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Frequent concomitant inactivation of miR-34a and miR-34b/c by CpG methylation in colorectal, pancreatic, mammary, ovarian, urothelial, and renal cell carcinomas and soft tissue sarcomas

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Abstract

The microRNA encoding genes miR-34a and miR-34b/c represent direct p53 target genes and possess tumor suppressive properties as they mediate apoptosis, cell cycle arrest, and senescence. We previously reported that the miR-34a gene is subject to epigenetic inactivation by CpG methylation of its promoter region in primary prostate cancer and melanomas, and in 110 different cancer cell lines of diverse origin. Here we analyzed the methylation status of miR-34a and miR-34b/c in additional primary tumors of divergent sites. We found methylation of miR-34a or miR-34b/c in formalin-fixed, paraffin-embedded (FFPE) tumor samples from 178 patients with the following frequencies: colorectal cancer (74% miR-34a, 99% miR-34b/c; n = 114), pancreatic cancer (64%, 100%; n = 11), mammary cancer (60%, 90%; n = 10), ovarian cancer (62%, 69%; n = 13), urothelial cancer (71%, 57%; n = 7), and renal cell cancer (58%, 100%; n = 12). Furthermore, soft tissue sarcomas showed methylation of miR-34 gene promoters in FFPE samples (64%, 45%; n = 11), in explanted, cultured cells (53%, 40%; n = 40), and in frozen tissue samples (75%, 75%, n = 8). In the colorectal cancer samples a statistically significant correlation of miR-34a methylation and the absence of p53 mutation was detected. With the exception of sarcoma cell lines, the inactivation of miR-34a and miR-34b/c was concomitant in most cases. These results show that miR-34 inactivation is a common event in tumor formation, and suggest that CpG methylation of miR-34a and miR-34-b/c may have diagnostic value. The mutual exclusiveness of miR-34a methylation and p53 mutation indicates that miR-34a inactivation may substitute for loss of p53 function in cancer.

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Acknowledgments

Work in Heiko Hermeking’s lab is supported by the Deutsche Krebshilfe, the Deutsche Forschungsgemeinschaft, the Friedrich-Baur-Stiftung, the Rudolf-Bartling-Stiftung, and the German-Israeli-Foundation for Scientific Research and Development (GIF).

Conflict of interest statement

We declare that we have no conflict of interest.

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Authors and Affiliations

  1. Institute of Pathology, Ruhr-University-Bochum, Bürkle-de-la-Camp-Platz 1, 44789, Bochum, Germany

    Markus Vogt, Johanna Munding, Martha Grüner, Sven-Thorsten Liffers, Berlinda Verdoodt, Andrea Tannapfel & Heiko Hermeking

  2. Department of Plastic and Reconstructive Surgery, Department of Hand Surgery, Soft Tissue Tumour Reference Centre, BG University Hospital Bergmannsheil, Ruhr University Bochum, Buerkle-de-la Camp-Platz 1, 44789, Bochum, Germany

    Jennifer Hauk & Lars Steinstraesser

  3. Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University Munich, Thalkirchner Str. 36, 80337, Munich, Germany

    Heiko Hermeking

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  1. Markus Vogt
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Correspondence to Heiko Hermeking.

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Markus Vogt and Johanna Munding equally contributed to this work

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Appendix

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Supplemental Legends

Table S1 Methylation-specific PCR conditions for the analysis of CpG methylation of the miR-34a and miR-34b/c promoter. Table S2 Association of methylation status of miR-34a and miR-34b/c promoter and p53 IHC datas with clinicopathological characteristics of the patients in 114 primary colon cancers. Abbreviations: T: infiltration depth, N: nodal status, M: distant metastasis status, L: lymphatic infiltration, V: infiltration of blood vessels, Pn: perineural infiltration.

Table S3 Association of methylation status of miR-34a and miR-34b/c with clinicopathological characteristics of the patients in different human tumors derived from FFPE, primary tissue culture cells and frozen tissue. Abbreviations: T: infiltration depth, N: nodal status, M: distant metastasis status, L: lymphatic infiltration, V: infiltration of blood vessels, Pn: perineural infiltration.

Figure S1 Methylation-specific PCR analyses of mir-34a and miR-34b/c in primary human colorectal cancer containing FFPE samples. a. miR-34a MSP of colon carcinomas (CoCa1-114). b. miR-34b/c MSP of colon carcinoma (CoCa1-114).

Figure S2 MSP analysis of miR-34a and miR-34b/c in FFPE tumor samples representing: a. pancreatic carcinoma (PaCa1-11), b. mammary carcinoma (MaCa1-10), c. renal cell carcinoma (RCCa1-12), d. urothelial carcinoma (UtCa1-7), e. ovarian carcinoma (OvCa1-13).

Figure S3 MSP analysis of miR-34a and miR-34b/c methylation in a. primary sarcoma FFPE (Sarc1-11), b. sarcoma cells cultured in vitro (Sarc1-40), c. frozen sarcoma tissue (Sarc41-48).

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Vogt, M., Munding, J., Grüner, M. et al. Frequent concomitant inactivation of miR-34a and miR-34b/c by CpG methylation in colorectal, pancreatic, mammary, ovarian, urothelial, and renal cell carcinomas and soft tissue sarcomas. Virchows Arch 458, 313–322 (2011). https://doi.org/10.1007/s00428-010-1030-5

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