An Epstein-Barr virus-encoded microRNA targets PUMA to promote host - - PowerPoint PPT Presentation

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An Epstein-Barr virus-encoded microRNA targets PUMA to promote host - - PowerPoint PPT Presentation

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1 An Epstein-Barr virus-encoded microRNA targets PUMA to promote host cell survival The Journal of Experimental Medicine 205(11): 2551-2560, 2008. Elizabeth Yee-Wai Choy, Kam-Leung Siu, Kin-Hang Kok, Raymond Wai-Ming Lung, Chi Man Tsang,

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

Elizabeth Yee-Wai Choy, Kam-Leung Siu, Kin-Hang Kok, Raymond Wai-Ming Lung, Chi Man Tsang, Ka-Fai To,

The Journal of Experimental Medicine 205(11): 2551-2560, 2008.

Student number: B94B02020 Speaker: Chien-Sin Chen Advisor: Li-Kwan Chang, Ph.D.

Raymond Wai-Ming Lung, Chi Man Tsang, Ka-Fai To, Dora Lai-Wan Kwong, Sai Wah Tsao, and Dong-Yan Jin

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Epstein-Barr virus (EBV)

Belongs to dsDNA γ-herpesvirus Capsid diameter: approx. 100 nm

Host:

Epithelial cells B lymphocytes

Chemical inducers (TPA, Na butyrate),

B lymphocytes

Life cycle:

Latent/lytic 2 stage

Pathogenicity:

90% people was infected Hodgkin’s disease, Burkitt’s lymphoma, and nasopharyngeal carcinoma (NPC)

(TPA, Na butyrate), Stress

Lytic phase Latent phase Latent protein, miRNA Tumor

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microRNA (miRNA)

miR-BART5 ˙Small regulatory RNAs ˙~22 nt in length ˙miRNA gene or intronic miRNA ˙Target 3’ UTR sequence ˙Target 3’ UTR sequence ˙Most target Imperfectly ˙Both cellular and viral miRNA are found 5’ UTR Gene 3’ UTR miRNA mRNA miR-BART5 PUMA

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Now-known microRNA of EBV

miR-BART5 Latent genes Lytic genes Previous known miRNA Newly identified miRNA Latent promoters Lytic promoters Intronic segments Exonic segments EBER: EBV-encoded small RNAs BART: Bam HI-A region rightward transcript

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p53 up-regulated modulator of apoptosis (PUMA)

˙Identified in 2001 ˙Bcl-2 family, “BH3 only” protein ˙PUMA-α, PUMA-β are 25, 20 kDa respectively p53 respectively ˙As a principal mediator of p53 apoptotic signal ˙p53 dependent and independent pathway are identified

Other than p53, PUMA is another hinge of apoptosis

apoptosis PUMA apoptosis

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The story is…

EBV miR-BART5

PUMA

  • immortalization
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Specific aim

p53 Normally… Cell death signal, Stress, … PUMA DNA PUMA mRNA PUMA Cell death EBV infection Latency miRNA Cell death signal, Stress, … p53 PUMA DNA PUMA mRNA Cell survival PUMA miR-BART5

? ?

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Framework

EBV infected NPC In silica prediction, confirming miR-BART5 PUMA Cell survival

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Luciferase reporter assay

Luciferase gene 3’ UTR GAPDH 4 binding sites

  • miR-BART5

Complementary sequence in PUMA 3’ UTR 4 binding sites Binding site mutant Perfectly match with miR-BART5 + in PUMA 3’ UTR (binding site) Luciferase gene

inhibition

PUMA 3’ UTR

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miR-BART5 binds 3’ UTR of PUMA

inhib Luciferase gene ibition

  • +

mutant

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PUMA is cellular target of miR-BART5

plasmid Luciferase gene Full length PUMA 3’ UTR Full length PUMA 3’ UTR inhibition Intact PUMA 3’ UTR

Anti-sense of PUMA 3’ UTR Chemical synthetic miRNA precursor

Full length PUMA 3’ UTR Luciferase gene Inverted PUMA 3’ UTR

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Framework

EBV infected NPC In silica prediction, confirming miR-BART5 PUMA Cell survival

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Assumption

PUMA EBV- infec

PUMA

EBV+ ection

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miR-BART5 was expressed in EBV infected NPC cells

NPC EBV- BART5- Detect directly (northern) HK1 EBV - + + -

  • EBV-

EBV+ BART5- BART5+ BART5+ C666-1 EBV-HK1

  • +
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PUMA was down-regulated in these EBV infected NPC cells

EBV - +

PUMA

PUMA

EBV+

N: noncancerous tissue (normal tissue); T: tumorous tissue (EBV infected NPC cells) HK1: EBV- NPC cell line

PUMA

EBV- EBV+

B

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EBV- infec

BART5 exogenous

PUMA

PUMA

EBV+ ction

Anti-BART5

PUMA

PUMA

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miR-BART5 negatively regulates PUMA expression

BART5 C666-1 is EBV+ cell line Endogenous miR-BART5 + +

PUMA

HeLa and HK1 are EBV- cell lines Endogenous miR-BART5 -

  • NC: negative control

PUMA

PUMA

Exogenous anti-miR-BART5 recovers PUMA-β and PUMA-α expression

PUMA

PUMA

Anti-BART5 Exogenous miR-BART5 suppresses PUMA-β expression

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Framework

EBV infected NPC In silica prediction, confirming miR-BART5 PUMA Cell survival

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PUMA

PUMA

EBV- EBV+

apoptosis

apoptosis

Chemical- triggered apoptosis

PARP Apoptotic molecular marker PARP be processed during apoptosis TUNEL assay be degraded during apoptosis termini labeling

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EBV+ Anti-BART5

EBV-encoded miR-BART5 represses PUMA to protect the host from death

PARP PARP

C666-1 is EBV+ cell line etoposide

EBV- (BART5-) EBV+ (BART5+)

PUMA

PUMA

siPUMA

PARP PARP

EBV- EBV+

PARP PARP

endogenous miR-BART5 + + +

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SUMMARY

p53 PUMA Cell death Luciferase gene

Full length PUMA 3’ UTR

miR-BART5

cell death rate

PUMA

PUMA

EBV- EBV+

Chemical- triggered apoptosis

High Low

EBV-encoded miR-BART5 protects host from apoptosis by repressing PUMA

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Thanks for your attention your attention and professor Chang’s advise

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Conclusion and significance

miR-BART5 miR-BART5

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Small RNA regulators: RNA

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Small RNA regulators: RNP

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siRISC

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Limitation of siRISC an example of TAR in HIV

2’-O-methyl oligonucleotide clamps

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miRISC

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Overview: biosynthesis and function

  • f miRNA
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Possible inhibition model of miRNA

Initiation complex inhibition complex

miRNA may inhibit initiation, elongation,

  • r induce mRNA degradation
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Molecular pathway of apoptosis

PUMA

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p53 network

Bcl-2 family Contains BH3 domain to neutralize BCL2 guard Cellular miRNA

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Death and survival balance

Apoptotic protein v.s. survival proteins

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Displacing Bak or Bax from its guards induces apoptosis

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BH3 (Bcl-2 homology 3) domain

  • “BH3 only” v.s. BID
  • BID is a pro-apoptotic Bcl-2 protein containing
  • nly the BH3 domain. In response to apoptotic

signaling, BID interacts with another Bcl-2 family protein, Bax (Bcl-2-associated X protein), leading to the insertion of Bax into organelle membranes, primarily the outer mitochondrial

  • membrane. Bax is believed to interact with,

and induce the opening of the mitochondrial voltage-dependent anion channel, VDAC. Alternatively, growing evidence suggest that Alternatively, growing evidence suggest that activated Bax and/or Bak (Bcl-2 homologous antagonist killer) form an oligomeric pore, MAC (the mitochondrial-induced apoptosis channel) in the outer membrane. This results in the release of cytochrome c and other pro- apoptotic factors from the mitochondria, often referred to as mitochondrial outer membrane permeabilization, leading to activation of

  • caspases. This defines BID as a direct

activator of Bax, a role common to some of the pro-apoptotic Bcl-2 proteins containing only the BH3 domain.

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Potential targets of miR-BART5 predicted by miRanda and RNAhybrid

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Expression of miR-BART5 in EBV-infected tissues and cells

Immortalized NP cell lines NPC xenografts Primary NPC cell lines Primary NPC cell lines Primary NPC cell lines EBV-infected Burkitt’s lymphoma cell lines NPC xenografts

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Comparison of PUMA 3’ UTR activity in HK1 and HK/EBV cells

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Influence of anti-miR-BART5 on PUMA 3’ UTR activity in C666-1 cells

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Inhibition of miR-BART5 results in results in apoptosis

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miR-BART5 and its target in different species

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The clinical sampling

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Down-regulate PUMA in mRNA level

HEK293 Plasmid co-transfection Plasmid co-transfection

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Etoposide and siPUMA activity test