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Identification of Deregulated Pathways, Key Regulators, and Novel miRNA-mRNA Interactions in HPV-Mediated Transformation

Authors
  • Babion, Iris1
  • Miok, Viktorian1, 2
  • Jaspers, Annelieke1
  • Huseinovic, Angelina1
  • Steenbergen, Renske D. M.1
  • van Wieringen, Wessel N.3
  • Wilting, Saskia M.
  • 1 (A.H.)
  • 2 Department of Functional Sciences, Faculty of Medicine, Victor Babeş University of Medicine and Pharmacy of Timişoara, 300041 Timişoara, Romania
  • 3 Department of Mathematics, VU University Amsterdam, 1081 HV Amsterdam, The Netherlands
Type
Published Article
Journal
Cancers
Publisher
MDPI AG
Publication Date
Mar 16, 2020
Volume
12
Issue
3
Identifiers
DOI: 10.3390/cancers12030700
PMID: 32188026
PMCID: PMC7140059
Source
PubMed Central
Keywords
License
Green

Abstract

Next to a persistent infection with high-risk human papillomavirus (HPV), molecular changes are required for the development of cervical cancer. To identify which molecular alterations drive carcinogenesis, we performed a comprehensive and longitudinal molecular characterization of HPV-transformed keratinocyte cell lines. Comparative genomic hybridization, mRNA, and miRNA expression analysis of four HPV-containing keratinocyte cell lines at eight different time points was performed. Data was analyzed using unsupervised hierarchical clustering, integrated longitudinal expression analysis, and pathway enrichment analysis. Biological relevance of identified key regulatory genes was evaluated in vitro and dual-luciferase assays were used to confirm predicted miRNA-mRNA interactions. We show that the acquisition of anchorage independence of HPV-containing keratinocyte cell lines is particularly associated with copy number alterations. Approximately one third of differentially expressed mRNAs and miRNAs was directly attributable to copy number alterations. Focal adhesion, TGF-beta signaling, and mTOR signaling pathways were enriched among these genes. PITX2 was identified as key regulator of TGF-beta signaling and inhibited cell growth in vitro, most likely by inducing cell cycle arrest and apoptosis. Predicted miRNA-mRNA interactions miR-221-3p_BRWD3, miR-221-3p_FOS, and miR-138-5p_PLXNB2 were confirmed in vitro. Integrated longitudinal analysis of our HPV-induced carcinogenesis model pinpointed relevant interconnected molecular changes and crucial signaling pathways in HPV-mediated transformation.

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