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. Oncogenic human papillomaviruses (HPVs) are major human carcinogens of broad biomedical importance. The growth of HPV-positive cervical cancer cells is critically dependent on sustained E6/E7 oncogene expression from endogenous viral DNA sequences. We here addressed the question of whether this process is linked to specific, E6/E7-dependent alterations of the cellular micro(mi)RNA network. By comprehensive deep sequencing analyses we show that endogenous E6/E7 expression significantly affects the concentrations of abundant intracellular miRNAs in HPV-positive cervical cancer cells, which are linked to the control of cell proliferation, senescence and apoptosis. These include members of the miR-17~92 cluster, which are expressed at increased levels by sustained E6/E7 expression and repress the anti-proliferative p21 gene in HPV-positive cancer cells. Moreover, we identified an E6/E7-dependent seven-miRNA-signature in exosomes secreted from HPV-positive cancer cells. These small vesicles are involved in intercellular communication and may serve as novel diagnostic markers. Taken together, our results show that continuous E6/E7 expression in HPV-positive cancer cells is linked to significant alterations in the amounts of intracellular and exosomal miRNAs with growth-promoting, anti-senescent and anti-apoptotic potential.