Supplementary Materials and Methods:
Patients
All human tissue samples used in this work were collected through the archive of the CNIO
Tumour Bank with the collaboration of the member hospitals of the tumour bank network. A first
series of 36 cases of DLBCL (frozen tissues) was used to analyse miRNA expression and a second
series of 149 DLBCL samples (formalin-fixed paraffin embedded tissues) including 36 plasmablastic
lymphoma, 2 primary effusion lymphoma and 2 EBV-positive DLBCL of the elderly, was used for
immunohistochemical analysis and for validating miRNA expression. All the studies were carried
out under the supervision of the institutional review board of the Instituto de Salud Carlos III,
Madrid, Spain.
Cell lines
DB, SU-DHL-4 and SU-DHL-6 are EBV- negative DLBCL derived cell lines of GC-type (1). Farage and
DoHH-2 were chose because they were EBV positive. BL derived cell lines Akata, Namalwa and Raji
are EBV-positive and Ramos is EBV-negative.
All cell lines were maintained at 37ºC in a humidified 5% CO2 incubator and cultured in RPMI-1640
medium supplemented with 1% L-Glutamine and either 10% (Akata, DoHH-2, Farage, Namalwa,
Raji, Ramos, SU-DHL-4 and SU-DHL-6) or 20% (DB) fetal bovine serum (Invitrogen).
HEK293T cell line was grown on DMEM medium supplemented with 1% L-Glutamine and 10% fetal
bovine serum (Invitrogen).
Immunohistochemistry
For BCL6 staining we used a mouse anti-BCL6 antibody produced at the Spanish National Cancer
Research Centre (clone GI 191E/A8). We used a mouse monoclonal anti-LMP1 antibody for
1
detection of LMP1 in FFPE tissues (DAKO, clone CS.1-4). EBV was detected by ISH using an EBVencoded RNA (EBER) specific probe (Bond ISH EBER Probe; Vision BioSystem, Germany) in human
samples
and
by
EBV-specific
PCR
in
cell
lines.
PCR
primers
used
were
5’-
CCAGAGGTAAGTGGACTTTA-3’ and 5’-GACCGGTGCCTTCTTAGGAG-3’.
MicroRNA microarrays
MiRNA expression profiling by miRNA microarrays in patients was performed as previously
described (2). Briefly, data from Agilent 8x15K human miRNA one-color microarrays (Agilent
Technologies Inc, Santa Clara, CA, USA) was normalized to the 75th percentile with GeneSpring
software (Agilent). MicroRNAs with more than two-fold change between EBV-positive and EBVnegative cases were subjected to t-test with correction for multi-test hypothesis. Corrected pvalues below 0.05 were considered significant. Data for each miRNA have been standardized (i.e.
mean subtracted and divided by standard deviation) for representation purposes.
MiRNA target prediction
MiRNA targets were predicted with the miRanda v3.0 miRNA Target Scanning Algorithm
(http://cbio.mskcc.org/microrna_data/miRanda-sept2008.tar.gz). Additional analyses were done
using
the
following
tools:
TargetScan
(http://www.targetscan.org/),
PITA
(http://genie.weizmann.ac.il/pubs/mir07/mir07_data.html) and miRgate, a platform developed by
CNIO’s Bioinformatic Unit that searches perfect matches between the miRNA seed region and the
3’ UTR of the target gene (unpublished).
2
Reporter assays
Luciferase assays were performed by cloning the BCL6 3’-UTR into the pGL3-Control vector
(Promega, Madison, WI) using the XbaI site. MiRNA control or miRNA precursor molecules
(Ambion, Austin, TX) were co-transfected in HEK293T cells at 50 nM with 400 ng of the reporter
vector by calcium phosphate precipitation. Luciferase activity was measured 48 hours after
transfection with the Dual-Glo Luciferase Assay System (Promega).
Transfection of miRNA mimics and miRNA inhibitors
For protein analysis, lymphoma cell lines were transfected with either 50 nM of miRNA scrambled
control or miRNA mimic or 200 nM of miRNA inhibitor scrambled control or miRNA inhibitor
(Ambion) using a microporator (NanoEnTek, Hopkinton, MA). BCL6 was detected using a mouse
anti-BCL6 antibody (Clone PG-B6p, DAKO, Denmark).
Real Time PCR
Levels of miRNAs were detected by RT-PCR using TaqMan MicroRNA Assays (Applied Biosystems
Foster City, CA, USA). RNU19 was used as endogenous control for cell lines and hsa-let-7a for
patient samples. Expression data are represented as 2-ΔCt. Differences in the expression of the four
viral miRNAs between EBV-positive and EBV-negative patients were analyzed using a t-test.
Results were plotted using the GraphPad Prism software (GraphPad Software, Inc, v. 5, La Jolla,
CA, USA). Average Ct values of endogenous controls for EBV-positive and EBV-negative samples
were 27.44 and 26.03 respectively.
3
References
1.
Davis RE, Brown KD, Siebenlist U, Staudt LM. Constitutive nuclear factor kappaB activity is
required for survival of activated B cell-like diffuse large B cell lymphoma cells. J Exp Med
2001 Dec 17; 194(12): 1861-1874.
2.
Di Lisio L, Gomez-Lopez G, Sanchez-Beato M, Gomez-Abad C, Rodriguez ME, Villuendas R,
et al. Mantle cell lymphoma: transcriptional regulation by microRNAs. Leukemia Jul; 24(7):
1335-1342.
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