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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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Modulation of PUMA expression by miR-BART5. (A) Down-regulation of PUMA-β protein expression by pre–miR-BART5. Pre–miR-BART5 and pre–miRNA-NC were introduced into HeLa and HK1 cells. Expression of PUMA-β and α-tubulin proteins was analyzed by Western blotting. Relative PUMA-β protein amounts are shown at the bottom. (B) Down-regulation of PUMA mRNA expression by miR-BART5. miR-BART3-5p or miR-BART5 expression vector was transfected into HEK293 cells. PUMA and GAPDH transcripts were analyzed by RT-PCR. (C and D) Up-regulation of PUMA expression by anti–miR-BART5 in C666-1 and AGS/BX1 cells. Anti–miR-BART5 oligonucleotide inhibitor was transfected into miR-BART5–expressing C666-1 cells. Endogenous PUMA-α and PUMA-β proteins were detected by Western blotting. An irrelevant anti-miRNA oligonucleotide (anti–miRNA-NC) was used as a negative control. Relative PUMA-β protein amounts are shown at the bottom.
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fig4: Modulation of PUMA expression by miR-BART5. (A) Down-regulation of PUMA-β protein expression by pre–miR-BART5. Pre–miR-BART5 and pre–miRNA-NC were introduced into HeLa and HK1 cells. Expression of PUMA-β and α-tubulin proteins was analyzed by Western blotting. Relative PUMA-β protein amounts are shown at the bottom. (B) Down-regulation of PUMA mRNA expression by miR-BART5. miR-BART3-5p or miR-BART5 expression vector was transfected into HEK293 cells. PUMA and GAPDH transcripts were analyzed by RT-PCR. (C and D) Up-regulation of PUMA expression by anti–miR-BART5 in C666-1 and AGS/BX1 cells. Anti–miR-BART5 oligonucleotide inhibitor was transfected into miR-BART5–expressing C666-1 cells. Endogenous PUMA-α and PUMA-β proteins were detected by Western blotting. An irrelevant anti-miRNA oligonucleotide (anti–miRNA-NC) was used as a negative control. Relative PUMA-β protein amounts are shown at the bottom.

Mentions: The above results provided the first experimental evidence for the regulation of PUMA expression by miR-BART5 of EBV. To test this idea more directly, we introduced pre–miR-BART5, the specificity of which had been verified in Fig. 1 D, into PUMA-expressing HeLa cells. When the miR-BART5 precursor RNA was expressed in the cells, substantial reduction in PUMA-β protein expression was observed, but this reduction was not seen in cells transfected with negative control precursor RNA (pre–miRNA-NC; Fig. 4 A, lanes 1 and 2). Likewise, a decline in PUMA-β expression was also observed when pre–miR-BART5 was overexpressed in HK1 cells (Fig. 4 A, lane 4 compared with lane 3). Because of difference in transfection efficiency (unpublished data), the magnitude of PUMA protein reduction in HK1 cells was not as great as in HeLa cells. To investigate the influence of miR-BART5 on the expression of PUMA mRNA, RT-PCR was performed with cells transfected with miR-BART5 expression vector. Although PUMA transcript was amply found in cells that expressed miR-BART3-5p, it was undetectable in miR-BART5-expressing cells (Fig. 4 B). Hence, regulation of PUMA protein expression by miR-BART5 was associated with decreased level of PUMA mRNA. This is consistent with the model in which mRNAs translationally repressed by miRNAs might be stored in the P-bodies and subsequently degraded (28, 29).


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)

Modulation of PUMA expression by miR-BART5. (A) Down-regulation of PUMA-β protein expression by pre–miR-BART5. Pre–miR-BART5 and pre–miRNA-NC were introduced into HeLa and HK1 cells. Expression of PUMA-β and α-tubulin proteins was analyzed by Western blotting. Relative PUMA-β protein amounts are shown at the bottom. (B) Down-regulation of PUMA mRNA expression by miR-BART5. miR-BART3-5p or miR-BART5 expression vector was transfected into HEK293 cells. PUMA and GAPDH transcripts were analyzed by RT-PCR. (C and D) Up-regulation of PUMA expression by anti–miR-BART5 in C666-1 and AGS/BX1 cells. Anti–miR-BART5 oligonucleotide inhibitor was transfected into miR-BART5–expressing C666-1 cells. Endogenous PUMA-α and PUMA-β proteins were detected by Western blotting. An irrelevant anti-miRNA oligonucleotide (anti–miRNA-NC) was used as a negative control. Relative PUMA-β protein amounts are shown at the bottom.
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fig4: Modulation of PUMA expression by miR-BART5. (A) Down-regulation of PUMA-β protein expression by pre–miR-BART5. Pre–miR-BART5 and pre–miRNA-NC were introduced into HeLa and HK1 cells. Expression of PUMA-β and α-tubulin proteins was analyzed by Western blotting. Relative PUMA-β protein amounts are shown at the bottom. (B) Down-regulation of PUMA mRNA expression by miR-BART5. miR-BART3-5p or miR-BART5 expression vector was transfected into HEK293 cells. PUMA and GAPDH transcripts were analyzed by RT-PCR. (C and D) Up-regulation of PUMA expression by anti–miR-BART5 in C666-1 and AGS/BX1 cells. Anti–miR-BART5 oligonucleotide inhibitor was transfected into miR-BART5–expressing C666-1 cells. Endogenous PUMA-α and PUMA-β proteins were detected by Western blotting. An irrelevant anti-miRNA oligonucleotide (anti–miRNA-NC) was used as a negative control. Relative PUMA-β protein amounts are shown at the bottom.
Mentions: The above results provided the first experimental evidence for the regulation of PUMA expression by miR-BART5 of EBV. To test this idea more directly, we introduced pre–miR-BART5, the specificity of which had been verified in Fig. 1 D, into PUMA-expressing HeLa cells. When the miR-BART5 precursor RNA was expressed in the cells, substantial reduction in PUMA-β protein expression was observed, but this reduction was not seen in cells transfected with negative control precursor RNA (pre–miRNA-NC; Fig. 4 A, lanes 1 and 2). Likewise, a decline in PUMA-β expression was also observed when pre–miR-BART5 was overexpressed in HK1 cells (Fig. 4 A, lane 4 compared with lane 3). Because of difference in transfection efficiency (unpublished data), the magnitude of PUMA protein reduction in HK1 cells was not as great as in HeLa cells. To investigate the influence of miR-BART5 on the expression of PUMA mRNA, RT-PCR was performed with cells transfected with miR-BART5 expression vector. Although PUMA transcript was amply found in cells that expressed miR-BART3-5p, it was undetectable in miR-BART5-expressing cells (Fig. 4 B). Hence, regulation of PUMA protein expression by miR-BART5 was associated with decreased level of PUMA mRNA. This is consistent with the model in which mRNAs translationally repressed by miRNAs might be stored in the P-bodies and subsequently degraded (28, 29).

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