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An Epstein-Barr virus-encoded microRNA targets PUMA to promote host cell survival.

Choy EY, Siu KL, Kok KH, Lung RW, Tsang CM, To KF, Kwong DL, Tsao SW, Jin DY - J. Exp. Med. (2008)

Bottom Line: In addition, PUMA was found to be significantly underexpressed in approximately 60% of human NPC tissues.Although expression of miR-BART5 rendered NPC and EBV-GC cells less sensitive to proapoptotic agents, apoptosis can be triggered by depleting miR-BART5 or inducing the expression of PUMA.Collectively, our findings suggest that EBV encodes an miRNA to facilitate the establishment of latent infection by promoting host cell survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, The University of Hong Kong, Pokfulam, Hong Kong.

ABSTRACT
Epstein-Barr virus (EBV) is a herpesvirus associated with nasopharyngeal carcinoma (NPC), gastric carcinoma (GC), and other malignancies. EBV is the first human virus found to express microRNAs (miRNAs), the functions of which remain largely unknown. We report on the regulation of a cellular protein named p53 up-regulated modulator of apoptosis (PUMA) by an EBV miRNA known as miR-BART5, which is abundantly expressed in NPC and EBV-GC cells. Modulation of PUMA expression by miR-BART5 and anti-miR-BART5 oligonucleotide was demonstrated in EBV-positive cells. In addition, PUMA was found to be significantly underexpressed in approximately 60% of human NPC tissues. Although expression of miR-BART5 rendered NPC and EBV-GC cells less sensitive to proapoptotic agents, apoptosis can be triggered by depleting miR-BART5 or inducing the expression of PUMA. Collectively, our findings suggest that EBV encodes an miRNA to facilitate the establishment of latent infection by promoting host cell survival.

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EBV confers resistance to apoptosis. (A) Sensitivity of NP69 and C666-1 cells to adriamycin. EBV− NP69 and EBV+ C666-1 cells were treated with indicated concentrations of adriamycin for 48 h. PUMA-β, PARP and α-tubulin proteins were analyzed by Western blotting. Shown at the bottom are relative expression levels of PUMA-β and percentages of cleaved PARP. (B) Sensitivity of AGS, AGS/BX1, HK1, and HK1/EBV cells to adriamycin. Cells were treated with 1.5 μg/ml (AGS and AGS/BX1 cells) or 1.2 μg/ml (HK1 and EBV/HK1) adriamycin for 48 h. Black lines indicate that intervening lanes have been spliced out.
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fig5: EBV confers resistance to apoptosis. (A) Sensitivity of NP69 and C666-1 cells to adriamycin. EBV− NP69 and EBV+ C666-1 cells were treated with indicated concentrations of adriamycin for 48 h. PUMA-β, PARP and α-tubulin proteins were analyzed by Western blotting. Shown at the bottom are relative expression levels of PUMA-β and percentages of cleaved PARP. (B) Sensitivity of AGS, AGS/BX1, HK1, and HK1/EBV cells to adriamycin. Cells were treated with 1.5 μg/ml (AGS and AGS/BX1 cells) or 1.2 μg/ml (HK1 and EBV/HK1) adriamycin for 48 h. Black lines indicate that intervening lanes have been spliced out.

Mentions: In the previous section, we presented several lines of evidence to support the regulation of PUMA by miR-BART5 in NPC cells. The importance of PUMA in mediating apoptosis prompted us to explore whether miR-BART5 inhibition of PUMA in NPC cells confers resistance to apoptosis. We compared the sensitivity of C666-1 and NP69 cells to adriamycin, a DNA-damaging agent that induces apoptosis. NP69 is an EBV− nasopharyngeal epithelial cell line, which is commonly used as a non-NPC counterpart of C666-1 (23, 30, 31). In line with our finding on the underexpression of PUMA in HK1/EBV cells (Fig. 3 A), the basal level of PUMA-β protein in C666-1 cells was significantly lower than in NP69 cells (Fig. 5 A). In addition, treatment with adriamycin induced PUMA-β expression in NP69 cells, but no significant induction was seen in C666-1 cells. Consistent with this, a majority of poly (ADP-ribose) polymerase (PARP) protein was found to be cleaved in NP69 cells treated with 0.8 μg/ml or 1.2 μg/ml adriamycin, but the percentages of cleaved PARP were much smaller in C666-1 cells treated with the same concentrations of adriamycin (Fig. 5 A). The cleavage of PARP by caspase 3 facilitates cellular disassembly and serves as a sensitive marker of apoptosis (32). Thus, our results suggested that C666-1 cells expressing less PUMA-β were more resistant to apoptosis. A similar observation was also made with AGS/BX1 and HK1/EBV cells, which were less prone to apoptosis than their respective parental cells (Fig. 5 B, lane 4 compared with lane 2 and lane 8 compared with lane 6). Thus, miR-BART5–expressing cells (C666-1, AGS/BX1 and HK1/EBV) are less susceptible to apoptosis-inducing drugs.


An Epstein-Barr virus-encoded microRNA targets PUMA to promote host cell survival.

Choy EY, Siu KL, Kok KH, Lung RW, Tsang CM, To KF, Kwong DL, Tsao SW, Jin DY - J. Exp. Med. (2008)

EBV confers resistance to apoptosis. (A) Sensitivity of NP69 and C666-1 cells to adriamycin. EBV− NP69 and EBV+ C666-1 cells were treated with indicated concentrations of adriamycin for 48 h. PUMA-β, PARP and α-tubulin proteins were analyzed by Western blotting. Shown at the bottom are relative expression levels of PUMA-β and percentages of cleaved PARP. (B) Sensitivity of AGS, AGS/BX1, HK1, and HK1/EBV cells to adriamycin. Cells were treated with 1.5 μg/ml (AGS and AGS/BX1 cells) or 1.2 μg/ml (HK1 and EBV/HK1) adriamycin for 48 h. Black lines indicate that intervening lanes have been spliced out.
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Related In: Results  -  Collection

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fig5: EBV confers resistance to apoptosis. (A) Sensitivity of NP69 and C666-1 cells to adriamycin. EBV− NP69 and EBV+ C666-1 cells were treated with indicated concentrations of adriamycin for 48 h. PUMA-β, PARP and α-tubulin proteins were analyzed by Western blotting. Shown at the bottom are relative expression levels of PUMA-β and percentages of cleaved PARP. (B) Sensitivity of AGS, AGS/BX1, HK1, and HK1/EBV cells to adriamycin. Cells were treated with 1.5 μg/ml (AGS and AGS/BX1 cells) or 1.2 μg/ml (HK1 and EBV/HK1) adriamycin for 48 h. Black lines indicate that intervening lanes have been spliced out.
Mentions: In the previous section, we presented several lines of evidence to support the regulation of PUMA by miR-BART5 in NPC cells. The importance of PUMA in mediating apoptosis prompted us to explore whether miR-BART5 inhibition of PUMA in NPC cells confers resistance to apoptosis. We compared the sensitivity of C666-1 and NP69 cells to adriamycin, a DNA-damaging agent that induces apoptosis. NP69 is an EBV− nasopharyngeal epithelial cell line, which is commonly used as a non-NPC counterpart of C666-1 (23, 30, 31). In line with our finding on the underexpression of PUMA in HK1/EBV cells (Fig. 3 A), the basal level of PUMA-β protein in C666-1 cells was significantly lower than in NP69 cells (Fig. 5 A). In addition, treatment with adriamycin induced PUMA-β expression in NP69 cells, but no significant induction was seen in C666-1 cells. Consistent with this, a majority of poly (ADP-ribose) polymerase (PARP) protein was found to be cleaved in NP69 cells treated with 0.8 μg/ml or 1.2 μg/ml adriamycin, but the percentages of cleaved PARP were much smaller in C666-1 cells treated with the same concentrations of adriamycin (Fig. 5 A). The cleavage of PARP by caspase 3 facilitates cellular disassembly and serves as a sensitive marker of apoptosis (32). Thus, our results suggested that C666-1 cells expressing less PUMA-β were more resistant to apoptosis. A similar observation was also made with AGS/BX1 and HK1/EBV cells, which were less prone to apoptosis than their respective parental cells (Fig. 5 B, lane 4 compared with lane 2 and lane 8 compared with lane 6). Thus, miR-BART5–expressing cells (C666-1, AGS/BX1 and HK1/EBV) are less susceptible to apoptosis-inducing drugs.

Bottom Line: In addition, PUMA was found to be significantly underexpressed in approximately 60% of human NPC tissues.Although expression of miR-BART5 rendered NPC and EBV-GC cells less sensitive to proapoptotic agents, apoptosis can be triggered by depleting miR-BART5 or inducing the expression of PUMA.Collectively, our findings suggest that EBV encodes an miRNA to facilitate the establishment of latent infection by promoting host cell survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, The University of Hong Kong, Pokfulam, Hong Kong.

ABSTRACT
Epstein-Barr virus (EBV) is a herpesvirus associated with nasopharyngeal carcinoma (NPC), gastric carcinoma (GC), and other malignancies. EBV is the first human virus found to express microRNAs (miRNAs), the functions of which remain largely unknown. We report on the regulation of a cellular protein named p53 up-regulated modulator of apoptosis (PUMA) by an EBV miRNA known as miR-BART5, which is abundantly expressed in NPC and EBV-GC cells. Modulation of PUMA expression by miR-BART5 and anti-miR-BART5 oligonucleotide was demonstrated in EBV-positive cells. In addition, PUMA was found to be significantly underexpressed in approximately 60% of human NPC tissues. Although expression of miR-BART5 rendered NPC and EBV-GC cells less sensitive to proapoptotic agents, apoptosis can be triggered by depleting miR-BART5 or inducing the expression of PUMA. Collectively, our findings suggest that EBV encodes an miRNA to facilitate the establishment of latent infection by promoting host cell survival.

Show MeSH
Related in: MedlinePlus