Targeting oncogenic microRNAs from the miR-371~373 and miR-302/367 clusters in malignant germ cell tumours causes growth inhibition through cell cycle disruption.

BACKGROUND: MiR-371~373 and miR-302/367 cluster over-expression occurs in all malignant germ cell tumours (GCTs), regardless of age (paediatric/adult), site (gonadal/extragonadal), or subtype [seminoma, yolk sac tumour (YST), embryonal carcinoma (EC)]. Six of eight microRNAs from these clusters contain the seed sequence 'AAGUGC', determining mRNA targeting. Here we sought to identify the significance of these observations by targeting these microRNAs functionally. METHODS: We targeted miR-371~373 and/or miR-302/367 clusters in malignant GCT cell lines, using CRISPR-Cas9, gapmer primary miR-302/367 transcript inhibition, and peptide nucleic acid (PNA) or locked nucleic acid (LNA)-DNA inhibition targeting miR-302a-d-3p, and undertook relevant functional assays. RESULTS: MiR-302/367 cluster microRNAs made the largest contribution to AAGUGC seed abundance in malignant GCT cells, regardless of subtype (seminoma/YST/EC). Following the unsuccessful use of CRISPR-Cas9, gapmer, and PNA systems, LNA-DNA-based targeting resulted in growth inhibition in seminoma and YST cells. This was associated with the de-repression of multiple mRNAs targeted by AAGUGC seed-containing microRNAs, with pathway analysis confirming predominant disruption of Rho-GTPase signalling, vesicle organisation/transport, and cell cycle regulation, findings corroborated in clinical samples. Further LNA-DNA inhibitor studies confirmed direct cell cycle effects, with an increase of cells in G0/G1-phase and a decrease in S-phase. CONCLUSION: Targeting of specific miR-371~373 and miR-302/367 microRNAs in malignant GCTs demonstrated their functional significance, with growth inhibition mediated through cell cycle disruption.

Keywords

32 Biomedical and Clinical Sciences, 3211 Oncology and Carcinogenesis, Genetics, Biotechnology, 2.1 Biological and endogenous factors, 2 Aetiology, Cancer

Journal Title

Br J Cancer

Journal ISSN

0007-0920
1532-1827

Publisher

Springer Science and Business Media LLC

Publisher DOI

https://doi.org/10.1038/s41416-023-02453-1

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 with Cancer UK (2014/168)
National Institute for Health and Care Research (IS-BRC-1215-20014)

We wish to thank Dr Mike Gait (Laboratory of Molecular Biology, Cambridge, UK) for technical assistance. We acknowledge grant funding from Action Medical Research (reference GN2244), Children with Cancer UK (14-168), and 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 (BRC-1215-20014). 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)