title: Dependence of intracellular and exosomal microRNAs on viral E6/E7 oncogene expression in HPV-positive tumor cells.
abstract: Specific types of human papillomaviruses (HPVs) cause cervical cancer. Cervical cancers exhibit aberrant cellular microRNA (miRNA) expression patterns. By genome-wide analyses, we investigate whether the intracellular and exosomal miRNA compositions of HPV-positive cancer cells are dependent on endogenous E6/E7 oncogene expression. Deep sequencing studies combined with qRT-PCR analyses show that E6/E7 silencing significantly affects ten of the 52 most abundant intracellular miRNAs in HPV18-positive HeLa cells, downregulating miR-17-5p, miR-186-5p, miR-378a-3p, miR-378f, miR-629-5p and miR-7-5p, and upregulating miR-143-3p, miR-23a-3p, miR-23b-3p and miR-27b-3p. The effects of E6/E7 silencing on miRNA levels are mainly not dependent on p53 and similarly observed in HPV16-positive SiHa cells. The E6/E7-regulated miRNAs are enriched for species involved in the control of cell proliferation, senescence and apoptosis, suggesting that they contribute to the growth of HPV-positive cancer cells. Consistently, we show that sustained E6/E7 expression is required to maintain the intracellular levels of members of the miR-17~92 cluster, which reduce expression of the anti-proliferative p21 gene in HPV-positive cancer cells. In exosomes secreted by HeLa cells, a distinct seven-miRNA-signature was identified among the most abundant miRNAs, with significant downregulation of let-7d-5p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-92a-3p and upregulation of miR-21-5p, upon E6/E7 silencing. Several of the E6/E7-dependent exosomal miRNAs have also been linked to the control of cell proliferation and apoptosis. This study represents the first global analysis of intracellular and exosomal miRNAs and shows that viral oncogene expression affects the abundance of multiple miRNAs likely contributing to the E6/E7-dependent growth of HPV-positive cancer cells.
authors.affiliation: Molecular Therapy of Virus-Associated Cancers (F065), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Honegger
authors.affiliation: Cancer Genome Research (B063), German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Heidelberg, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Schilling
authors.affiliation: Molecular Therapy of Virus-Associated Cancers (F065), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Bastian
authors.affiliation: Molecular Therapy of Virus-Associated Cancers (F065), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Jasmin
authors.initials: J
authors.lastname: Sponagel
authors.affiliation: Cancer Genome Research (B063), German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Heidelberg, Germany.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Kuryshev
authors.affiliation: Cancer Genome Research (B063), German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Heidelberg, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Sültmann
authors.affiliation: Department of Biology, University of Konstanz, Konstanz, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Scheffner
authors.affiliation: Molecular Therapy of Virus-Associated Cancers (F065), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Hoppe-Seyler
authors.affiliation: Molecular Therapy of Virus-Associated Cancers (F065), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Hoppe-Seyler
mesh_terms: D017209:Apoptosis / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics; D016922:Cellular Senescence / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D055354:Exosomes / Q000235:genetics / Q000378:metabolism; D005260:Female; D055106:Genome-Wide Association Study; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D015151:Immunoblotting; D035683:MicroRNAs / Q000096:biosynthesis*; D046529:Microscopy, Electron, Transmission; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D030361:Papillomavirus Infections / Q000150:complications / Q000235:genetics; D012097:Repressor Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000821:virology*
chemical_list: D004268:DNA-Binding Proteins; C068651:E6 protein, Human papillomavirus type 16; C052603:E6 protein, Human papillomavirus type 18; D035683:MicroRNAs; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins