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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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Specific detection of SncmtRNA-1 and SncmtRNA-2.A, sequence of probes 12 (specific for SncmtRNA-1) and 13 (specific for SncmtRNA-2) targeted to the linker region between the IR and the 5′-end of the 16S mtrRNA (see Fig. 3F, showing the position of these primers and control primers 14 and 15). B, PCR amplification of cDNA obtained from the indicated cells using primers 12 and 2 (SncmtRNA-1; Fig. 3F). A single fragment of 190 bp was obtained in all samples. C, same as B but using primers 13 and 2 (SncmtRNA-2; Fig. 3F). A 130-bp fragment corresponding to SncmtRNA-2 was obtained only with cDNA from HFK698 and 18Nco cells. M, 100-bp ladder. D, PCR amplification of SncmtRNA-1 and -2 from HFK698 cDNA. The expected fragments of 190 and 130 bp were obtained using primer 2 in combination with primers 12 or 13 (lanes 1–4). No amplification was observed when primer 2 was used in combination with primer 14 (9 nt complementary to the 5′-end of the 16S rRNA) or primer 15 (9 nt complementary to the 3′-end of the IR) of the SncmtRNA-1. Lane M, 100-bp ladder. E, differential expression of the SncmtRNA-1 and SncmtRNA-2. The expression of SncmtRNA-1 (S-1) and SncmtRNA-2 (S-2) in the indicated cells was determined by ISH using probe 12 and probe 13, respectively. The SncmtRNA-2 was expressed only in the immortalized cells HFK698 and 18Nco. Notice that only HFK expresses the ASncmtRNAs (AS). Magnification was ×40.
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Figure 4: Specific detection of SncmtRNA-1 and SncmtRNA-2.A, sequence of probes 12 (specific for SncmtRNA-1) and 13 (specific for SncmtRNA-2) targeted to the linker region between the IR and the 5′-end of the 16S mtrRNA (see Fig. 3F, showing the position of these primers and control primers 14 and 15). B, PCR amplification of cDNA obtained from the indicated cells using primers 12 and 2 (SncmtRNA-1; Fig. 3F). A single fragment of 190 bp was obtained in all samples. C, same as B but using primers 13 and 2 (SncmtRNA-2; Fig. 3F). A 130-bp fragment corresponding to SncmtRNA-2 was obtained only with cDNA from HFK698 and 18Nco cells. M, 100-bp ladder. D, PCR amplification of SncmtRNA-1 and -2 from HFK698 cDNA. The expected fragments of 190 and 130 bp were obtained using primer 2 in combination with primers 12 or 13 (lanes 1–4). No amplification was observed when primer 2 was used in combination with primer 14 (9 nt complementary to the 5′-end of the 16S rRNA) or primer 15 (9 nt complementary to the 3′-end of the IR) of the SncmtRNA-1. Lane M, 100-bp ladder. E, differential expression of the SncmtRNA-1 and SncmtRNA-2. The expression of SncmtRNA-1 (S-1) and SncmtRNA-2 (S-2) in the indicated cells was determined by ISH using probe 12 and probe 13, respectively. The SncmtRNA-2 was expressed only in the immortalized cells HFK698 and 18Nco. Notice that only HFK expresses the ASncmtRNAs (AS). Magnification was ×40.

Mentions: The ISH shown in Fig. 1A was carried out with probe 8 (Fig. 3A), which hybridizes to SncmtRNA-1 and SncmtRNA-2. To differentiate between these two transcripts, probes targeted to the linker region between the IR of each transcript and the 5′-end of the 16S mtrRNA were used. Probes 12 and 13 (Fig. 2F), specific for SncmtRNA-1 and SncmtRNA-2, respectively, contain 9 nt complementary to the 3′-end of the IR of each transcript followed by 9 nt targeted to the first 9 positions of the 16S mtrRNA (Fig. 4A). To test the specificity of these probes, cDNA from HFK, HFK698, 18Nco, SiHa, and HeLa cells was amplified using primers 12 and 2, resulting in a 190-bp amplicon (Fig. 4B), which corresponds to SncmtRNA-1. Amplification with primers 13 and 2 yielded an amplicon of about 130 bp only with cDNA from HFK698 and 18Nco cells (Fig. 4C). The sequence of this amplicon confirmed the structure of the SncmtRNA-2 (data not shown). As expected, no amplification was obtained with primer 2 and a reverse primer of 9 nt (Fig. 3F, primer 14) complementary to the 3′-end of the IR of the SncmtRNA-1 (Fig. 4D, lanes 5 and 6) or the SncmtRNA-2 (primer 15) (Fig. 4D, lanes 7 and 8). Therefore, probes 12 and 13 were used to distinguish SncmtRNA-1 and SncmtRNA-2 by ISH. As shown in Fig. 4E, ISH with probe 12 revealed the expression of SncmtRNA-1 (S-1) in HFK, HFK698, 18Nco, SiHa, and HeLa cells. On the other hand, hybridization with probe 13 revealed the expression of SncmtRNA-2, only in HFK698 and 18Nco cells (Fig. 4E, S-2). The ASncmtRNAs were expressed only in HFK (Fig. 4E, AS).


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)

Specific detection of SncmtRNA-1 and SncmtRNA-2.A, sequence of probes 12 (specific for SncmtRNA-1) and 13 (specific for SncmtRNA-2) targeted to the linker region between the IR and the 5′-end of the 16S mtrRNA (see Fig. 3F, showing the position of these primers and control primers 14 and 15). B, PCR amplification of cDNA obtained from the indicated cells using primers 12 and 2 (SncmtRNA-1; Fig. 3F). A single fragment of 190 bp was obtained in all samples. C, same as B but using primers 13 and 2 (SncmtRNA-2; Fig. 3F). A 130-bp fragment corresponding to SncmtRNA-2 was obtained only with cDNA from HFK698 and 18Nco cells. M, 100-bp ladder. D, PCR amplification of SncmtRNA-1 and -2 from HFK698 cDNA. The expected fragments of 190 and 130 bp were obtained using primer 2 in combination with primers 12 or 13 (lanes 1–4). No amplification was observed when primer 2 was used in combination with primer 14 (9 nt complementary to the 5′-end of the 16S rRNA) or primer 15 (9 nt complementary to the 3′-end of the IR) of the SncmtRNA-1. Lane M, 100-bp ladder. E, differential expression of the SncmtRNA-1 and SncmtRNA-2. The expression of SncmtRNA-1 (S-1) and SncmtRNA-2 (S-2) in the indicated cells was determined by ISH using probe 12 and probe 13, respectively. The SncmtRNA-2 was expressed only in the immortalized cells HFK698 and 18Nco. Notice that only HFK expresses the ASncmtRNAs (AS). Magnification was ×40.
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Figure 4: Specific detection of SncmtRNA-1 and SncmtRNA-2.A, sequence of probes 12 (specific for SncmtRNA-1) and 13 (specific for SncmtRNA-2) targeted to the linker region between the IR and the 5′-end of the 16S mtrRNA (see Fig. 3F, showing the position of these primers and control primers 14 and 15). B, PCR amplification of cDNA obtained from the indicated cells using primers 12 and 2 (SncmtRNA-1; Fig. 3F). A single fragment of 190 bp was obtained in all samples. C, same as B but using primers 13 and 2 (SncmtRNA-2; Fig. 3F). A 130-bp fragment corresponding to SncmtRNA-2 was obtained only with cDNA from HFK698 and 18Nco cells. M, 100-bp ladder. D, PCR amplification of SncmtRNA-1 and -2 from HFK698 cDNA. The expected fragments of 190 and 130 bp were obtained using primer 2 in combination with primers 12 or 13 (lanes 1–4). No amplification was observed when primer 2 was used in combination with primer 14 (9 nt complementary to the 5′-end of the 16S rRNA) or primer 15 (9 nt complementary to the 3′-end of the IR) of the SncmtRNA-1. Lane M, 100-bp ladder. E, differential expression of the SncmtRNA-1 and SncmtRNA-2. The expression of SncmtRNA-1 (S-1) and SncmtRNA-2 (S-2) in the indicated cells was determined by ISH using probe 12 and probe 13, respectively. The SncmtRNA-2 was expressed only in the immortalized cells HFK698 and 18Nco. Notice that only HFK expresses the ASncmtRNAs (AS). Magnification was ×40.
Mentions: The ISH shown in Fig. 1A was carried out with probe 8 (Fig. 3A), which hybridizes to SncmtRNA-1 and SncmtRNA-2. To differentiate between these two transcripts, probes targeted to the linker region between the IR of each transcript and the 5′-end of the 16S mtrRNA were used. Probes 12 and 13 (Fig. 2F), specific for SncmtRNA-1 and SncmtRNA-2, respectively, contain 9 nt complementary to the 3′-end of the IR of each transcript followed by 9 nt targeted to the first 9 positions of the 16S mtrRNA (Fig. 4A). To test the specificity of these probes, cDNA from HFK, HFK698, 18Nco, SiHa, and HeLa cells was amplified using primers 12 and 2, resulting in a 190-bp amplicon (Fig. 4B), which corresponds to SncmtRNA-1. Amplification with primers 13 and 2 yielded an amplicon of about 130 bp only with cDNA from HFK698 and 18Nco cells (Fig. 4C). The sequence of this amplicon confirmed the structure of the SncmtRNA-2 (data not shown). As expected, no amplification was obtained with primer 2 and a reverse primer of 9 nt (Fig. 3F, primer 14) complementary to the 3′-end of the IR of the SncmtRNA-1 (Fig. 4D, lanes 5 and 6) or the SncmtRNA-2 (primer 15) (Fig. 4D, lanes 7 and 8). Therefore, probes 12 and 13 were used to distinguish SncmtRNA-1 and SncmtRNA-2 by ISH. As shown in Fig. 4E, ISH with probe 12 revealed the expression of SncmtRNA-1 (S-1) in HFK, HFK698, 18Nco, SiHa, and HeLa cells. On the other hand, hybridization with probe 13 revealed the expression of SncmtRNA-2, only in HFK698 and 18Nco cells (Fig. 4E, S-2). The ASncmtRNAs were expressed only in HFK (Fig. 4E, AS).

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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Related in: MedlinePlus