Replenishing co-downregulated miR-100-5p and miR-125b-5p in malignant germ cell tumors causes growth inhibition through cell cycle disruption.

MicroRNAs (miRNAs) are short, nonprotein-coding RNAs, and their expression is dysregulated in malignant germ cell tumors (GCTs). Here, we investigated the causes and consequences of downregulated miR-99a-5p/miR-100-5p (functionally identical) and miR-125b-5p levels in malignant GCTs regardless of age, site, or subtype. Quantitative RT-PCR was used to assess miR-99a-5p/miR-100-5p, miR-125b-5p, and associated gene expression in malignant GCT tissues/cell lines [seminoma (Sem), yolk sac tumor (YST), embryonal carcinoma (EC)]. Cells were treated with demethylating 5-azacytidine and pyrosequencing was performed. Combination miR-100-5p/miR-125b-5p mimic replenishment was used to treat malignant GCT cells. Global messenger RNA (mRNA) targets of the replenished miRNAs were identified and Metascape used to study pathway effects. We found that expression levels of miR-99a-5p/miR-100-5p and miR-125b-5p, their respective pri-miRNAs, and associated genes from chromosomes 11 and 21 (chr11/chr21) were downregulated and highly correlated in malignant GCT cells. Treatment with 5-azacytidine caused upregulation of these miRNAs, with pyrosequencing revealing hypermethylation of their chr11/chr21 loci, likely contributing to miR-100-5p/miR-125b-5p downregulation. Combination miR-100-5p/miR-125b-5p mimic replenishment resulted in growth inhibition in Sem/YST cells, with miR-100-5p/miR-125b-5p mRNA targets enriched in downregulated genes, which were involved in cell cycle (confirmed by flow cytometry) and signaling pathways. Knockdown of the miR-100-5p/miR-125b-5p target tripartite motif containing 71 (TRIM71kd) recapitulated miR-100-5p/miR-125b-5p replenishment, with growth inhibition and cell cycle disruption of Sem/YST/EC cells. Further, replenishment led to reduced lin-28 homolog A (LIN28A) levels and concomitant increases in let-7 (MIRLET7B) tumor suppressor miRNAs, creating a sustained reversion of cell phenotype. In summary, combination miR-100-5p/miR-125b-5p mimic replenishment or TRIM71kd caused growth inhibition in malignant GCT cells via cell cycle disruption. Further studies are now warranted, including mimic treatment alongside conventional platinum-based chemotherapy.

Keywords

germ cell tumor, in vitro models, mRNA, methylation, microRNA, testis, MicroRNAs, Humans, Cell Line, Tumor, Neoplasms, Germ Cell and Embryonal, Down-Regulation, Gene Expression Regulation, Neoplastic, Male, Cell Proliferation, Azacitidine, Cell Cycle

Journal Title

Mol Oncol

Journal ISSN

1574-7891
1878-0261

Publisher

Wiley

Publisher DOI

https://doi.org/10.1002/1878-0261.13757

Rights and licensing

Except where otherwised noted, this item's license is described as Attribution 4.0 International

Sponsorship

St Baldrick's Foundation (via Dana-Farber Cancer Institute) (2015-0743)
Children s Cancer and Leukaemia Group (CCLGA 2020 02)
St. Baldrick's Foundation (358099)

We acknowledge grant funding from the Waldmann Fund, Department of Pathology, University of Cambridge and from the St. Baldrick’s Foundation (358099). We thank the CCLG Tissue Bank and CCLG Principal Treatment Centres for the collection and provision of tissue samples (under CCLG project numbers 2002 BS 03 and 2020 BS 02). The CCLG Tissue Bank is funded by Cancer Research UK and CCLG. Some tissues were acquired from the Human Research Tissue Bank at Cambridge University Hospitals NHS Foundation Trust, whom we must additionally thank for their provision of histological services, and which is supported by the NIHR Cambridge Biomedical Research Centre (NIHR203312). We are grateful for financial support from the Max Williamson Fund and from Christiane and Alan Hodson, in memory of their daughter Olivia. The funders were not involved in study design, data collection or interpretation, or decision to submit for publication.

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University of Cambridge Research Outputs (Articles and Conferences)