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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 E2 is involved in down-regulation of the ASncmtRNAs.A, schematic illustration of the HPV-16 genome, indicating the position of E6, E7, E1, E2, E4, E5, and L2 genes. The absence of L1 is for simplicity. The transplicing reaction between a fragment of E1 with a fragment of E2 to generate E1^E4 is shown. B, expression of E2 mRNA in 18Nco cells was determined by RT-PCR amplification. A fragment of 330 bp was obtained with cDNA of 18Nco cells (lanes 1 and 2). E2 was knocked down by an ASO complementary to the NH2-terminal coding region (18 E2-ASO; lanes 3 and 4) but not by a control oligonucleotide (C-ASO; lanes 5 and 6). 24 h after transfection with the 18E2-ASO, the cells were cultured in fresh medium for another 24 h. The expression of E2 was reestablished (Recovery; lanes 7 and 8). C, analysis of the expression of SncmtRNA-1 and -2 (S) and ASncmtRNAs (AS) by fluorescent in situ hybridization. Hybridization revealed that the expression of the ASncmtRNAs was recovered in 18Nco cells transfected with the 18E2-ASO. Once the expression of E2 was reestablished, expression of the ASncmtRNAs was down-regulated (Recovery). D, the same results were obtained with HFK698 cells transfected with an E2-ASO complementary to HPV-16 E2 (16 E2-ASO). The expression of the ASncmtRNAs was reestablished after knocking down HPV-16 E2. Recovery of E2 expression resulted in down-regulation of the ASncmtRNAs (Recovery). Magnification was ×20.
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Figure 8: HPV E2 is involved in down-regulation of the ASncmtRNAs.A, schematic illustration of the HPV-16 genome, indicating the position of E6, E7, E1, E2, E4, E5, and L2 genes. The absence of L1 is for simplicity. The transplicing reaction between a fragment of E1 with a fragment of E2 to generate E1^E4 is shown. B, expression of E2 mRNA in 18Nco cells was determined by RT-PCR amplification. A fragment of 330 bp was obtained with cDNA of 18Nco cells (lanes 1 and 2). E2 was knocked down by an ASO complementary to the NH2-terminal coding region (18 E2-ASO; lanes 3 and 4) but not by a control oligonucleotide (C-ASO; lanes 5 and 6). 24 h after transfection with the 18E2-ASO, the cells were cultured in fresh medium for another 24 h. The expression of E2 was reestablished (Recovery; lanes 7 and 8). C, analysis of the expression of SncmtRNA-1 and -2 (S) and ASncmtRNAs (AS) by fluorescent in situ hybridization. Hybridization revealed that the expression of the ASncmtRNAs was recovered in 18Nco cells transfected with the 18E2-ASO. Once the expression of E2 was reestablished, expression of the ASncmtRNAs was down-regulated (Recovery). D, the same results were obtained with HFK698 cells transfected with an E2-ASO complementary to HPV-16 E2 (16 E2-ASO). The expression of the ASncmtRNAs was reestablished after knocking down HPV-16 E2. Recovery of E2 expression resulted in down-regulation of the ASncmtRNAs (Recovery). Magnification was ×20.

Mentions: E1^E4 results from a transplicing reaction between the first 15 nt of E1 and a fragment of 264 nt positioned close to the 3′-end of E2, as illustrated in Fig. 8A (25). Therefore, the E4-shRNA should also induce knockdown of E2 (Fig. 8A, shRNA, red arrow). To explore whether E2 is indeed involved in down-regulation of the ASncmtRNAs, 18Nco cells were transfected with an ASO complementary to the NH2-terminal region of HPV-18 E2 (Fig. 6A, E2-ASO, blue arrow) or with ASO-C. Twenty-four hours after transfection, cells were used to prepare total RNA or fixed to determine the expression of the ASncmtRNAs. As shown in Fig. 8B, E2-ASO induced knockdown of E2 in 18Nco cells, whereas the control ASO had no effect (Fig. 8B, C-ASO). Interestingly, fluorescent in situ hybridization revealed that the expression of the ASncmtRNAs was reestablished in 18Nco cells upon knockdown of E2 (Fig. 8C, 18 E2-ASO). Transfection with ASO-C had no effect (Fig. 8C, C-ASO). To determine whether the effect on the expression of the ASncmtRNAs was reversible, E2-ASO was removed, and the cells were further cultured in normal medium for an additional 24 h. Together with the recovery of E2 expression (Fig. 6B, Recovery), the ASncmtRNAs were again down-regulated (Fig. 8C, Recovery). The same results were obtained with HFK698 (Fig. 8D and supplemental Fig. 2A) and HF18 cells (keratinocytes immortalized with HPV-18) (see supplemental Fig. 3, B and C). Transfection of 18Nco or HFK698 cell lines with E2-ASO did not affect the expression of E6, E7, E1, and E5 (see supplemental Fig. 4, A and B).


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 E2 is involved in down-regulation of the ASncmtRNAs.A, schematic illustration of the HPV-16 genome, indicating the position of E6, E7, E1, E2, E4, E5, and L2 genes. The absence of L1 is for simplicity. The transplicing reaction between a fragment of E1 with a fragment of E2 to generate E1^E4 is shown. B, expression of E2 mRNA in 18Nco cells was determined by RT-PCR amplification. A fragment of 330 bp was obtained with cDNA of 18Nco cells (lanes 1 and 2). E2 was knocked down by an ASO complementary to the NH2-terminal coding region (18 E2-ASO; lanes 3 and 4) but not by a control oligonucleotide (C-ASO; lanes 5 and 6). 24 h after transfection with the 18E2-ASO, the cells were cultured in fresh medium for another 24 h. The expression of E2 was reestablished (Recovery; lanes 7 and 8). C, analysis of the expression of SncmtRNA-1 and -2 (S) and ASncmtRNAs (AS) by fluorescent in situ hybridization. Hybridization revealed that the expression of the ASncmtRNAs was recovered in 18Nco cells transfected with the 18E2-ASO. Once the expression of E2 was reestablished, expression of the ASncmtRNAs was down-regulated (Recovery). D, the same results were obtained with HFK698 cells transfected with an E2-ASO complementary to HPV-16 E2 (16 E2-ASO). The expression of the ASncmtRNAs was reestablished after knocking down HPV-16 E2. Recovery of E2 expression resulted in down-regulation of the ASncmtRNAs (Recovery). Magnification was ×20.
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Figure 8: HPV E2 is involved in down-regulation of the ASncmtRNAs.A, schematic illustration of the HPV-16 genome, indicating the position of E6, E7, E1, E2, E4, E5, and L2 genes. The absence of L1 is for simplicity. The transplicing reaction between a fragment of E1 with a fragment of E2 to generate E1^E4 is shown. B, expression of E2 mRNA in 18Nco cells was determined by RT-PCR amplification. A fragment of 330 bp was obtained with cDNA of 18Nco cells (lanes 1 and 2). E2 was knocked down by an ASO complementary to the NH2-terminal coding region (18 E2-ASO; lanes 3 and 4) but not by a control oligonucleotide (C-ASO; lanes 5 and 6). 24 h after transfection with the 18E2-ASO, the cells were cultured in fresh medium for another 24 h. The expression of E2 was reestablished (Recovery; lanes 7 and 8). C, analysis of the expression of SncmtRNA-1 and -2 (S) and ASncmtRNAs (AS) by fluorescent in situ hybridization. Hybridization revealed that the expression of the ASncmtRNAs was recovered in 18Nco cells transfected with the 18E2-ASO. Once the expression of E2 was reestablished, expression of the ASncmtRNAs was down-regulated (Recovery). D, the same results were obtained with HFK698 cells transfected with an E2-ASO complementary to HPV-16 E2 (16 E2-ASO). The expression of the ASncmtRNAs was reestablished after knocking down HPV-16 E2. Recovery of E2 expression resulted in down-regulation of the ASncmtRNAs (Recovery). Magnification was ×20.
Mentions: E1^E4 results from a transplicing reaction between the first 15 nt of E1 and a fragment of 264 nt positioned close to the 3′-end of E2, as illustrated in Fig. 8A (25). Therefore, the E4-shRNA should also induce knockdown of E2 (Fig. 8A, shRNA, red arrow). To explore whether E2 is indeed involved in down-regulation of the ASncmtRNAs, 18Nco cells were transfected with an ASO complementary to the NH2-terminal region of HPV-18 E2 (Fig. 6A, E2-ASO, blue arrow) or with ASO-C. Twenty-four hours after transfection, cells were used to prepare total RNA or fixed to determine the expression of the ASncmtRNAs. As shown in Fig. 8B, E2-ASO induced knockdown of E2 in 18Nco cells, whereas the control ASO had no effect (Fig. 8B, C-ASO). Interestingly, fluorescent in situ hybridization revealed that the expression of the ASncmtRNAs was reestablished in 18Nco cells upon knockdown of E2 (Fig. 8C, 18 E2-ASO). Transfection with ASO-C had no effect (Fig. 8C, C-ASO). To determine whether the effect on the expression of the ASncmtRNAs was reversible, E2-ASO was removed, and the cells were further cultured in normal medium for an additional 24 h. Together with the recovery of E2 expression (Fig. 6B, Recovery), the ASncmtRNAs were again down-regulated (Fig. 8C, Recovery). The same results were obtained with HFK698 (Fig. 8D and supplemental Fig. 2A) and HF18 cells (keratinocytes immortalized with HPV-18) (see supplemental Fig. 3, B and C). Transfection of 18Nco or HFK698 cell lines with E2-ASO did not affect the expression of E6, E7, E1, and E5 (see supplemental Fig. 4, A and B).

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