Dependence of Intracellular and Exosomal microRNAs on Viral <i>E6/E7</i> Oncogene Expression in HPV-positive Tumor Cells

<div><p>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 <i>E6/E7</i> oncogene expression. Deep sequencing studies combined with qRT-PCR analyses show that <i>E6/E7</i> 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 <i>E6/E7</i> silencing on miRNA levels are mainly not dependent on p53 and similarly observed in HPV16-positive SiHa cells. The <i>E6/E7</i>-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 <i>E6/E7</i> expression is required to maintain the intracellular levels of members of the miR-17~92 cluster, which reduce expression of the anti-proliferative <i>p21</i> 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 <i>E6/E7</i> silencing. Several of the <i>E6/E7</i>-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 <i>E6/E7</i>-dependent growth of HPV-positive cancer cells.</p>