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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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Expression of miR-BART5 in EBV-infected epithelial cells (A) Northern blot analysis of EBV miRNAs in NPC and EBV-GC cell lines. HK1, HK1/EBV, and C666-1 are NPC cell lines (lanes 1–3). AGS/BX1 is an EBV-GC cell line (lane 6). Untransfected HEK293 cells (293; lane 4), HEK293 cells stably expressing miR-BART5 (293/pmiR-BART5; lane 5), and AGS cells (lane 7) were included as controls. Expression signal of miR-BART5 in HK1/EBV cells was visible only after long exposure (inset). The 5S rRNA served as a loading control. Although lanes 1–5 in the middle were from the same exposure of one experiment, lanes 6 and 7 were from another experiment. (B) Northern blot analysis of miR-BART5 in human NPC tissue samples. Three normal nasopharyngeal biopsies (NP01, NP02, and NP03; lanes 2–4) and nine NPC tumor samples (NPC01 to NPC09; lanes 5–8 and 11–15) were analyzed. C666-1 and latency III B95-8 lymphoblastoid cells (lanes 1, 9, and 10) were included as positive and negative controls, respectively. U6 small nuclear RNA served as a loading control.
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fig2: Expression of miR-BART5 in EBV-infected epithelial cells (A) Northern blot analysis of EBV miRNAs in NPC and EBV-GC cell lines. HK1, HK1/EBV, and C666-1 are NPC cell lines (lanes 1–3). AGS/BX1 is an EBV-GC cell line (lane 6). Untransfected HEK293 cells (293; lane 4), HEK293 cells stably expressing miR-BART5 (293/pmiR-BART5; lane 5), and AGS cells (lane 7) were included as controls. Expression signal of miR-BART5 in HK1/EBV cells was visible only after long exposure (inset). The 5S rRNA served as a loading control. Although lanes 1–5 in the middle were from the same exposure of one experiment, lanes 6 and 7 were from another experiment. (B) Northern blot analysis of miR-BART5 in human NPC tissue samples. Three normal nasopharyngeal biopsies (NP01, NP02, and NP03; lanes 2–4) and nine NPC tumor samples (NPC01 to NPC09; lanes 5–8 and 11–15) were analyzed. C666-1 and latency III B95-8 lymphoblastoid cells (lanes 1, 9, and 10) were included as positive and negative controls, respectively. U6 small nuclear RNA served as a loading control.

Mentions: For miR-BART5 to fulfill a gene regulatory function, it has to be expressed in EBV-infected cells. Because miR-BARTs were thought to be expressed preferentially in EBV-infected epithelial cells (4, 7), we tested miR-BART5 expression in C666-1, an NPC cell line that constitutively harbors EBV (23). For comparison, we also examined HK1, another NPC cell line that does not carry EBV (24). In addition, the HK1/EBV cell line, to which EBV was reintroduced by coculture with infected Akata cells (25), was also included. Consistent with previous reports (4, 11), ample amount of miR-BART5 was found in C666-1 cells (Fig. 2 A, lane 3). The expression level of miR-BART5 in C666-1 cells was comparable to that in HEK293 cells stably transfected with miR-BART5 expression vector (Fig. 2 A, lane 5). As a control, miR-BART3-5p, another miRNA encoded by EBV, was also detected in C666-1 cells. Although no miR-BART5 was found in HK1 cells, the expression signal of miR-BART5 in HK1/EBV was visible after longer exposure of the blot (Fig. 2 A, lanes 1 and 2).


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)

Expression of miR-BART5 in EBV-infected epithelial cells (A) Northern blot analysis of EBV miRNAs in NPC and EBV-GC cell lines. HK1, HK1/EBV, and C666-1 are NPC cell lines (lanes 1–3). AGS/BX1 is an EBV-GC cell line (lane 6). Untransfected HEK293 cells (293; lane 4), HEK293 cells stably expressing miR-BART5 (293/pmiR-BART5; lane 5), and AGS cells (lane 7) were included as controls. Expression signal of miR-BART5 in HK1/EBV cells was visible only after long exposure (inset). The 5S rRNA served as a loading control. Although lanes 1–5 in the middle were from the same exposure of one experiment, lanes 6 and 7 were from another experiment. (B) Northern blot analysis of miR-BART5 in human NPC tissue samples. Three normal nasopharyngeal biopsies (NP01, NP02, and NP03; lanes 2–4) and nine NPC tumor samples (NPC01 to NPC09; lanes 5–8 and 11–15) were analyzed. C666-1 and latency III B95-8 lymphoblastoid cells (lanes 1, 9, and 10) were included as positive and negative controls, respectively. U6 small nuclear RNA served as a loading control.
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fig2: Expression of miR-BART5 in EBV-infected epithelial cells (A) Northern blot analysis of EBV miRNAs in NPC and EBV-GC cell lines. HK1, HK1/EBV, and C666-1 are NPC cell lines (lanes 1–3). AGS/BX1 is an EBV-GC cell line (lane 6). Untransfected HEK293 cells (293; lane 4), HEK293 cells stably expressing miR-BART5 (293/pmiR-BART5; lane 5), and AGS cells (lane 7) were included as controls. Expression signal of miR-BART5 in HK1/EBV cells was visible only after long exposure (inset). The 5S rRNA served as a loading control. Although lanes 1–5 in the middle were from the same exposure of one experiment, lanes 6 and 7 were from another experiment. (B) Northern blot analysis of miR-BART5 in human NPC tissue samples. Three normal nasopharyngeal biopsies (NP01, NP02, and NP03; lanes 2–4) and nine NPC tumor samples (NPC01 to NPC09; lanes 5–8 and 11–15) were analyzed. C666-1 and latency III B95-8 lymphoblastoid cells (lanes 1, 9, and 10) were included as positive and negative controls, respectively. U6 small nuclear RNA served as a loading control.
Mentions: For miR-BART5 to fulfill a gene regulatory function, it has to be expressed in EBV-infected cells. Because miR-BARTs were thought to be expressed preferentially in EBV-infected epithelial cells (4, 7), we tested miR-BART5 expression in C666-1, an NPC cell line that constitutively harbors EBV (23). For comparison, we also examined HK1, another NPC cell line that does not carry EBV (24). In addition, the HK1/EBV cell line, to which EBV was reintroduced by coculture with infected Akata cells (25), was also included. Consistent with previous reports (4, 11), ample amount of miR-BART5 was found in C666-1 cells (Fig. 2 A, lane 3). The expression level of miR-BART5 in C666-1 cells was comparable to that in HEK293 cells stably transfected with miR-BART5 expression vector (Fig. 2 A, lane 5). As a control, miR-BART3-5p, another miRNA encoded by EBV, was also detected in C666-1 cells. Although no miR-BART5 was found in HK1 cells, the expression signal of miR-BART5 in HK1/EBV was visible after longer exposure of the blot (Fig. 2 A, lanes 1 and 2).

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