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Dependence of intracellular and exosomal microRNAs on viral E6/E7 oncogene expression in HPV-positive tumor cells.

Honegger A, Schilling D, Bastian S, Sponagel J, Kuryshev V, Sültmann H, Scheffner M, Hoppe-Seyler K, Hoppe-Seyler F - PLoS Pathog. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Therapy of Virus-Associated Cancers (F065), German Cancer Research Center (DKFZ), Heidelberg, Germany.

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.

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Silencing of HPV18 E6/E7 expression by RNA interference.(A) qRT-PCR analysis of HPV18 E6/E7 (left panel) and p21 (right panel) mRNA expression, 72 h after transfection of HeLa cells with si18E6/E7, control siRNA siContr-1, or upon mock treatment. mRNA levels were normalized to ACTB and calculated relative to the mock control. Data represent mean ± SEM (n = 4). Asterisks above columns indicate statistically significant differences from siContr-1-treated cells (p ≤ 0.05 (*), p ≤ 0.001 (***)). (B) Immunoblot analysis of HPV18 E6, p53, and p21 protein levels, 72 h after transfection of HeLa cells with si18E6/E7 or siContr-1. α-Tubulin: loading control. (C) Immunoblot analysis of HPV18 E7, total pRb (pRb), phosphorylated pRb (pRb-P), and Cyclin A1 protein levels, 72 h after transfection of HeLa cells with si18E6/E7 or siContr-1. α-Tubulin: loading control.
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ppat.1004712.g001: Silencing of HPV18 E6/E7 expression by RNA interference.(A) qRT-PCR analysis of HPV18 E6/E7 (left panel) and p21 (right panel) mRNA expression, 72 h after transfection of HeLa cells with si18E6/E7, control siRNA siContr-1, or upon mock treatment. mRNA levels were normalized to ACTB and calculated relative to the mock control. Data represent mean ± SEM (n = 4). Asterisks above columns indicate statistically significant differences from siContr-1-treated cells (p ≤ 0.05 (*), p ≤ 0.001 (***)). (B) Immunoblot analysis of HPV18 E6, p53, and p21 protein levels, 72 h after transfection of HeLa cells with si18E6/E7 or siContr-1. α-Tubulin: loading control. (C) Immunoblot analysis of HPV18 E7, total pRb (pRb), phosphorylated pRb (pRb-P), and Cyclin A1 protein levels, 72 h after transfection of HeLa cells with si18E6/E7 or siContr-1. α-Tubulin: loading control.

Mentions: In order to investigate the influence of the HPV oncogenes on the intracellular miRNA composition of HPV-positive cancer cells, endogenous HPV18 E6/E7 expression in HeLa cervical carcinoma cells was blocked by RNA interference (RNAi) for subsequent deep sequencing analyses. Treatment with E6/E7-targeting siRNAs (si18E6/E7) led to efficient downregulation of E6/E7 mRNA levels as shown by qRT-PCR analyses, using primers recognizing all three transcript classes [8] coding for HPV18 E6 and E7 (Fig. 1A, left panel). A substantial reduction of the HPV18 E6 and E7 protein levels 72 h after transfection with si18E6/E7 was observed (Fig. 1B/C). This was linked to increased p53 protein levels (Fig. 1B), as expected from the ability of E6 to induce the degradation of p53 [4], and increased p21 mRNA and protein levels (Fig. 1A/B), representing a downstream transcriptional target gene for p53 [62]. Further, E6/E7 silencing was associated with an increase of total pRb protein levels, consistent with the ability of E7 to induce pRb degradation [6], as well as with decreased amounts of phosphorylated pRb and of Cyclin A1, indicating reactivation of the pRb cascade (Fig. 1C).


Dependence of intracellular and exosomal microRNAs on viral E6/E7 oncogene expression in HPV-positive tumor cells.

Honegger A, Schilling D, Bastian S, Sponagel J, Kuryshev V, Sültmann H, Scheffner M, Hoppe-Seyler K, Hoppe-Seyler F - PLoS Pathog. (2015)

Silencing of HPV18 E6/E7 expression by RNA interference.(A) qRT-PCR analysis of HPV18 E6/E7 (left panel) and p21 (right panel) mRNA expression, 72 h after transfection of HeLa cells with si18E6/E7, control siRNA siContr-1, or upon mock treatment. mRNA levels were normalized to ACTB and calculated relative to the mock control. Data represent mean ± SEM (n = 4). Asterisks above columns indicate statistically significant differences from siContr-1-treated cells (p ≤ 0.05 (*), p ≤ 0.001 (***)). (B) Immunoblot analysis of HPV18 E6, p53, and p21 protein levels, 72 h after transfection of HeLa cells with si18E6/E7 or siContr-1. α-Tubulin: loading control. (C) Immunoblot analysis of HPV18 E7, total pRb (pRb), phosphorylated pRb (pRb-P), and Cyclin A1 protein levels, 72 h after transfection of HeLa cells with si18E6/E7 or siContr-1. α-Tubulin: loading control.
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ppat.1004712.g001: Silencing of HPV18 E6/E7 expression by RNA interference.(A) qRT-PCR analysis of HPV18 E6/E7 (left panel) and p21 (right panel) mRNA expression, 72 h after transfection of HeLa cells with si18E6/E7, control siRNA siContr-1, or upon mock treatment. mRNA levels were normalized to ACTB and calculated relative to the mock control. Data represent mean ± SEM (n = 4). Asterisks above columns indicate statistically significant differences from siContr-1-treated cells (p ≤ 0.05 (*), p ≤ 0.001 (***)). (B) Immunoblot analysis of HPV18 E6, p53, and p21 protein levels, 72 h after transfection of HeLa cells with si18E6/E7 or siContr-1. α-Tubulin: loading control. (C) Immunoblot analysis of HPV18 E7, total pRb (pRb), phosphorylated pRb (pRb-P), and Cyclin A1 protein levels, 72 h after transfection of HeLa cells with si18E6/E7 or siContr-1. α-Tubulin: loading control.
Mentions: In order to investigate the influence of the HPV oncogenes on the intracellular miRNA composition of HPV-positive cancer cells, endogenous HPV18 E6/E7 expression in HeLa cervical carcinoma cells was blocked by RNA interference (RNAi) for subsequent deep sequencing analyses. Treatment with E6/E7-targeting siRNAs (si18E6/E7) led to efficient downregulation of E6/E7 mRNA levels as shown by qRT-PCR analyses, using primers recognizing all three transcript classes [8] coding for HPV18 E6 and E7 (Fig. 1A, left panel). A substantial reduction of the HPV18 E6 and E7 protein levels 72 h after transfection with si18E6/E7 was observed (Fig. 1B/C). This was linked to increased p53 protein levels (Fig. 1B), as expected from the ability of E6 to induce the degradation of p53 [4], and increased p21 mRNA and protein levels (Fig. 1A/B), representing a downstream transcriptional target gene for p53 [62]. Further, E6/E7 silencing was associated with an increase of total pRb protein levels, consistent with the ability of E7 to induce pRb degradation [6], as well as with decreased amounts of phosphorylated pRb and of Cyclin A1, indicating reactivation of the pRb cascade (Fig. 1C).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Therapy of Virus-Associated Cancers (F065), German Cancer Research Center (DKFZ), Heidelberg, Germany.

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.

Show MeSH
Related in: MedlinePlus