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.

Exosomic microRNAs as emerging key regulators of intercellular communication in the tumor microenvironment and beyond

“…Honegger et al determined how the intracellular and exosomal miR pool in HPVpositive tumor cells is affected by sustained expression of the viral E6/E7 oncogenes. RNA deep sequencing analysis showed abundance of pro-tumorigenic miRs such as miR-7-5p and miR-378a-3p in exosomes from cells expressing high levels E6/E7, further supporting the significant role of oncogenes in promoting miR loading onto exosomes [47]. Shen et al showed that epidermal growth factor receptor (EGFR), a known oncogene in several cancers, suppresses maturation of several tumor suppressor miRs under hypoxic conditions by phosphorylating Ago2 [48].…”

miR-143 is associated with proliferation and apoptosis involving ERK5 in HeLa cells

“…miR-143 expression has been reported to be downregulated in human cervical cancer tissues (13). Furthermore, previous studies have revealed that miR-143 is downregulated in HPV-induced pre-neoplastic lesions, which suggests that miR-143 may have a significant role in the early stages of cervical cancer development (13,14).…”

“…miRs are able to regulate multiple target genes simultaneously (15). miR-143 has been demonstrated to target Kirsten rat sarcoma viral oncogene homolog (KRAS) (16), matrix metalloproteinase-13 (7), epithelial-mesenchymal transition (17), cyclooxygenase-2 (6) and cluster of differentiation 44v3 (3), and is able to suppress cell growth and metastasis in vitro and in vivo in several types of tumor (13). Among the target genes regulated by miR-143, in silico screening of gene targets for miR-143 was performed using TargetScan (www.targetscan.org).…”

A New Method Using Mean Value Matrix for Risk Types Prediction of Human Papillomaviruses

“…HPVs16 and HPVs18 are the most common forms of cervical cancers in HPVs types around the world, which were found about 50% and 20% of the cases, respectively [6,7]. However, the E6 and E7 proteins of high-risk HPVs determines the behavior of deteriorating development to the potential host cells [8][9][10]. The deteriorative E6 oncogene is commonly accepted to determine the tumorigenic character of HPVs positive cancer cells [11].…”