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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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HPV-16- or HPV-18-immortalized cells express a novel sense transcript or SncmtRNA-2.A, schematic structure of the SncmtRNA-1 and strategy to amplify the transcript between the loop and the IR. Red line, 16S mitochondrial rRNA; blue line, IR. The primers used for amplification are indicated. B, PCR amplification of cDNA from the indicated cells, using primers 1 and 2. The 210-bp fragment corresponds to the SncmtRNA-1, whereas the 150-bp fragment corresponds to the SncmtRNA-2. Lanes labeled 698 correspond to HFK698 cells. C, PCR amplification of HFK698 cDNA between primer 1 and primers 2, 3, 4, 5, 6, 7, and 8 (lanes 1–7, respectively). No amplification was observed when using primers 1 and 8 (lane 7). The inset shows the amplification fragments obtained with HFK698 cDNA and corresponding to lanes 5 and 6. M, 100-bp ladder. D and E, SncmtRNA-1 and SncmtRNA-2 contain a double-stranded region. D, RNA from SiHa cells in 2× SCC was incubated in the presence (even lanes) or absence (odd lanes) of 50 μg/ml RNase A for 15 s at room temperature. RNA was recovered and amplified by RT-PCR using primers 1 and 6 (lanes 1 and 2), primers 11 and 6 covering the double-stranded region (lanes 3 and 4), and primers 9 and 10 (lanes 5 and 6) targeted to the 3′ single-stranded region (see A). Only the double-stranded region was resistant to RNase digestion (lanes 3 and 4). E, similar results were obtained with total RNA from HFK698 cells. Amplification of the double amplicons with primers 1 and 6 (lanes 1 and 2) was abolished after digestion, and a similar result was obtained with primers 9 and 10 (lanes 5 and 6). These results indicate that the single-stranded regions (the loop and the 3′ region) of the SncmtRNA-2 were digested by RNase A. Amplification between primers 11 and 6, covering the double-stranded region of the SncmtRNA-2, was not affected by the enzyme (lanes 3 and 4). M, 100-bp ladder. F, schematic structure of the SncmtRNA-1 and SncmtRNA-2, indicating the primers used for amplification. Oligonucleotides 12 and 13 were used for specific RT-PCR amplification or for ISH. Primers 14 and 15 were used as controls.
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Figure 3: HPV-16- or HPV-18-immortalized cells express a novel sense transcript or SncmtRNA-2.A, schematic structure of the SncmtRNA-1 and strategy to amplify the transcript between the loop and the IR. Red line, 16S mitochondrial rRNA; blue line, IR. The primers used for amplification are indicated. B, PCR amplification of cDNA from the indicated cells, using primers 1 and 2. The 210-bp fragment corresponds to the SncmtRNA-1, whereas the 150-bp fragment corresponds to the SncmtRNA-2. Lanes labeled 698 correspond to HFK698 cells. C, PCR amplification of HFK698 cDNA between primer 1 and primers 2, 3, 4, 5, 6, 7, and 8 (lanes 1–7, respectively). No amplification was observed when using primers 1 and 8 (lane 7). The inset shows the amplification fragments obtained with HFK698 cDNA and corresponding to lanes 5 and 6. M, 100-bp ladder. D and E, SncmtRNA-1 and SncmtRNA-2 contain a double-stranded region. D, RNA from SiHa cells in 2× SCC was incubated in the presence (even lanes) or absence (odd lanes) of 50 μg/ml RNase A for 15 s at room temperature. RNA was recovered and amplified by RT-PCR using primers 1 and 6 (lanes 1 and 2), primers 11 and 6 covering the double-stranded region (lanes 3 and 4), and primers 9 and 10 (lanes 5 and 6) targeted to the 3′ single-stranded region (see A). Only the double-stranded region was resistant to RNase digestion (lanes 3 and 4). E, similar results were obtained with total RNA from HFK698 cells. Amplification of the double amplicons with primers 1 and 6 (lanes 1 and 2) was abolished after digestion, and a similar result was obtained with primers 9 and 10 (lanes 5 and 6). These results indicate that the single-stranded regions (the loop and the 3′ region) of the SncmtRNA-2 were digested by RNase A. Amplification between primers 11 and 6, covering the double-stranded region of the SncmtRNA-2, was not affected by the enzyme (lanes 3 and 4). M, 100-bp ladder. F, schematic structure of the SncmtRNA-1 and SncmtRNA-2, indicating the primers used for amplification. Oligonucleotides 12 and 13 were used for specific RT-PCR amplification or for ISH. Primers 14 and 15 were used as controls.

Mentions: The differential expression of these mitochondrial transcripts assessed by ISH was confirmed by RT-PCR (see “Experimental Procedures”). The SncmtRNA-1 was amplified between the loop and the IR, between primers 1 and 2 (see Fig. 3A), to generate a fragment of 210 bp (9, 10) (Fig. 1B, S). The density of each band was analyzed by densitometry measurements (see “Experimental Procedures”). The 210-bp amplicon was normalized to the 150-bp amplicon from 18S rRNA, used as a loading control (Fig. 1B, 18S). The normalized amplicon of the SncmtRNA-1 indicates that the level of this transcript was similar in the five cell lines studied (Fig. 1C). Notice that only the RNA of HFK698 and 18Nco cells yielded an amplicon of about 150 bp with the same primers (Fig. 1B, S).


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)

HPV-16- or HPV-18-immortalized cells express a novel sense transcript or SncmtRNA-2.A, schematic structure of the SncmtRNA-1 and strategy to amplify the transcript between the loop and the IR. Red line, 16S mitochondrial rRNA; blue line, IR. The primers used for amplification are indicated. B, PCR amplification of cDNA from the indicated cells, using primers 1 and 2. The 210-bp fragment corresponds to the SncmtRNA-1, whereas the 150-bp fragment corresponds to the SncmtRNA-2. Lanes labeled 698 correspond to HFK698 cells. C, PCR amplification of HFK698 cDNA between primer 1 and primers 2, 3, 4, 5, 6, 7, and 8 (lanes 1–7, respectively). No amplification was observed when using primers 1 and 8 (lane 7). The inset shows the amplification fragments obtained with HFK698 cDNA and corresponding to lanes 5 and 6. M, 100-bp ladder. D and E, SncmtRNA-1 and SncmtRNA-2 contain a double-stranded region. D, RNA from SiHa cells in 2× SCC was incubated in the presence (even lanes) or absence (odd lanes) of 50 μg/ml RNase A for 15 s at room temperature. RNA was recovered and amplified by RT-PCR using primers 1 and 6 (lanes 1 and 2), primers 11 and 6 covering the double-stranded region (lanes 3 and 4), and primers 9 and 10 (lanes 5 and 6) targeted to the 3′ single-stranded region (see A). Only the double-stranded region was resistant to RNase digestion (lanes 3 and 4). E, similar results were obtained with total RNA from HFK698 cells. Amplification of the double amplicons with primers 1 and 6 (lanes 1 and 2) was abolished after digestion, and a similar result was obtained with primers 9 and 10 (lanes 5 and 6). These results indicate that the single-stranded regions (the loop and the 3′ region) of the SncmtRNA-2 were digested by RNase A. Amplification between primers 11 and 6, covering the double-stranded region of the SncmtRNA-2, was not affected by the enzyme (lanes 3 and 4). M, 100-bp ladder. F, schematic structure of the SncmtRNA-1 and SncmtRNA-2, indicating the primers used for amplification. Oligonucleotides 12 and 13 were used for specific RT-PCR amplification or for ISH. Primers 14 and 15 were used as controls.
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Figure 3: HPV-16- or HPV-18-immortalized cells express a novel sense transcript or SncmtRNA-2.A, schematic structure of the SncmtRNA-1 and strategy to amplify the transcript between the loop and the IR. Red line, 16S mitochondrial rRNA; blue line, IR. The primers used for amplification are indicated. B, PCR amplification of cDNA from the indicated cells, using primers 1 and 2. The 210-bp fragment corresponds to the SncmtRNA-1, whereas the 150-bp fragment corresponds to the SncmtRNA-2. Lanes labeled 698 correspond to HFK698 cells. C, PCR amplification of HFK698 cDNA between primer 1 and primers 2, 3, 4, 5, 6, 7, and 8 (lanes 1–7, respectively). No amplification was observed when using primers 1 and 8 (lane 7). The inset shows the amplification fragments obtained with HFK698 cDNA and corresponding to lanes 5 and 6. M, 100-bp ladder. D and E, SncmtRNA-1 and SncmtRNA-2 contain a double-stranded region. D, RNA from SiHa cells in 2× SCC was incubated in the presence (even lanes) or absence (odd lanes) of 50 μg/ml RNase A for 15 s at room temperature. RNA was recovered and amplified by RT-PCR using primers 1 and 6 (lanes 1 and 2), primers 11 and 6 covering the double-stranded region (lanes 3 and 4), and primers 9 and 10 (lanes 5 and 6) targeted to the 3′ single-stranded region (see A). Only the double-stranded region was resistant to RNase digestion (lanes 3 and 4). E, similar results were obtained with total RNA from HFK698 cells. Amplification of the double amplicons with primers 1 and 6 (lanes 1 and 2) was abolished after digestion, and a similar result was obtained with primers 9 and 10 (lanes 5 and 6). These results indicate that the single-stranded regions (the loop and the 3′ region) of the SncmtRNA-2 were digested by RNase A. Amplification between primers 11 and 6, covering the double-stranded region of the SncmtRNA-2, was not affected by the enzyme (lanes 3 and 4). M, 100-bp ladder. F, schematic structure of the SncmtRNA-1 and SncmtRNA-2, indicating the primers used for amplification. Oligonucleotides 12 and 13 were used for specific RT-PCR amplification or for ISH. Primers 14 and 15 were used as controls.
Mentions: The differential expression of these mitochondrial transcripts assessed by ISH was confirmed by RT-PCR (see “Experimental Procedures”). The SncmtRNA-1 was amplified between the loop and the IR, between primers 1 and 2 (see Fig. 3A), to generate a fragment of 210 bp (9, 10) (Fig. 1B, S). The density of each band was analyzed by densitometry measurements (see “Experimental Procedures”). The 210-bp amplicon was normalized to the 150-bp amplicon from 18S rRNA, used as a loading control (Fig. 1B, 18S). The normalized amplicon of the SncmtRNA-1 indicates that the level of this transcript was similar in the five cell lines studied (Fig. 1C). Notice that only the RNA of HFK698 and 18Nco cells yielded an amplicon of about 150 bp with the same primers (Fig. 1B, S).

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