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PROGRESS REPORT (II YEAR)
“Role of Cellular microRNAs in EBV Induced DNA Damage Response”
Student: SHIVANGI YADAV (XXVIII ciclo)
Supervisor: Prof. Pankaj Trivedi
Prof. Alberto Faggioni Lab
Department of Experimental Medicine
La Sapienza University, Rome
BACKGROUND:
Epstein-Barr virus (EBV) was the first human tumor virus originally identified in cultured Burkitt
lymphoma (BL) cells by Epstein and Barr. It belongs to the γ-herpesvirus family. EBV is the
causative agent of infectious mononucleosis and B-cell lymphoproliferative disorders in
immunocompromised individuals.1
EBV transforms primary B-cells into indefinitely proliferating lymphoblasts. In order to keep them
proliferating the virus expresses a subset of genes, including 6 nuclear antigens (EBNA-1,-2,-3A,-3B,3C and –LP), 3 latent membrane proteins (LMP-1,-2A, and-2B) , 2 non-coding RNAs (EBER-1 and
2), BamHI-A rightward transcripts (BART) and 25 microRNAs.
Genomic instability and chromosomal aberrations are hallmarks of malignant transformation. It has
been reported that EBV carriage promotes genomic instability in BL2. To preserve genomic stability,
cells have elaborate response pathways called DNA damage response (DDR) pathways. Many
proteins involved in DDR are subjected to precise regulation at transcriptional and post-transcriptional
levels. MicroRNAs (miRNAs) are a class of small non-coding RNAs, that negatively regulate the
expression of DNA repair genes and thus debilitate the DNA repair phenomenon.3
RATIONALE:
We aim to study how EBV through its latent proteins EBNA2 and LMP1 contributes to genomic
instability by regulating miRNAs.
PRELIMINARY RESULTS:
Virus infection is perceived as a DNA damage by the target cell, as a result DDR pathway is
activated. During double stranded break (DSB) repair mechanism, H2AX - a key DNA repair protein
is phosphorylated by Ataxia telangiectasia mutated (ATM) to form γ-H2AX foci at nascent DSBs
sites. After H2AX is phosphorylated, γ-H2AX foci can be visualized as a marker of ongoing DNA
damage.
We studied γ-H2AX signals in U2932 EBNA2 transfected cells. Indeed there was a significant
increase in number of γ-H2AX foci in EBNA2 expressing U2932 cells. Since H2AX is
phosphorylated by ATM, we next analyzed the ATM expression by western blotting and found that
indeed in EBNA2 and LMP1 transfected U2932 cells, this protein is downregulated.
We then examined PTEN expression in LMP1 transfected U2932 lymphoma cells. PTEN is one of
the guardians of the genome. It was severely downregulated in U2932 LMP1 transfected cells.
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Next, we investigated if downregulation of ATM and PTEN by EBV latent proteins is related to
increase in cellular miRs. Therefore, we studied miR-26a and miR-335 expression by q-PCR. We
found miR-26a and miR-335 significantly upregulated in the presence of LMP1 and EBNA2 viral
proteins respectively. miR-26a and miR-335 has been implicated in DDR mechanism by targeting
ATM.4,5
In order to see if ATM and PTEN downregulation is due to miR-26a over-expression, we silenced
miR-26a with antisense LNA oligonucleotides. We did not see the reconstitution of ATM or PTEN
expression. This suggest that other microRNAs could be involved in ATM and PTEN regulation.
FUTURE PLANS:
1. Gamma-H2AX foci counting: To score number of γ-H2AX foci at different times intervals
post irradiation using flow cytometry in order to quantify the efficiency of DSB repair in
U2932 EBNA2 and LMP1 transfected lymphoma cells.
2. Micro-Nucleus Assay: Micro nuclei are good markers of chromosome instability and depicts
illegitimate repair.
3. To validate if miR-335 targets ATM in U2932 EBNA2 transfected clones.
4. To study other DNA repair protein kinase- Ataxia telangiectasia mutated and Rad-3 related
(ATR) and check downstream targets of ATM and ATR activation- CHK2, CHK1, and
claspin protein in U2932 EBNA2 and LMP1 cells.
5. BrdU Incorporation Assay: ATM regulates DNA damage-induced cell cycle checkpoints at
G1-S and intra-S phase . A hallmark of A-T cells is the failure to arrest DNA synthesis in Sphase following DNA damage and continuous incorporation of nucleotides into DNA despite
damage .
Overall, our preliminary data suggest that EBV can influence cellular miRNAs that unstabilize the
DDR response in infected cells. It is envisaged that such study could achieve more depth into novel
mechanisms of EBV mediated B-cell transformation which may provide novel miRNA based
therapeutic strategies for EBV associated B-cell lymphomas.
REFERENCES
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5.
Saha et al ; Clin Cancer Research (2011) , 17(10) : 3056-63.
Kamranvar et al ; Oncogene (2007 ) , 26(35) : 5115-23.
Wang et al ; Cell Cycle (2013) , 12(1) : 32-42.
Martin et al ; PLoS Genet (2013) , 9 (5) : e1003505.
Pin Guo et al ; Exp Cell Research ( 2014) : 320(2):200-8.
Shivangi Yadav
Prof. Pankaj Trivedi