Targeting of DICE1 tumor suppressor by Epstein–Barrvirus-encoded miR-BART3* microRNA innasopharyngeal carcinoma

Ting Lei1,2*, Kit-San Yuen2*, Rui Xu3, Sai Wah Tsao4, Honglin Chen5, Mengfeng Li6, Kin-Hang Kok2 and Dong-Yan Jin2

1 Department of Pathology, School of Medicine, Xi’an Jiaotong University, Xi’an, China2 Department of Biochemistry, The University of Hong Kong, Pokfulam, Hong Kong SAR, China3 Department of Ear, Nose and Throat, The First Affiliated Hospital, Sun Yatsen University, Guangzhou, China4 Department of Anatomy, The University of Hong Kong, Pokfulam, Hong Kong SAR, China5 Department of Microbiology, The University of Hong Kong, Pokfulam, Hong Kong SAR, China6 Department of Microbiology, Zhongshan School of Medicine, Sun Yatsen University, Guangzhou, China

Latent infection with Epstein–Barr virus (EBV) is associated with several types of malignancies including nasopharyngeal

carcinoma (NPC), which is particularly more prevalent in Southern China. EBV expresses at least 44 mature microRNAs

(miRNAs) to modulate the activity of viral and cellular RNAs, but the targets of these EBV-encoded miRNAs in NPC are not well

understood. In this report, we characterized DICE1 tumor suppressor to be a cellular target of EBV miR-BART3* miRNA. miR-

BART3* was abundantly expressed in NPC cells. The target site of miR-BART3* located in the 30-untranslated region of DICE1

transcript was identified and characterized. Enforced expression of miR-BART3* or its precursor pre-miR-BART3 led to down-

regulation of endogenous DICE1 expression. Inhibition of endogenous miR-BART3* in NPC cells with anti-miR-BART3* oligonu-

cleotide inhibitor resulted in increased expression of DICE1 protein. On the contrary, expression of miR-BART3* overcame the

growth suppressive activity of DICE1 and stimulated cell proliferation. Consistent with its tumor suppressive function, DICE1

was underexpressed in EBV-expressing NPC tumor tissues. Taken together, our findings suggest that EBV encoded miR-BART3*

miRNA targets DICE1 tumor suppressor to promote cellular growth and transformation in NPC.

MicroRNAs (miRNAs) have emerged as critical regulators ofgene expression in all multicellular organisms and someviruses.1,2 The current release of the miRNA registry lists 23viruses which encode more than 240 miRNA hairpins.Increasing evidence suggests that these viral miRNAs play piv-otal roles in viral infection, persistence and pathogenesis.1–4

Epstein–Barr virus (EBV) successfully and persistentlyinfects more than 90% of the world’s adult population. EBVinfection is causally associated with multiple types of human

cancers of both lymphoid and epithelial origin, includingBurkitt’s lymphoma, Hodgkin’s disease, extranodal nasal nat-ural killer/T cell lymphoma, post-transplant lymphoid prolif-eration disease, nasopharyngeal carcinoma (NPC) and asubset of gastric cancer. NPC is particularly more prevalentin Southern China including Hong Kong, where it accountsfor approximately 20% of all adult cancers. The link betweenEBV and NPC was corroborated by the detection of EBVDNA in almost all cases of undifferentiated NPC, taken fromhigh, intermediate and low incidence areas.5,6 But the exactroles of EBV and its oncoproteins in NPC tumorigenesisremain poorly understood.7

EBV is the first human virus known to encode miRNAswithin the BHRF1 and BART regions of its genome.8 At least25 EBV miRNA hairpins and 44 mature miRNAs have beenidentified. Among them miR-BARTs are particularly abun-dant in epithelial carcinoma including NPC.9–11 The biologi-cal functions of some EBV-encoded miRNAs in post-transcriptional regulation of some viral and cellular RNAshave been characterized.12–14 Particularly, the roles of BHRF1miRNAs in cell cycle progression, cell survival and oncogenictransformation have been documented in B cells usingBHRF1-disrupted recombinant viruses.15,16 Global analyses ofviral and cellular targets of EBV miRNAs in B cells suggestedthat EBV miRNAs target hundreds of oncogenic and

Key words: nasopharyngeal carcinoma, Epstein–Barr virus (EBV),

DICE1 tumor suppressor, EBV-encoded microRNAs

Grant sponsor: National Natural Science Foundation of China;

Grand number: 31070146; Grant sponsor: Hong Kong Research

Grants Council; Grand numbers: HKU7668/09M; HKU1/CRF/11G;

AoE/M-06/08; Grant sponsor: Research Fund for the Control of

Infectious Diseases of Hong Kong; Grand numbers: 11100602;

12110962; Grant sponsor: S.K. Yee Medical Research Fund (2011);

Grant sponsor: Hong Kong Research Grants Council;

DOI: 10.1002/ijc.28007

History: Received 31 July 2012; Accepted 13 Dec 2012; Online 27

Dec 2012

Correspondence to: Dong-Yan Jin, Department of Biochemistry, The

University of Hong Kong, 3/F Laboratory Block, Faculty of Medicine

Building, 21 Sassoon Road, Pokfulam, Hong Kong, Tel.:

[1852-2819-9491], Fax: [1852-2855-1254], E-mail: dyjin@hku.hk

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International Journal of Cancer

IJC

apoptotic genes.17–19 Microarray analysis of RNA expressionin an EBV-infected gastric carcinoma cell line also demon-strated critical involvement of miR-BARTs in cellulartransformation.20

A couple of viral and cellular targets of EBV miRNAs inlymphoblastoid and epithelial cells have been validated exper-imentally. In particular, on the basis of perfect complemen-tarity to the 30 untranslated region (UTR) of BALF5 mRNA,miR-BART2 was found to target BALF5 mRNA for degrada-tion, thereby silencing the expression of BALF5 viral DNApolymerase and inhibiting lytic replication.21 Several miR-BARTs recognizing the 30 UTR of the LMP1 transcript wasshown to negatively regulate LMP1 oncoprotein expression,leading to attenuation of LMP1-induced NF-jB signaling andalleviation of the sensitivity of LMP1-expressing NPC cells tocisplatin.22 EBV LMP2A, a highly immunogenic viral antigen,is the target of newly identified miR-BART22. Down-regula-tion of LMP2A might facilitate the evasion of EBV-infectedcells from host immune surveillance.23 Additionally, onerecent study implicated miR-BHRF1-2 in the cleavage ofBHRF1 RNA in the cytoplasm.24 While EBV miRNAs areimportant regulators of viral gene expression, they also targetcellular transcripts to promote viral persistence and oncogen-esis. An inverse correlation between expression levels ofmiR-BHRF1-3 and CXCL-11, an interferon-induced T-cellattracting chemokine, suggested an immuno-modulatoryfunction of the EBV miRNA in B cells.25 We have previouslyidentified p53 upregulated modulator of apoptosis (PUMA)to be a cellular target of miR-BART5. Modulation of PUMAexpression by miR-BART5 had antiapoptotic effect in EBV1

cells and NPC tissues.10 miR-BART2-5p was found to becapable of directly targeting MICB mRNA to evade recogni-tion and subsequent elimination by natural killer cells.26

Other cellular targets of EBV miRNAs include Dicer, Bim,TOMM22, p53, NLRP3 and IPO7.17,27–31 Nevertheless, thereis scarce experimental evidence for cellular targets of EBVmiRNAs, particularly in NPC cells. The roles of most EBVmiRNAs in EBV persistence and NPC tumorigenesis remainelusive.

In this study, we identified and characterized DICE1/INTS6 to be a cellular target of EBV miR-BART3*, also knownas miR-BART3-5p. DICE1 is a tumor suppressor genefrequently inactivated genetically and epigenetically in humancancers.32,33 Suppression of DICE1 expression by EBV miR-BART3* might be implicated in NPC tumorigenesis.

Material and MethodsTarget prediction

miRanda and RNAhybrid were used to predict the potentialtargets of EBV miRNAs as previously described.10 The top 50potential targets of miR-BART3* predicted by miRanda werethen reanalyzed using RNAhybrid. Potential targets with ahigh minimum free energy and perfect complementary sitesto the miR-BART3* seed region were shortlisted for func-tional screening with luciferase reporter assays. DICE1 wasthe top prediction for miR-BART3* and the targeting wasvalidated by reporter assay.

Cell culture and reporter assay

HeLa and HEK293 cells, human EBV1 GC cell line AGS/BX1 and its parental line AGS were maintained in Dulbecco’smodified Eagle medium (DMEM) supplemented with 10% fe-tal bovine serum (FBS) and antibiotics; human NPC cell linesC666-1, HK1 and HK1/EBV were cultured in RPMI 1640medium supplemented with 10% FBS and antibiotics. Dualluciferase reporter assay was performed, as previouslydescribed,34 using a reagent kit supplied by Promega (Madi-son, WI). The readouts of luciferase activity were obtainedusing an LB 96 V microplate luminometer (EG&G Berthold,Quebec, Canada).

Plasmid construction

miR-BART expression vectors (pSuper-BARTs) wereconstructed by inserting cDNAs for primary miR-BART(pri-miR-BART) transcripts into pSuper vector (provided byR. Agami, Netherlands Cancer Institute, Amsterdam, TheNetherlands). miRNA expression was driven by H1 promoter.Pri-miR-BART cDNAs were PCR-amplified using genomicDNA of C666-1 cells as template. The long pri-miR-BARTtranscripts expressed from these vectors are processed byDrosha in the nucleus to generate precursor miR-BARTs(pre-miR-BARTs) of �70 nucleotides, which are furthercleaved by Dicer in the cytoplasm to produce mature miR-BARTs of �21 nucleotides.1,2 Firefly luciferase reporter plas-mids were constructed by inserting different sequences intothe XbaI site of pGL3-Control, which is located immediatelyafter the stop codon of the luciferase gene (Promega). pGL3-DICE1.UTR contains four copies of the miR-BART3* targetsite found in 30 UTR of DICE1. As controls, pGL3-GAPD-H.UTR contains four copies of irrelevant sequences derived

What’s new?

While there is a known link between Epstein-Barr virus (EBV) and nasopharyngeal carcinoma (NPC), the roles of EBV and its

oncoproteins in NPC tumorigenesis remain poorly understood. Here the authors looked at EBV-encoded microRNAs and their

potential targets in NPC. They found that DICE1 tumor suppressor was a cellular target of an EBV miRNA abundantly expressed

in transfected and infected cells as well as in NPC tumor samples. This is the first report of an EBV microRNA targeting a tu-

mor suppressor in NPC. The findings provide a new mechanism in NPC tumorigenesis and reveal potential new targets for

therapy.

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from GAPDH, pGL3-BART3.UTR carries four repeats ofsequences perfectly complementary to miR-BART3*, whereaspGL3-revBART3 contains four reverse repeats of sequence per-fectly complementary to miR-BART3*. pGL3-FL-DICE1.UTRcontains a single copy of the entire 30 UTR of DICE1. pGL3-rFL-DICE1.UTR contains the reverse sequence of the entireDICE1 30UTR. pGL3-mFL-DICE1.UTR was derived frompGL3-FL-DICE1.UTR, with the change of three nucleotidesintroduced using PCR. DICE1 cDNA IRATp970C0471D waspurchased from imaGenes (Berlin, Germany), and was sub-cloned into expression vectors pcDNA3.1 and pEGFP-C1.

RT-PCR and Northern blotting

Total RNA was extracted from cultured cells and tissues usingTrizol reagent (Invitrogen, Carlsbad, CA) according to the man-ufacturer’s protocol, and 50 mg of total RNA was subjected toNorthern blotting as detailed elsewhere.34 Chemical crosslinkingwith 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC)was used to improve the sensitivity of small RNA detection.35

cDNA was also synthesized from Trizol-extracted RNA withThermoScript reagents (Invitrogen). As for mature miRNAs,reverse transcription (RT) of cDNA was performed with totalRNA samples using a small RNA-specific, stem-loop RT primerand TaqMan MicroRNA Reverse Transcription reagents(Applied Biosystems, Carlsbad, CA). Semi-quantitative PCR wascarried out using the following thermal cycling profile: 94 �C for

5 min, 35 cycles of amplification (94�C for 30 sec, 62�C for 1min), and 72�C for 7 min.

Tumor and non-tumorous samples

Nasopharyngeal biopsy samples voluntarily donated by 22individual patients were obtained from the Department of On-cology at the First Affiliated Hospital, Sun Yatsen University,Guangzhou, China. Informed consent and approval from theInstitutional Research Ethics Committee were obtained. Theage of these patients ranged from 26 to 76 years old. Amongthem 11 patients were diagnosed with primary NPC and 11patients were diagnosed with chronic nasopharyngeal inflam-mation. All NPC samples were histologically evaluated to beundifferentiated carcinoma. All samples were snap frozen inliquid nitrogen and stored at 280�C until lysed with RIPAbuffer for protein separation or with Trizol for RNA extraction.Each sample was separated into two parts, one part was testedfor DICE1 expression with Western blotting, and the other wastested for miR-BART3* expression using RT-PCR.

Western blotting

Western blot analysis was performed as described.36 In brief,RIPA buffer was used to lyse the cells. The primary antibod-ies included mouse monoclonal (H-6) and goat polyclonal(V-20) against DICE1 (Santa Cruz Biotechnology, SantaCruz, CA), mouse monoclonal against green fluorescent pro-tein (GFP; Santa Cruz Biotechnology), as well as mouse

Figure 1. DICE1 targeting by EBV miR-BART3*. (a) A miR-BART3* target site in the 30 UTR of DICE1. Shown at the top is the sequence in the

30 UTR of DICE1 which base pairs with miR-BART3*. Plasmid pGL3-DICE1.UTR has four copies of miR-BART3* target sites inserted into the 30

UTR of luciferase gene. pGL3-BART3 contains target sequences that match perfectly with miR-BART3*, whereas pGL3-revBART3 has the

reverse sequence. pGL3-GAPDH.UTR harbors irrelevant GAPDH sequences. HEK293 cells were co-transfected with an empty pSuper expres-

sion vector (vector), pSuper-BART3 (BART3) or pSuper-BART5 (BART5). Relative luciferase activity was obtained by normalizing the readings

of the firefly luciferase (Fluc) activity recovered from the pGL3 vector with those of the Renilla luciferase activity from pRL-CMV. Results rep-

resent mean6SD from three independent experiments. #The difference between the two groups is statistically very significant (p<0.001 by

Student’s t test). Similar results were also obtained in HeLa cells. (b) Functionality of miR-BART3* target site in DICE1 30 UTR. A single copy

of the entire DICE1 30 UTR sequence was inserted into the 30 UTR of luciferase gene to generate pGL3-FL-DICE1.UTR. Plasmid pGL3-rFL-

DICE1.UTR contains a reversed single copy of the DICE1 30 UTR sequence. pGL3-mFL-DICE1.UTR has a single copy of mutated DICE1 30 UTR

sequence with the substitution of three nucleotides as indicated above. Experiments were done in HEK293 cells. #The difference between

the two groups is statistically very significant (p<0.001 by Student’s t test).

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monoclonal antibody against b-actin (Sigma-Aldrich, St.Louis, MO). These antibodies were detected with horseradishperoxidase-conjugated secondary antibodies and ECLreagents (GE Healthcare, Piscataway, NJ).

Growth suppression assays

Growth suppression assays were carried out as described.37 Forb-galactosidase (b-gal) staining experiments, HeLa cells werecultured in 6-well plate, and co-transfected at Day 0 with pSV-bgal (0.2 mg), pcDNA3.1-DICE1 or pcDNA3.1-mDICE1 (2mg) and pSuper-BART3. On Days 1, 2 and 3 post-transfection,cells were stained with X-gal for b-gal activity as follows: thecells were washed three times with phosphate-buffered saline(PBS) and then fixed by adding 0.2% glutaraldehyde in PBS for5 min at room temperature. Cells were washed again three timeswith PBS and then X-gal/PBS was added. b-gal staining was per-formed at 37�C in a humidified atmosphere at 5% CO2 for atleast 2 hr. Cell growth was calculated by counting the number ofblue cell clusters in ten different fields under microscope.

For BrdU staining experiment, HeLa cells were cultured oncover-slides in 6-well plate. Cells were transfected with eitherpEGFP-C1 or pEGFP-DICE1 for 16 hr. Cells were then incor-porated with BrdU (10 mM) for 6 hr, washed three times withPBS and fixed with ice-cold 70% ethanol. DNA was denaturedwith 2 N HCl for 30 min at 37�C, and then neutralized with0.1 M sodium borate (pH 8.5). Cells were subsequently stainedwith rabbit anti-GFP and mouse anti-BrdU. A Texas red-con-jugated secondary antibody against rabbit IgG and a fluores-cein isothiocyanate-conjugated secondary against mouse IgGwere used to visualize the GFP and BrdU signals.

ResultsDICE1 is a cellular target of EBV miR-BART3*

As previously described,10 we used miRanda and RNAhybridprograms to predict the cellular targets of EBV miR-BARTs.DICE1 was chosen for further validation because it was notonly a predicted target of miR-BART3* on the top of the list,but was also one of the only three targets that were con-firmed by luciferase reporter assay. The predicted target siteof miR-BART3* in the 30 UTR of DICE1 exhibited goodcomplementarity to miR-BART3* (Fig. 1a). To validate therecognition of this site by miR-BART3*, additional luciferaseassays were performed with a set of reporter constructs. Inplasmid pGL3-DICE1.UTR, four copies of a DICE1 30 UTRfragment containing the predicted miR-BART3* target sitewere inserted into the 30 UTR of the firefly luciferase gene.Cotransfection of this plasmid and a pri-miR-BART3 expres-sion vector named pSuper-BART3 into HEK293 and HeLacells resulted in a significant reduction of luciferase activity,which was not seen when pri-miR-BART5 was expressed(Fig. 1a). As positive and negative controls, the perfectlymatched pre-miR-BART3 and its reverse sequence were alsocloned into the 30 UTR of luciferase gene, giving rise toreporter plasmids pGL3-BART3 and pGL3-revBART3.Expression of pri-miR-BART3 dampened the activity of

pGL3-BART3 but not pGL3-revBART3. Furthermore, noinhibition was observed when luciferase expression was regu-lated by the control glyceraldehyde 3-phosphate dehydrogen-ase (GAPDH) sequence in the 30 UTR (Fig. 1a). Collectively,these data supported that the miR-BART3* target sequencein DICE1 30 UTR was functional and specifically recognized.

To verify that the miR-BART3* target site in DICE1 30

UTR operates in a more physiological context, we alsoinserted a single copy of the full-length 30 UTR of DICE1downstream of the luciferase gene and repeated the reporterassays. Indeed, the luciferase activity was significantlydecreased in cells expressing miR-BART3*. In contrast, whenthe reversed or mutated copy of the full-length 30 UTR ofDICE1 was cloned into the same site, the luciferase activitywas not affected by miR-BART3* (Fig. 1b). Thus, DICE1expression is likely modulated by miR-BART3*.

Modulation of DICE1 expression by miR-BART3*

We next investigated whether miR-BART3* might influencethe expression of DICE1 protein. The full-length DICE1

Figure 2. Down-regulation of DICE1 protein expression by miR-

BART3*. HEK293 cells were co-transfected with the indicated com-

binations of plasmids (a-c). The predicted miR-BART3* target site

was mutated in mDICE1 (c). Cells were collected for Western blot

analysis with mouse monoclonal anti-DICE1 at 48 hr post-transfec-

tion. Indicated at the bottom of the panels are relative amounts of

DICE1 normalized to b-actin as determined by densitometry.

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cDNA containing a complete 30 UTR was cloned into pEGFP-C1 so that an EGFP-DICE1 fusion protein could be expressedand readily detected. We co-transfected pEGFP-DICE1 andpSuper-BART3 into HEK293 cells and found that the expres-sion of EGFP-DICE1 was progressively diminished as the doseof pSuper-BART3 was elevated (Fig. 2a, lanes 3–6 comparedto lane 2). In contrast, expression of pri-miR-BART5 did notinfluence the expression of EGFP-DICE1 protein (Fig. 2b,lanes 2–6). Furthermore, the expression of EGFP-DICE1 frompEGFP-mDICE1, in which the predicted miR-BART3* targetsite in the 30 UTR was mutated, was uninhibitable by miR-BART3* (Fig. 2c, lanes 2–6). Hence, miR-BART3* is capableof modulating the expression of DICE1 protein.

We went on to explore whether miR-BART3* might havean impact on the expression of endogenous DICE1 mRNAand protein. HEK293 cells stably expressing miR-BART3*and miR-BART5 (293B3 and 293B5) were established andmiRNA expression was examined by Northern blotting.Mature miR-BART3* was found to be abundantly expressedin 293B3 cells (Fig. 3a, lane 4). It was also detectable inEBV1 NPC cell lines C666-1 and HK1/EBV, but absent inHK1 cells (Fig. 3a, lanes 2 and 3 compared to lane 1). C666-1 is an NPC cell line that constitutively harbors EBV.38 HK1is another NPC cell line that does not carry EBV,39 whereasEBV was reintroduced into the HK1/EBV cell line throughco-culture with EBV-infected Akata cells. Our results on theexpression pattern of miR-BART3* are generally consistentwith the notion that miR-BARTs were preferentiallyexpressed in EBV-infected epithelial cells.9–11

Notably, the expression of endogenous DICE1 protein in293B3 stable cells was reduced compared to the control and293B5 cells (Fig. 3b, lane 4 compared to lanes 3 and 5). Con-sistent with this, the steady-state amounts of DICE1 proteinin HK1/EBV and C666-1 cells were remarkably lower thanthe level in HK1 cells (Fig. 3b, lanes 2 and 6 compared tolane 1). In addition, DICE1 mRNA level was also down-regu-lated in miR-BART3*-expressing 293B3 and HK1/EBV cells(Fig. 3c, lane 2 compared to 1 and 3, and lane 5 compared tolane 4). On the other hand, elevation of DICE1 protein wasobserved in 293B3 and C666-1 cells when miR-BART3* wasspecifically inhibited with an anti-miR-BART3* oligonucleo-tide (Fig. 4a, lane 4 compared to lane 2; Fig. 4b, lane 2 com-pared to lanes 1 and 3). Hence, miR-BART3* expressed in293B3 and C666-1 cells might exert a suppressive role on theexpression of DICE1.

miR-BART3* promotes cell proliferation

Inactivation of DICE1 tumor suppressor in human cancers iscommon32,33 and enforced expression of DICE1 inhibits col-ony formation of human tumor cells.40 To investigate howmiR-BART3* might affect tumor suppressor function ofDICE1, we employed a well-established cell growth assaybased on X-gal staining.37,41 HeLa cells were transiently co-transfected with pSV-bgal plus 10-fold excess amount ofpcDNA3.1-DICE1/mDICE1 and pSuper-BART3. Differencesin blue cell numbers among the groups were evident on Days2 and 3 (Fig. 5a). The numbers of blue cells in the groupsexpressing DICE1 and mDICE1 were significantly decreased

Figure 3. Suppression of endogenous DICE1 expression by miR-BART3*. (a) Northern blot analysis of miR-BART3* (mB3*) expression in

C666-1, HK1, HK1/EBV and 293B3 cells. pri-miR-BART3 was stably expressed in 293B3 cells. Equal loading was verified by staining of 5S

RNA with ethidium bromide. (b, c) Expression of endogenous DICE1 was suppressed by miR-BART3*. pri-miR-BART5 was stably expressed in

293B5 cells. Indicated at the bottom of the panels are relative ratios of DICE1 normalized to b-actin protein or GAPDH transcript as deter-

mined by densitometry.

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compared with that of the control group receiving pcDNA3.1empty vector. Since the mutations in mDICE1 were intro-duced to the 30 UTR of DICE1, the basal expression of afunctional DICE1 protein was unaffected (Fig. 2c). Uponexpression of miR-BART3*, the decrease of blue cell numberinduced by DICE1 was reversed in cells receiving pSuper-BART3. However, this reversal was not seen in cells trans-fected with pcDNA3.1-mDICE1 expressing DICE1 in a miR-BART3*-uninhibitable manner (Fig. 5b). In keeping with this,similar results were obtained in our analysis of BrdU incor-poration in DICE1 expressing cells (data not shown). Thus,miR-BART3* was able to stimulate cell proliferation by coun-teracting the growth suppressive function of DICE1.

DICE1 and miR-BART3* expression in NPC tumor samples

EBV is consistently detected in NPC samples5,6 and EBV-encoded miR-BARTs were also abundantly expressed.9–11 If

DICE1 is indeed targeted by miR-BART3*, its expressionshould be compromised in NPC tumor samples. To test thishypothesis and to get a glimpse of the status of DICE1 inNPC, we determined the expression levels of DICE1 proteinand miR-BART3* miRNA in 22 nasopharyngeal biopsy sam-ples collected from 11 patients diagnosed with primary NPCand 11 non-NPC subjects who had chronic nasopharyngealinflammation. Western blot and RT-PCR were performed inparallel for each sample. In 1 out of 11 NPC tumor samples(T5) and 6 out of 11 non-NPC samples (N1, N2, N6, N8, N9and N10), DICE1 protein was found to be abundantlyexpressed with the ratio of DICE1 versus b-actin beinggreater than 0.2 (Fig. 6). miR-BART3* was highly expressedin all NPC tumor samples, but its expression was low in 6out of 11 non-NPC samples in which the ratio of miR-BART3* versus GAPDH mRNA was less than 0.2. Notewor-thily, in 4 out of 6 non-NPC samples in which miR-BART3*was underexpressed, DICE1 protein was abundant (N1, N2,N6 and N9). Based on our analysis of a small number ofclinical samples, DICE1 was underexpressed in NPC tumortissues and there might be an inverse correlation betweenmiR-BART3* and DICE1 expression (Fig. 6).

DiscussionIn this study, we have obtained several lines of evidence thatconsistently support specific targeting of DICE1 by miR-BART3*. First, miR-BART3* specifically repressed reporterexpression driven by either miR-BART3* target site in the 30

UTR of DICE1 (Fig. 1a) or the complete 30 UTR of DICE1(Fig. 1b). Second, miR-BART3* specifically repressed DICE1protein expression in cultured cells (Fig. 2). Third, miR-BART3* in 293B3 and HK1/EBV cells suppressed the expres-sion of endogenous DICE1 mRNA and protein (Fig. 3).Finally, compromising miR-BART3* in C666-1 and 293B3cells enhanced the expression of DICE1 protein (Fig. 4).

The miR-BART3* target site in 30 UTR of DICE1 imper-fectly matches miR-BART3* sequence (Fig. 1a), thus miR-BART3* is thought to repress the translation of DICE1mRNA. However, we noted that the steady-state levels ofDICE1 mRNA were also decreased in cells expressing miR-BART3* or infected with EBV (Fig. 3c). This is compatiblewith the notion that RNA transcripts translationally silencedby miRNAs are enriched in cytoplasmic P-bodies and subse-quently degraded.42

The role of miRNAs in gene regulation is largely to finetune the availability of key proteins.1,2 It was therefore not sur-prising that in most cases miR-BART3* exhibited only a mildinhibitory effect on DICE1 (Figs. 1). However, miR-BART3*was constitutively expressed to a relatively high level in EBV-infected NPC cells.9–11 Hence the accumulated action of miR-BART3* could still make a difference in the activity of DICE1.On the other hand, it is not uncommon that multiple EBVand cellular miRNAs co-target the same cellular mRNAs.19,28

Genetic and epigenetic inactivation of DICE1 tumor sup-pressor occurs commonly in cancer cells,30,31 but the

Figure 4. Inhibition of miR-BART3* by anti-miR-BART3* oligonucleo-

tide in 293B3 (a) and C666-1 (b) cells led to elevation of DICE1

protein expression. Anti-miR-NC is an irrelevant oligonucleotide

serving as negative control. Indicated at the bottom of the panels

are relative ratios of DICE1 normalized to b-actin protein as deter-

mined by densitometry. Cells were collected for Western blotting

with mouse monoclonal anti-DICE1 at 48 hr post-transfection.

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mechanism by which DICE1 performs its tumor suppressorfunction remains elusive. DICE1 is also known as INTS6,which is a subunit of the integrator complex that mediatessmall nuclear RNA processing.43 Whether the activity of inte-grator complex is linked to the tumor suppressor function ofDICE1 is not understood. However, the integrator complexwas recently found to be employed by a primate herpesvirusknown as Herpervirus saimiri to process its viral miRNAs.44

In this regard, it will be of interest to determine whether theintegrator complex might also be involved in the processingof some EBV RNAs and how miR-BART3* might affect thefunction of the integrator complex.

DICE1 has growth suppressive activity which is counter-acted by miR-BART3* (Fig. 5). Although the DICE1-inhibi-tory effect of miR-BART3* might not be dramatic (Figs. 1),persistent expression of miR-BART3* to high level in naso-pharyngeal epithelial cells could still be critical in tipping thebalance toward cell proliferation in the initiation of NPC tu-morigenesis. As the first step to shed light on this, we haveassessed the expression patterns of miR-BART3* and DICE1in NPC clinical samples (Fig. 6). We noted that there werevariations in the detection of DICE1 in the clinical samples.Particularly, DICE1 was expressed in one NPC tumor sample(T5) and two non-NPC samples (N8 and N10) in which

Figure 5. Alleviation of the growth suppressive activity of DICE1 by miR-BART3*. (a) X-gal staining of cells. HeLa cells were transiently co-

transfected with pSV-b-gal and excess amount (103) of the indicated plasmids. (b) Cell counting. X-gal-positive cells were counted in ten

different fields and the means were shown. Three independent experiments were carried out and error bars indicate SD. The difference

between the pcDNA3.1-DICE1 and pcDNA3.1-DICE11pSuper-BART3 groups is statistically significant (p<0.05) by Student’s t test.

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miR-BART3* was also expressed. We wondered whether het-erogeneity of the clinical samples obtained and particularlythe different proportions of non-cancerous or non-EBV-infected cells in the samples might account for the variationsobserved (Fig. 6), but the trend of DICE1 underexpression inNPC and the inverse correlation between DICE1 andmiR-BART3* were still evident. Coexpression of DICE1 andmiR-BART3* in one NPC tumor sample (T5) as well as theabsence of DICE1 and concurrent underexpression of miR-BART3* in two non-NPC samples (N3 and N4) might alsobe explained by alternative regulatory mechanisms that gov-ern DICE1 expression. Particularly, it will be of interestto see whether the DICE1 promoter is hypermethylated inNPC as in prostate cancer.33 The protein bands reactive togoat polyclonal anti-DICE1 antibodies in clinical samplesappeared as a doublet (Fig. 6). Both species were diminished

in most miR-BART3*-positive tissues. More experiments arerequired to determine whether the smaller form might bederived from a degradation product. Although NPC biopsysamples suitable for both protein and RNA analysis were lim-ited, it is of importance to verify our findings on the under-expression of DICE1 and the inverse correlation betweenmiR-BART3* and DICE1 levels in a larger cohort of NPCpatients and control subjects. Further investigations are alsorequired to derive new mechanistic insights on how compro-mising DICE1 might contribute to NPC tumorigenesis.

AcknowledgementsAuthors thank members of Jin laboratory for critical reading of manuscriptand members of Mengfeng Li and Jun Li’s laboratory at Sun Yatsen Univer-sity for help with the collection of clinical samples. EBV research in S.W.T.’slaboratory was also supported by Hong Kong Research Grants Council(HKU7766/08M and HKU7809/11M).

Figure 6. Expression of DICE1 protein and miR-BART3* in NPC tumor samples. Western blotting with rabbit polyclonal anti-DICE1 and RT-

PCR were performed for each sample. Relative ratios of DICE1 normalized to b-actin and mature miR-BART3* normalized to GAPDH were

determined by densitometry and indicated below the panels. T: NPC tumor sample. N: nasopharyngeal biopsy sample from non-NPC

subjects.

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