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Expression of mitochondrial non-coding RNAs (ncRNAs) is modulated by high risk human papillomavirus (HPV) oncogenes.

Villota C, Campos A, Vidaurre S, Oliveira-Cruz L, Boccardo E, Burzio VA, Varas M, Villegas J, Villa LL, Valenzuela PD, Socías M, Roberts S, Burzio LO - J. Biol. Chem. (2012)

Bottom Line: Transduction of HFK with both E6 and E7 is sufficient to induce expression of SncmtRNA-2.Moreover, E2 oncogene is involved in down-regulation of the ASncmtRNAs.Knockdown of E2 in immortalized cells reestablishes in a reversible manner the expression of the ASncmtRNAs, suggesting that endogenous cellular factors(s) could play functions analogous to E2 during non-HPV-induced oncogenesis.

View Article: PubMed Central - PubMed

Affiliation: Andes Biotechnologies SA, Fundación Ciencia para la Vida, Zanartu 1482 7782272, Chile. claudio.villota@gmail.com

ABSTRACT
The study of RNA and DNA oncogenic viruses has proved invaluable in the discovery of key cellular pathways that are rendered dysfunctional during cancer progression. An example is high risk human papillomavirus (HPV), the etiological agent of cervical cancer. The role of HPV oncogenes in cellular immortalization and transformation has been extensively investigated. We reported the differential expression of a family of human mitochondrial non-coding RNAs (ncRNAs) between normal and cancer cells. Normal cells express a sense mitochondrial ncRNA (SncmtRNA) that seems to be required for cell proliferation and two antisense transcripts (ASncmtRNAs). In contrast, the ASncmtRNAs are down-regulated in cancer cells. To shed some light on the mechanisms that trigger down-regulation of the ASncmtRNAs, we studied human keratinocytes (HFK) immortalized with HPV. Here we show that immortalization of HFK with HPV-16 or 18 causes down-regulation of the ASncmtRNAs and induces the expression of a new sense transcript named SncmtRNA-2. Transduction of HFK with both E6 and E7 is sufficient to induce expression of SncmtRNA-2. Moreover, E2 oncogene is involved in down-regulation of the ASncmtRNAs. Knockdown of E2 in immortalized cells reestablishes in a reversible manner the expression of the ASncmtRNAs, suggesting that endogenous cellular factors(s) could play functions analogous to E2 during non-HPV-induced oncogenesis.

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Expression of SncmtRNA-2 is induced by HPV-16 E6 and E7.A, expression of the SncmtRNA-1, SncmtRNA-2, and the ASncmtRNAs was determined in HFK transduced with E6, E7, or E6/E7. The SncmtRNA-1 was expressed in all cells, including HFK698. The SncmtRNA-2 was expressed only in HFK transduced with E6 and E7 and in HFK698. Expression of the ASncmtRNAs was maintained in all cells except HFK698. Magnification was ×20. B, PCR amplification confirmed that the SncmtRNA-2 was expressed only in HFK698 and HFK transduced with E6 and E7. C, total RNA from HFK698 cells, HFK, and HFK transduced with E6, E7, and E6/E7 was used for RT-PCR amplification of HPV-16 E6 mRNA. E6 is expressed in HFK transduced with E6 or E6/E7 and HFK698 cells. D, same as C, but amplification was carried out with primers corresponding to HPV-16 E7 mRNA. Lane M, 100-bp ladder.
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Figure 6: Expression of SncmtRNA-2 is induced by HPV-16 E6 and E7.A, expression of the SncmtRNA-1, SncmtRNA-2, and the ASncmtRNAs was determined in HFK transduced with E6, E7, or E6/E7. The SncmtRNA-1 was expressed in all cells, including HFK698. The SncmtRNA-2 was expressed only in HFK transduced with E6 and E7 and in HFK698. Expression of the ASncmtRNAs was maintained in all cells except HFK698. Magnification was ×20. B, PCR amplification confirmed that the SncmtRNA-2 was expressed only in HFK698 and HFK transduced with E6 and E7. C, total RNA from HFK698 cells, HFK, and HFK transduced with E6, E7, and E6/E7 was used for RT-PCR amplification of HPV-16 E6 mRNA. E6 is expressed in HFK transduced with E6 or E6/E7 and HFK698 cells. D, same as C, but amplification was carried out with primers corresponding to HPV-16 E7 mRNA. Lane M, 100-bp ladder.

Mentions: We next examined which oncogene(s) of HPV-16 is involved in down-regulation of the ASncmtRNAs and in the expression of the SncmtRNA-2. HFK were transduced with retroviral vectors containing the coding sequences of E6, E7, or E6 and E7 (16, 17). Probes 12 and 13 were used to determine the expression of SncmtRNA-1 and SncmtRNA-2, respectively, by ISH. HFK transduced with E6 or E7 express the SncmtRNA-1 (S-1) and the ASncmtRNAs (AS) (Fig. 6A), but not SncmtRNA-2 (Fig. 6A, S-2), just as normal keratinocytes. Interestingly, however, transduction of HFK with E6 and E7 induced expression of the SncmtRNA-2, just like the immortalized cell line HFK698 (Fig. 6A, S-2). RT-PCR amplification using primers 1 and 2 (see Fig. 3A) confirmed that the 150-bp fragment corresponding to SncmtRNA-2 was amplified only in HFK transduced with E6/E7 as well as in HFK698 cells (Fig. 6B). Expression of E6 and E7 mRNA was confirmed by RT-PCR amplification. E6 is expressed in HFK698 cells, or in HFK transduced with E6 or with E6/E7 (Fig. 6C), whereas E7 mRNA is expressed in HFK transduced with E7 or E6/E7 and in HFK698 cells (Fig. 6D).


Expression of mitochondrial non-coding RNAs (ncRNAs) is modulated by high risk human papillomavirus (HPV) oncogenes.

Villota C, Campos A, Vidaurre S, Oliveira-Cruz L, Boccardo E, Burzio VA, Varas M, Villegas J, Villa LL, Valenzuela PD, Socías M, Roberts S, Burzio LO - J. Biol. Chem. (2012)

Expression of SncmtRNA-2 is induced by HPV-16 E6 and E7.A, expression of the SncmtRNA-1, SncmtRNA-2, and the ASncmtRNAs was determined in HFK transduced with E6, E7, or E6/E7. The SncmtRNA-1 was expressed in all cells, including HFK698. The SncmtRNA-2 was expressed only in HFK transduced with E6 and E7 and in HFK698. Expression of the ASncmtRNAs was maintained in all cells except HFK698. Magnification was ×20. B, PCR amplification confirmed that the SncmtRNA-2 was expressed only in HFK698 and HFK transduced with E6 and E7. C, total RNA from HFK698 cells, HFK, and HFK transduced with E6, E7, and E6/E7 was used for RT-PCR amplification of HPV-16 E6 mRNA. E6 is expressed in HFK transduced with E6 or E6/E7 and HFK698 cells. D, same as C, but amplification was carried out with primers corresponding to HPV-16 E7 mRNA. Lane M, 100-bp ladder.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375551&req=5

Figure 6: Expression of SncmtRNA-2 is induced by HPV-16 E6 and E7.A, expression of the SncmtRNA-1, SncmtRNA-2, and the ASncmtRNAs was determined in HFK transduced with E6, E7, or E6/E7. The SncmtRNA-1 was expressed in all cells, including HFK698. The SncmtRNA-2 was expressed only in HFK transduced with E6 and E7 and in HFK698. Expression of the ASncmtRNAs was maintained in all cells except HFK698. Magnification was ×20. B, PCR amplification confirmed that the SncmtRNA-2 was expressed only in HFK698 and HFK transduced with E6 and E7. C, total RNA from HFK698 cells, HFK, and HFK transduced with E6, E7, and E6/E7 was used for RT-PCR amplification of HPV-16 E6 mRNA. E6 is expressed in HFK transduced with E6 or E6/E7 and HFK698 cells. D, same as C, but amplification was carried out with primers corresponding to HPV-16 E7 mRNA. Lane M, 100-bp ladder.
Mentions: We next examined which oncogene(s) of HPV-16 is involved in down-regulation of the ASncmtRNAs and in the expression of the SncmtRNA-2. HFK were transduced with retroviral vectors containing the coding sequences of E6, E7, or E6 and E7 (16, 17). Probes 12 and 13 were used to determine the expression of SncmtRNA-1 and SncmtRNA-2, respectively, by ISH. HFK transduced with E6 or E7 express the SncmtRNA-1 (S-1) and the ASncmtRNAs (AS) (Fig. 6A), but not SncmtRNA-2 (Fig. 6A, S-2), just as normal keratinocytes. Interestingly, however, transduction of HFK with E6 and E7 induced expression of the SncmtRNA-2, just like the immortalized cell line HFK698 (Fig. 6A, S-2). RT-PCR amplification using primers 1 and 2 (see Fig. 3A) confirmed that the 150-bp fragment corresponding to SncmtRNA-2 was amplified only in HFK transduced with E6/E7 as well as in HFK698 cells (Fig. 6B). Expression of E6 and E7 mRNA was confirmed by RT-PCR amplification. E6 is expressed in HFK698 cells, or in HFK transduced with E6 or with E6/E7 (Fig. 6C), whereas E7 mRNA is expressed in HFK transduced with E7 or E6/E7 and in HFK698 cells (Fig. 6D).

Bottom Line: Transduction of HFK with both E6 and E7 is sufficient to induce expression of SncmtRNA-2.Moreover, E2 oncogene is involved in down-regulation of the ASncmtRNAs.Knockdown of E2 in immortalized cells reestablishes in a reversible manner the expression of the ASncmtRNAs, suggesting that endogenous cellular factors(s) could play functions analogous to E2 during non-HPV-induced oncogenesis.

View Article: PubMed Central - PubMed

Affiliation: Andes Biotechnologies SA, Fundación Ciencia para la Vida, Zanartu 1482 7782272, Chile. claudio.villota@gmail.com

ABSTRACT
The study of RNA and DNA oncogenic viruses has proved invaluable in the discovery of key cellular pathways that are rendered dysfunctional during cancer progression. An example is high risk human papillomavirus (HPV), the etiological agent of cervical cancer. The role of HPV oncogenes in cellular immortalization and transformation has been extensively investigated. We reported the differential expression of a family of human mitochondrial non-coding RNAs (ncRNAs) between normal and cancer cells. Normal cells express a sense mitochondrial ncRNA (SncmtRNA) that seems to be required for cell proliferation and two antisense transcripts (ASncmtRNAs). In contrast, the ASncmtRNAs are down-regulated in cancer cells. To shed some light on the mechanisms that trigger down-regulation of the ASncmtRNAs, we studied human keratinocytes (HFK) immortalized with HPV. Here we show that immortalization of HFK with HPV-16 or 18 causes down-regulation of the ASncmtRNAs and induces the expression of a new sense transcript named SncmtRNA-2. Transduction of HFK with both E6 and E7 is sufficient to induce expression of SncmtRNA-2. Moreover, E2 oncogene is involved in down-regulation of the ASncmtRNAs. Knockdown of E2 in immortalized cells reestablishes in a reversible manner the expression of the ASncmtRNAs, suggesting that endogenous cellular factors(s) could play functions analogous to E2 during non-HPV-induced oncogenesis.

Show MeSH
Related in: MedlinePlus