi, K., Goel, A., Singhai, A. et al. Promoter hypomethylation as potential confounder of Ras gene overexpression and their clinical significance in subsets of urothelial carcinoma of bladder. Mol Biol Rep 48, 2183–2199 (2021). https://doi.org/10.1007/s11033-021-06227-x

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