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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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The SncmtRNA-2 is not present in isolated mitochondria. Mitochondria were isolated from ∼5 × 108 HFK698 or 18Nco cells as described under “Experimental Procedures.” The final mitochondrial fraction was treated with RNase A to eliminate contamination from cytoplasmic RNA, followed by extraction of mitochondrial RNA with TRIzol. In parallel, total RNA was also extracted from HFK698 and 18Nco cells. Amplification of fragments of 190 bp (SncmtRNA-1), 130 bp (SncmtRNA-2), 320 bp (COX I mRNA), and 150 bp corresponding to 18S rRNA was observed in total RNA from both immortalized cell lines (T lanes). Only the amplicons of 190 bp of the SncmtRNA-1 and 320 bp of the COX I mRNA were amplified from mitochondrial RNA (Mt lanes) of HFK698 and 18Nco cells, whereas no amplification was obtained of the SncmtRNA-2 or 18S rRNA. M, 100-bp ladder.
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Figure 5: The SncmtRNA-2 is not present in isolated mitochondria. Mitochondria were isolated from ∼5 × 108 HFK698 or 18Nco cells as described under “Experimental Procedures.” The final mitochondrial fraction was treated with RNase A to eliminate contamination from cytoplasmic RNA, followed by extraction of mitochondrial RNA with TRIzol. In parallel, total RNA was also extracted from HFK698 and 18Nco cells. Amplification of fragments of 190 bp (SncmtRNA-1), 130 bp (SncmtRNA-2), 320 bp (COX I mRNA), and 150 bp corresponding to 18S rRNA was observed in total RNA from both immortalized cell lines (T lanes). Only the amplicons of 190 bp of the SncmtRNA-1 and 320 bp of the COX I mRNA were amplified from mitochondrial RNA (Mt lanes) of HFK698 and 18Nco cells, whereas no amplification was obtained of the SncmtRNA-2 or 18S rRNA. M, 100-bp ladder.

Mentions: We reported before that the SncmtRNA-1 and the ASncmtRNAs were localized in mitochondria isolated from HFK cells and mitogen-activated human lymphocytes (10). SncmtRNA-2 exhibits 99,7% identity with the sequence of the human 16S mitochondrial gene, and therefore one would expect that this transcript is also found localized in the organelle. Mitochondria were isolated from HFK698 and 18Nco cells and treated externally with RNase A to eliminate cytoplasmic RNA contamination (9, 10, 19, 20). RNA was extracted from the isolated mitochondria and amplified by RT-PCR. To ascertain that RNase treatment eliminated cytoplasmic RNA contamination, total RNA was also extracted from HFK698 and 18Nco cells. Amplification was carried out using primer pairs for SncmtRNA-1, SncmtRNA-2, COX I, and 18S rRNA (see “Experimental Procedures”). SncmtRNA-1 and COX I mRNA were readily amplified from RNA of nuclease-treated mitochondria and total RNA from HFK698 and 18Nco cells (Fig. 5). As expected, the 18S rRNA was amplified from total RNA but not from nuclease-treated mitochondria (Fig. 5). Interestingly, and in contrast to SncmtRNA-1, SncmtRNA-2 was amplified from total RNA of HFK698 and 18Nco but not from mitochondrial RNA (Fig. 5, SncmtRNA-2). These results indicate that the SncmtRNA-2 is found only outside the organelle.


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)

The SncmtRNA-2 is not present in isolated mitochondria. Mitochondria were isolated from ∼5 × 108 HFK698 or 18Nco cells as described under “Experimental Procedures.” The final mitochondrial fraction was treated with RNase A to eliminate contamination from cytoplasmic RNA, followed by extraction of mitochondrial RNA with TRIzol. In parallel, total RNA was also extracted from HFK698 and 18Nco cells. Amplification of fragments of 190 bp (SncmtRNA-1), 130 bp (SncmtRNA-2), 320 bp (COX I mRNA), and 150 bp corresponding to 18S rRNA was observed in total RNA from both immortalized cell lines (T lanes). Only the amplicons of 190 bp of the SncmtRNA-1 and 320 bp of the COX I mRNA were amplified from mitochondrial RNA (Mt lanes) of HFK698 and 18Nco cells, whereas no amplification was obtained of the SncmtRNA-2 or 18S rRNA. M, 100-bp ladder.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3375551&req=5

Figure 5: The SncmtRNA-2 is not present in isolated mitochondria. Mitochondria were isolated from ∼5 × 108 HFK698 or 18Nco cells as described under “Experimental Procedures.” The final mitochondrial fraction was treated with RNase A to eliminate contamination from cytoplasmic RNA, followed by extraction of mitochondrial RNA with TRIzol. In parallel, total RNA was also extracted from HFK698 and 18Nco cells. Amplification of fragments of 190 bp (SncmtRNA-1), 130 bp (SncmtRNA-2), 320 bp (COX I mRNA), and 150 bp corresponding to 18S rRNA was observed in total RNA from both immortalized cell lines (T lanes). Only the amplicons of 190 bp of the SncmtRNA-1 and 320 bp of the COX I mRNA were amplified from mitochondrial RNA (Mt lanes) of HFK698 and 18Nco cells, whereas no amplification was obtained of the SncmtRNA-2 or 18S rRNA. M, 100-bp ladder.
Mentions: We reported before that the SncmtRNA-1 and the ASncmtRNAs were localized in mitochondria isolated from HFK cells and mitogen-activated human lymphocytes (10). SncmtRNA-2 exhibits 99,7% identity with the sequence of the human 16S mitochondrial gene, and therefore one would expect that this transcript is also found localized in the organelle. Mitochondria were isolated from HFK698 and 18Nco cells and treated externally with RNase A to eliminate cytoplasmic RNA contamination (9, 10, 19, 20). RNA was extracted from the isolated mitochondria and amplified by RT-PCR. To ascertain that RNase treatment eliminated cytoplasmic RNA contamination, total RNA was also extracted from HFK698 and 18Nco cells. Amplification was carried out using primer pairs for SncmtRNA-1, SncmtRNA-2, COX I, and 18S rRNA (see “Experimental Procedures”). SncmtRNA-1 and COX I mRNA were readily amplified from RNA of nuclease-treated mitochondria and total RNA from HFK698 and 18Nco cells (Fig. 5). As expected, the 18S rRNA was amplified from total RNA but not from nuclease-treated mitochondria (Fig. 5). Interestingly, and in contrast to SncmtRNA-1, SncmtRNA-2 was amplified from total RNA of HFK698 and 18Nco but not from mitochondrial RNA (Fig. 5, SncmtRNA-2). These results indicate that the SncmtRNA-2 is found only outside the organelle.

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