ORIGINAL ARTICLE MiR-663, a microRNA targeting p21 WAF1/CIP1 , promotes the proliferation and tumorigenesis of nasopharyngeal carcinoma C Yi 1,2,5 , Q Wang 1,2,5 , L Wang 1,2 , Y Huang 1,2 , L Li 1,2 , L Liu 1,2 , X Zhou 1,2 , G Xie 1,2 , T Kang 1,3 , H Wang 1,3 , M Zeng 1,3 , J Ma 1,4 , Y Zeng 1,3 and J-P Yun 1,2 1 State Key Laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou, China; 2 Department of Pathology, Sun Yat-Sen University Cancer Center, Guangzhou, China; 3 Department of Experimental Research, Sun Yat-Sen University Cancer Center, Guangzhou, China and 4 Department of Radiation Oncology, Cancer Center, Sun Yat-Sen University, Guangzhou, China MicroRNAs (miRNAs) may function as either oncogenes or tumor suppressors in the malignant progression of different tumor types. MiR-663 was recently reported to be decreased and identified as a tumor suppressor in gastric cancer. We also verified its role in repressing cell proliferation of a gastric cancer cell line. In this study, however, miR-663 was found to be upregulated in nasopharyngeal carcinoma (NPC) cells compared with human immortalized nasopharyngeal epithelium cells, using a miRNA microarray, and this higher expression was confirmed in NPC tissue samples. Indeed, inhibition of miR-663 impaired the proliferation of NPC cells in vitro and the NPC tumor growth of xenografts in nude mice. Mechanistically, miR-663 directly targeted p21 WAF1/CIP1 to promote the cellular G1/S transition, as the inhibitory effects of miR-663 on the G1/S transition could be rescued by p21 WAF1/CIP1 silencing. Our results imply that miR-663 may act as an oncogene in NPC. The newly identified miR-663/p21 WAF1/CIP1 axis clarifies the molecu- lar mechanism of NPC cell proliferation and represents a novel strategy for the diagnosis and treatment of patients with NPC. Oncogene (2012) 31, 4421–4433; doi:10.1038/onc.2011.629; published online 16 January 2012 Keywords: microRNAs; miR-663; cell cycle; p21; nasopharyngeal carcinoma Introduction Nasopharyngeal carcinoma (NPC) is a highly malignant cancer derived from the nasopharyngeal epithelium. NPC has a particularly high incidence in Southeast Asia and Northern Africa, with a distinct ethnic predilection (Chou et al., 2008). Accumulating evidence revealed that NPC is a multifactorial malignancy associated with Epstein–Barr virus (EBV) infection, genetic susceptibil- ity and environmental factors (McDermott et al., 2001; Zeng and Jia, 2002; Wei and Sham, 2005; Bei et al., 2010). Although NPC tumors are sensitive to radio- therapy and chemotherapy, the prognosis is poor due to recurrence and metastases. However, the precise mole- cular mechanism of its pathogenesis and progression is still largely unknown. Better understanding of the cellular and molecular mechanisms of its pathogenesis and progression is essential for the development of novel strategies for the diagnosis and treatment of NPC. MicroRNAs (miRNAs) belong to a class of conserved endogenous non-coding small RNAs, which negatively regulate gene expression at the post-transcriptional level by annealing with the 3 0 -untranslated region (3 0 UTR; Bartel, 2004; Valencia-Sanchez et al., 2006). They are important regulators of basic cellular functions, includ- ing proliferation, migration, differentiation and apop- tosis (Felli et al., 2005; Esquela-Kerscher and Slack, 2006; Fontana et al., 2007; He et al., 2007; Wang et al., 2007). Many types of human disease, especially cancer, have been linked to a deregulation of the expression of miRNAs (Calin et al., 2004). To date, several groups have suggested that human endogenous miRNAs are associated with oncogenesis and tumor progression in NPC. MiR-29c is downregulated in NPC and regulates genes encoding extracellular matrix proteins (Sengupta et al., 2008). MiR-26a has been reported to suppress cell proliferation and tumorigenesis of NPC through repres- sion of EZH2 (Lu et al., 2011). MiR-218 can cause significant toxicity in NPC cells and delay tumor growth, as well as regulate NPC cell migration through downregulation of survivin and the SLIT2-ROBO1 pathway (Alajez et al., 2011). Expression levels of 35 miRNAs have been found to be significantly altered in NPC and these miRNAs target several oncogenic pathways in NPC (Chen et al., 2009). Moreover, human miRNAs, such as miR-141, miR-200a, let-7, miR-100 and several EBV-encoded miRNAs, including miR- BART5 and miR-BART22, have been shown to be aberrantly expressed and to contribute to the develop- ment and progression of NPC (Choy et al., 2008; Lung et al., 2009; Shi et al., 2010; Xia et al., 2010; Zhang et al., 2010; Wong et al., 2011). Received 26 March 2011; revised 4 November 2011; accepted 2 December 2011; published online 16 January 2012 Correspondence: Dr J-P Yun, Department of Pathology of Cancer Center of Sun Yat-Sen University, No. 651 Dongfeng Road East, Guangzhou 510060, China. E-mail: [email protected]5 These authors contributed equally to this work. Oncogene (2012) 31, 4421–4433 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc
Transcript
ORIGINAL ARTICLE
MiR-663, a microRNA targeting p21WAF1/CIP1, promotes the proliferation
and tumorigenesis of nasopharyngeal carcinoma
C Yi1,2,5, Q Wang1,2,5, L Wang1,2, Y Huang1,2, L Li1,2, L Liu1,2, X Zhou1,2, G Xie1,2, T Kang1,3,H Wang1,3, M Zeng1,3, J Ma1,4, Y Zeng1,3 and J-P Yun1,2
1State Key Laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou, China; 2Department ofPathology, Sun Yat-Sen University Cancer Center, Guangzhou, China; 3Department of Experimental Research, Sun Yat-SenUniversity Cancer Center, Guangzhou, China and 4Department of Radiation Oncology, Cancer Center, Sun Yat-Sen University,Guangzhou, China
MicroRNAs (miRNAs) may function as either oncogenesor tumor suppressors in the malignant progression ofdifferent tumor types. MiR-663 was recently reported tobe decreased and identified as a tumor suppressor ingastric cancer. We also verified its role in repressing cellproliferation of a gastric cancer cell line. In this study,however, miR-663 was found to be upregulated innasopharyngeal carcinoma (NPC) cells compared withhuman immortalized nasopharyngeal epithelium cells,using a miRNA microarray, and this higher expressionwas confirmed in NPC tissue samples. Indeed, inhibitionof miR-663 impaired the proliferation of NPC cellsin vitro and the NPC tumor growth of xenografts innude mice. Mechanistically, miR-663 directly targetedp21WAF1/CIP1 to promote the cellular G1/S transition, as theinhibitory effects of miR-663 on the G1/S transition couldbe rescued by p21WAF1/CIP1 silencing. Our results imply thatmiR-663 may act as an oncogene in NPC. The newlyidentified miR-663/p21WAF1/CIP1 axis clarifies the molecu-lar mechanism of NPC cell proliferation and represents anovel strategy for the diagnosis and treatment of patientswith NPC.Oncogene (2012) 31, 4421–4433; doi:10.1038/onc.2011.629;published online 16 January 2012
Nasopharyngeal carcinoma (NPC) is a highly malignantcancer derived from the nasopharyngeal epithelium.NPC has a particularly high incidence in Southeast Asiaand Northern Africa, with a distinct ethnic predilection(Chou et al., 2008). Accumulating evidence revealed that
NPC is a multifactorial malignancy associated withEpstein–Barr virus (EBV) infection, genetic susceptibil-ity and environmental factors (McDermott et al., 2001;Zeng and Jia, 2002; Wei and Sham, 2005; Bei et al.,2010). Although NPC tumors are sensitive to radio-therapy and chemotherapy, the prognosis is poor due torecurrence and metastases. However, the precise mole-cular mechanism of its pathogenesis and progressionis still largely unknown. Better understanding of thecellular and molecular mechanisms of its pathogenesisand progression is essential for the development of novelstrategies for the diagnosis and treatment of NPC.
MicroRNAs (miRNAs) belong to a class of conservedendogenous non-coding small RNAs, which negativelyregulate gene expression at the post-transcriptional levelby annealing with the 30-untranslated region (30UTR;Bartel, 2004; Valencia-Sanchez et al., 2006). They areimportant regulators of basic cellular functions, includ-ing proliferation, migration, differentiation and apop-tosis (Felli et al., 2005; Esquela-Kerscher and Slack,2006; Fontana et al., 2007; He et al., 2007; Wang et al.,2007). Many types of human disease, especially cancer,have been linked to a deregulation of the expression ofmiRNAs (Calin et al., 2004). To date, several groupshave suggested that human endogenous miRNAs areassociated with oncogenesis and tumor progression inNPC. MiR-29c is downregulated in NPC and regulatesgenes encoding extracellular matrix proteins (Senguptaet al., 2008). MiR-26a has been reported to suppress cellproliferation and tumorigenesis of NPC through repres-sion of EZH2 (Lu et al., 2011). MiR-218 can causesignificant toxicity in NPC cells and delay tumorgrowth, as well as regulate NPC cell migration throughdownregulation of survivin and the SLIT2-ROBO1pathway (Alajez et al., 2011). Expression levels of 35miRNAs have been found to be significantly altered inNPC and these miRNAs target several oncogenicpathways in NPC (Chen et al., 2009). Moreover, humanmiRNAs, such as miR-141, miR-200a, let-7, miR-100and several EBV-encoded miRNAs, including miR-BART5 and miR-BART22, have been shown to beaberrantly expressed and to contribute to the develop-ment and progression of NPC (Choy et al., 2008; Lunget al., 2009; Shi et al., 2010; Xia et al., 2010; Zhang et al.,2010; Wong et al., 2011).
Received 26 March 2011; revised 4 November 2011; accepted 2December 2011; published online 16 January 2012
Correspondence: Dr J-P Yun, Department of Pathology of CancerCenter of Sun Yat-Sen University, No. 651 Dongfeng Road East,Guangzhou 510060, China.E-mail: [email protected] authors contributed equally to this work.
Oncogene (2012) 31, 4421–4433& 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12
MiR-663, which is located at human chromosome20q11.1, is associated with cellular senescence, immunityand cancer (Lehmann et al., 2007; Pizzimenti et al.,2009; Pan et al., 2010). MiR-663 is upregulated inreplicative senescence in WI-38 human fibroblasts and iscorrelated with the repression of Id1, as well as insulin-like growth factor 1 receptor (Maes et al., 2009). Aresearch of global changes in miRNAs between early-passage and senescent WI-38 human diploid fibroblastsshowed that miR-663 is markedly more abundant insenescent cells (Marasa et al., 2010). Furthermore, miR-663 was identified to be differentially expressed inexperiments using EBV-transformed B-cell lines andperipheral blood mononuclear cells obtained from lupusnephritis affected patients, suggesting that it is asso-ciated with lupus nephritis (Te et al., 2010). It isupregulated by oscillatory shear stress and has a keyrole in oscillatory shear-induced inflammatory responsesby mediating the expression of the inflammatory genenetwork in human umbilical vein endothelial cells(Ni et al., 2011). In human THP-1 monocytic cells andhuman blood monocytes, resveratrol upregulates miR-663, which decreases endogenous activator protein-1activity and impairs its upregulation by lipopolysac-charides by targeting Jun B and Jun D (Tili et al.,2010a). Furthermore, it is involved in a resveratrol-relevant pathway by targeting transforming growthfactor-b1 (Tili et al., 2010b). This miRNA is increasinglythought to act as a tumor suppressor and has beenshown to be downregulated in human gastric cancercells and induce mitotic catastrophe growth arrest (Panet al., 2010). However, the role of miR-663 in NPCremains unknown.
Cip1/Waf1/Sdi1 (p21) is a member of the Cip/Kipfamily of cyclin-dependent kinase inhibitors. It hasfunctions in multiple cellular processes, includingdifferentiation, senescence, apoptosis, DNA replicationand repair, cell proliferation and cell cycle arrest. Theexpression of p21 is likely to be tightly regulated at thetranscriptional and post-transcriptional level. For ex-ample, at the post-translational level, p21 is mainlyregulated by phosphorylation, localization and ubiqui-tination. We have recently demonstrated that nucleo-phosmin/B23 and hSSB1 may function as chaperones toprotect p21 from degradation by the ubiquitin–protea-some pathway (Xiao et al., 2009; Xu et al., 2011).Interestingly, miRNAs such as the miR-17 family andhuman pathogenic gamma herpes virus Kaposi’s sarco-ma-associated herpes virus miR-K1 have also beenreported to regulate p21 expression negatively at thepost-transcriptional level via the p21 30UTR (Gottweinand Cullen, 2010; Wang et al., 2010).
In this study, we generated a specific miRNAexpression profile in NPC cells with the use of a miRNAmicroarray that contained 686 miRNAs. We demon-strated that miR-663 was upregulated in NPC cells. Inthese cells, miR-663 may function as an oncogene bypromoting cell growth and tumorigenesis. Furthermore,p21 was characterized as a direct and functional targetof miR-663. The newly identified miR-663/p21WAF1/CIP1
axis elucidates the molecular mechanism of NPC cell
proliferation and represents a novel strategy for thediagnosis and treatment of patients with NPC.
Results
MiR-663 is overexpressed in human NPCAs an initial step to establish a correlation between NPCtumorigenesis and miRNA gene expression, miRNAexpression profiling was conducted with a miRNAmicroarray using RNA isolated from HONE1 and 5-8Fcells, two NPC cell lines, and N5-Bmi-1, a humanimmortalized nasopharyngeal epithelial cell line (Songet al., 2006; Kong et al., 2010). Overall, 686 miRNAs weredetected in these cell lines, of which 27 were upregulatedand 18 were downregulated in NPC cells compared withthe immortalized epithelial cells (Figure 1a). To oursurprise, upregulation of miR-663, a miRNA recentlyidentified to be a tumor suppressor in gastric cancer, wasobserved in NPC cells, which indicated that miR-663 mayfunction differently in NPC cells.
To validate this unexpected finding, we then detectedmiR-663 expression in 12 tissue samples of human NPCand 12 samples of nasopharyngitis. Consistent with thedata we observed in the NPC cell lines, the averageexpression level of miR-663 was significantly higher inthe NPC specimens compared with the nasopharyngitistissue samples (Po0.05; Figure 1b).
MiR-663 promotes cell proliferation in vitroThe upregulation of miR-663 in NPC cells prompted usto examine its potential role in cell proliferation. Asshown in Figure 2a, the growth rate was significantlydecreased in both CNE1 and 5-8F cells as a result of thedownregulation of miR-663 using a locked nucleic acid(LNA)-modified miR-663-specific anti-sense oligonu-cleotide (LNA anti-miR). This result suggested thatmiR-663 may promote the proliferation of NPC cells.This notion was further supported by the colonyformation assay using both CNE1 and 5-8F cellsin vitro (Figure 2c).
To confirm this phenomenon, the miR-663 spongeplasmid vector was constructed to stably inhibit miR-663(Supplementary Figures S1a and b). Additionally, asshown in Figures 2b and d, both the cell growthand the clone formation were suppressed by the stableinhibition of miR-663 in both CNE1 and 5-8F cells.As shown in Supplementary Figure S2, proliferationwas promoted by transfecting both CNE1 and 5-8F cellswith a precursor miR-663 oligonucleotide (pre-miR-663).
In addition, to investigate the effect of miR-663 onproliferation of NP69 cells (a human immortalizednasopharyngeal epithelial cell line), a cell proliferationassay was performed after miR-663 was overexpressedin the cells. As shown in Supplementary Figure S3, miR-663 dramatically promoted the cell growth and cloneformation in NP69 cells.
Collectively, our results indicate that miR-663 maypromote the proliferation of both NPC cells andnon-tumor-derived cells in vitro.
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MiR-663 regulates cellular G1/S transitionAs miR-663 promotes cell proliferation of NPC cellsin vitro, we tested whether it affects the cell cycle profilein NPC cells. As shown in Figures 3a and b, thepercentage of cells in the G1 phase was increased afterthe transfection of miR-663 LNA or miR-663 spongevectors in both CNE1 and 5-8F cells. A decrease of thepercentage of cells in the G1 phase was observed after
transfection of pre-miR-663 in NP69 cells (Supplemen-tary Figure S7a). However, the cell cycle profile wasmarginally altered by the overexpression of miR-663 inNPC cells (Supplementary Figure S4a). The endogenousmiR-663 level may have been high enough to execute itsfunctions in NPC cells (Figure 4d). Furthermore, aftercells were synchronized by serum starvation, we furtherdetected that the percentage of cells that re-entered S-phase after serum stimulation was less when miR-663was inhibited by LNA anti-miR or miR-663 spongevectors in both CNE1 and 5-8F cells (Figures 3c and d).When the same serum-starvation stimulation assaywas used to compare cells that were transfected withpre-miR-663 and controls, there was no apparentdifference in the percentage of CNE1 and 5-8F cellsentering the S-phase between the experimental andcontrol cells (Supplementary Figure S4b). These resultssuggest that miR-663 may enhance cell proliferation bypromoting the G1/S transition.
p21 is a direct target of miR-663p21 is one of the key regulators of G1/S transition.Interestingly, we found two putative binding sites formiR-663 at 664–670 and 132–138 in the 30UTR of p21,with the use of two online algorithms, Target Scanand microrna.org (Figure 4a). Indeed, as shown inFigure 4b, pre-miR-663 was efficient in downregulatingthe luciferase activity of p21-30UTR-wt, but not of p21-30UTR-mt, in which two putative binding sites for miR-663 were individually or jointly mutated. Moreover, asexpected, upregulation of miR-663 caused a significantdecrease of p21 expression at both mRNA and proteinlevels in CNE1, 5-8F and NP69 cells (Figure 4c andSupplementary Figure S7b). Consistently, an obviousincrease of endogenous p21 was observed by either thetransient or stable inhibition of miR-663 in both CNE1and 5-8F cells (Figure 4c and Supplementary FigureS1c). To further confirm that the effects of miR-663were specifically due to the downregulation of p21through its 30UTR, we overexpressed miR-663 alongwith a p21 construct lacking its 30UTR and thus,unresponsive to inhibition by the miRNA. As expected,overexpression of the p21 construct rescued the in-creased proliferation phenotype and the decreasedpercentage of cells in the G1 phase in the miR-663-transfected NP69 cells (Supplementary Figure S9).However, alteration of p21 localization was notobserved by immunofluorescence after overexpressionand knockdown of miR-663 (Supplementary Figure S5).We further analyzed the endogenous expression levels ofmiR-663 and p21 in NPC cell lines. Quantitative real-time RT-PCR analyses revealed that miR-663 wasmarkedly upregulated in NPC cell lines when comparedwith N5-Bmi-1 or NP69 (Figure 4d). Protein expressionlevels for p21 were downregulated in NPC cell linescompared with N5-Bmi-1 and NP69 (Figure 4e). Ourresults reveal an inverse relationship between highexpression of miR-663 and low expression of p21 inNPC cells.
However, no inverse correlation was observed be-tween miR-663 and p21 in the 12 samples of NPC tissues
Figure 1 MiR-663 is overexpressed in human NPC cells and tissuesamples. (a) The miRNA microarray analysis showed that 45miRNAs were differentially expressed between the NPC cells(HONE1 and 5-8F) and the immortalized nasopharyngeal epithe-lial cells (N5-Bmi-1). (b) Real-time PCR showed the averageexpression level of miR-663 in human NPC (n¼ 12) andnasopharyngitis (n¼ 12) tissue samples. The expression of miR-663 was normalized to U6 RNA. Data shown are from at leastthree independent experiments. Error bars, s.d.; n¼ 3.
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and 12 samples of nasopharyngitis tissues (Figure 1band Supplementary Figure S6). We speculated that p21may be controlled by various mechanisms in the humantissues, especially in different conditions of diseases suchas cancer or inflammation.
p21 is involved in miR-663-regulated G1/S transitionNext, we examined the role of p21 in the miR-663-regulated G1/S transition. The percentage of cells at G1phase was decreased when p21 was singly knocked down(Supplementary Figure S4c). More importantly, boththe transient and stable inhibition of miR-663 resultedin the lack of cells accumulating in the G1 phase afterp21 was silenced by siRNA in both the CNE1 and 5-8F
cell lines (Figures 5a and b). This finding indicates thatthe promotion of the G1/S transition by miR-663 isdependent on p21.
Furthermore, to detect alteration of expression ofthe p21 pathway proteins after inhibition or over-expression of miR-663 in CNE1 and NP69 cells, wecharacterized phosphorylated-Rb protein (ppRb),total Rb protein (pRb), E2F1, cyclin E, cyclin A andp27 at the RNA and protein levels (Figures 5c and dand Supplementary Figure S7). Expressions of ppRband E2F1 changed inversely with p21 expressionafter inhibition or overexpression of miR-663 in CNE1or NP69 cells. Reductions of ppRb and E2F1 expressionand an increase of p21 expression were observed
Figure 2 Inhibition of miR-663 suppresses cell proliferation in vitro. (a) (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) cell viability assays was performed on days 1 to 5 after the transfection of CNE1 and 5-8F cells with either LNA anti-miR or the negative control. (b) MTT cell viability assays were performed on days 1 to 5 of CNE1 and 5-8F cells stably expressing themiR-663 sponge or control sponge vectors. (c) The colony formation assay was performed in CNE1 and 5-8F cells after they weretransfected with miR-663 LNA or the negative control and incubated for 14 days. (d) The colony formation assay was performed onCNE1 and 5-8F cells stably expressing the miR-663 sponge or control sponge vectors that were incubated for 14 days. All results arethe average of at least three independent experiments. Error bars, s.d.; n¼ 3. *Po0.05; **Po0.01; ***Po0.001, compared withcontrol.
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after transfection with miR-663 sponge or LNA miR-663 in CNE1 cells (Figures 5c and d). ppRb andE2F1 were increased after transfection with p21 siRNAin CNE1 cells (Figures 5c and d). Increases of ppRband E2F1 expression, together with a decrease of cellsin the G1 phase were detected after transfectionwith pre-miR-663 or p21 siRNA in NP69 cells(Supplementary Figure S7). However, cyclin E andcyclin A were not observed to change at the RNA orprotein levels. As p21 regulates the G1/S transitionby activating cyclin-dependent kinases and cyclincomplexes, it is reasonable that expression of thecyclins may not change during the dissociation. Anotherkey regulator of the G1/S transition, p27, was notaltered with overexpression and inhibition of miR-663or silencing of p21 (Figure 5 and SupplementaryFigure S7).
Together, these results suggest that miR-663 pro-motes the G1/S transition through the p21-pRb
pathway in both NPC cells and non-tumor-derivedcells.
Inhibition of miR-663 suppresses tumor growth in vivoAs inhibition of miR-663 suppresses cell proliferation ofNPC cells in vitro, we hypothesized that miR-663 mayalso have a similar action in vivo. After nude mice wereinjected with CNE1 cells stably transfected with miR-663sponge (CNE1-sponge-663) or control sponge (CNE1-NC), respectively, we found that tumors grew at a slowerrate and had smaller sizes when miR-663 was inhibited inthis animal model of tumorigenicity (Figures 6a–d).Consistently, both the downregulation of Ki67 and theupregulation of p21 were observed in the tumorsgenerated from the CNE1-sponge-663 vector comparedwith those generated from CNE1-NC according to theresults of immunohistochemical analysis (Figure 6e).These results indicate that inhibition of miR-663 maysuppress tumor growth by upregulating p21 in vivo.
840LNA control LNA anti-miR sponge control miR-663 sponge
LNA control LNA anti-miR sponge control miR-663 sponge
Figure 3 MiR-663 is involved in regulation of the G1/S transition. (a) Cell cycle profiles were analyzed after CNE1 and 5-8F cells weretransfected with miR-663 LNA anti-miR or the negative control for 48 h. (b) Cell cycle analysis of CNE1 and 5-8F cells stablyexpressing the miR-663 sponge or control sponge vectors. (c) The effects of miR-663 inhibition on S-phase entry upon serumstimulation. LNA anti-miR or control transfected cells were serum-deprived for 48 h, and then serum was re-added. The cells wereharvested at 8 h after the re-addition of serum and then analyzed for cell cycle profiles. (d) The serum-starvation stimulation assay ofCNE1 and 5-8F cells stably expressing the miR-663 sponge or control sponge vectors. Data are from at least three independentexperiments and are expressed as means±s.d.
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Discussion
The abnormal expression of miRNAs has been reportedin many types of cancer, and much attention has focusedon understanding the roles of miRNAs in modulating
the process of cancer development (Lu et al., 2005;Landgraf et al., 2007). In this report, the specificmiRNA expression profile of NPC cells was generatedusing a miRNA microarray that contained 686miRNAs. Furthermore, miR-663 was determined to
Figure 4 p21 is a direct target of miR-663. (a) Putative miR-663-binding sequence in the 30UTR of p21 mRNA. Mutations weregenerated in the p21 30UTR sequence at the complementary site for the seed region of miR-663, as indicated. A human p21 30UTRfragment containing either the wild-type or mutant miR-663-binding sequences was cloned downstream of the luciferase reporter gene.(b) Analysis of luciferase activity. 293T cells were co-transfected with a Renilla luciferase expression construct psiCHECK-2 vector (asan internal control), a firefly luciferase reporter plasmid containing either the wild-type or mutant p21 30-UTR (indicated as WT orMUT on the x axis), and either the pre-miR-663 or the negative control. Luciferase activity was normalized to Renilla luciferaseactivity. Data are from at least three independent experiments. Error bars, s.d.; n¼ 3. **Po0.01, compared with control. (c) miR-663negatively regulates p21 expression. Overexpression of miR-663 suppressed the expression of endogenous p21, and antagonism of miR-663 elevated p21 expression. Expressions of p21 were analyzed by western blotting and RT–PCR 48 h after transfection with pre-miR-663, LNA or the negative control in CNE1 and 5-8F cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and b-actin wereinternal controls for western blotting and RT–PCR, respectively. (d) Real-time PCR showed high expression of miR-663 in the NPCcell lines (CNE1, CNE2, HONE1, SUNE1, 5-8F, 6-10B and C666), compared with the immortalized nasopharyngeal epithelial celllines (N5-bmi-1 and NP69); miR-663 expression was normalized to U6 RNA. Data are from at least three independent experiments.Error bars, s.d.; n¼ 3. (e) Western blotting showing endogenous expression of p21 in NPC and nasopharyngeal epithelial cells, withGAPDH as the internal control.
Figure 5 p21 is involved in miR-663-regulated transition from G1 to the S phase. (a) Knockdown of p21 rescues miR-663-LNA-induced G1 arrest. CNE1 and 5-8F cells were first transfected with the negative control or miR-663 LNA anti-miR for 24 h, followedby transfection with scrambled or p21 siRNA for 24 h. Cell cycle profiles were analyzed using fluorescence-activated cell sorting(FACS). (b) Knockdown of p21 rescues miR-663 sponge-induced G1 arrest. CNE1 and 5-8F cells stably expressing the miR-663sponge or control sponge vectors were transfected with scrambled or p21 siRNA for 24 h. Cells were harvested and cell cycle profileswere analyzed using FACS. Data are from three independent experiments and expressed as means±s.d. (c) Expressions of p21, ppRb,pRb, E2F1, cyclin E, cyclin A and p27 were analyzed after co-transfection of miR-663 LNA anti-miR and p21 siRNA in CNE1 cells.CNE1 cells were first transfected with the negative control or miR-663 LNA anti-miR for 24 h, followed by transfection with scrambledor p21 siRNA for 24 h. Western blotting and RT-PCR were used to detect expressions of the indicated proteins and RNA, respectively.Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and b-actin were internal controls for western blotting and RT–PCR,respectively. (d) Expressions of p21, ppRb, pRb, E2F1, cyclin E, cyclin A and p27 were analyzed after co-transfection of the miR-663sponge vector and p21 siRNA in CNE1 cells. CNE1 cells stably expressing the miR-663 sponge or control sponge vector weretransfected with scrambled control or p21 siRNA for 24 h, and western blotting and RT–PCR were used to detect expressions of theindicated proteins and RNA, respectively. GAPDH and b-actin were internal controls for western blotting and RT–PCR, respectively.
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Figure 6 Antagonism of miR-663 suppresses tumor growth in vivo. (a) Tumor formation in nude mice 6 weeks after injection with CNE1-sponge-663 and CNE1-NC. (b) Growth curves of CNE1-sponge-663- and CNE1-NC-generated tumors. Volumes of the tumors weremeasured every 3 days. Error bars, s.d. (n¼ 7 for CNE1-sponge-663; n¼ 7 for CNE1-NC). (c) Volumes of the CNE1-sponge-663- andCNE1-NC-generated tumors 6 weeks after the initial injection. (d) Weights of the CNE1-sponge-663- and CNE1-NC-generated tumors 6weeks after the initial injection. (e) Immunohistochemical analysis of seven CNE1-sponge-663- and seven CNE1-NC-generated tumors 3weeks after injection. Sections derived from tumors were incubated with anti-p21 and anti-ki67 antibody. Representative fields are shown(� 400). Of each generated tumor, five fields were randomly selected according to semi-quantitative scales. p21- and Ki67-positive cells per1000 cells were counted by three independent experienced pathologists. The bar graph shows average expression levels of p21 and Ki67 ofCNE1-sponge-663- and CNE1-NC-generated tumors. Error bars, s.d.; n¼ 3. *Po0.05; ***Po0.001, compared with the control.
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function as an oncogene in NPC by directly targetingp21. In these cells, the inhibition of miR-663 impededthe proliferation of NPC cells in vitro and the growth ofNPC xenograft tumors in nude mice. Our resultsindicate that miR-663 may be a novel future therapeutictarget for patients with NPC.
As miRNAs are important regulators of basic cellfunctions (Felli et al., 2005; Esquela-Kerscher and Slack,2006; Fontana et al., 2007; He et al., 2007; Wang et al.,2007), the roles of miRNAs in cancer are consequentlyvery complicated. A single miRNA may be upregulatedin one type of cancer, but downregulated in another.For example, miR-125b is markedly downregulated inhuman breast cancers and breast cancer cell lines (Iorioet al., 2005), whereas high levels of miR-125b have beendetected in prostate cancer cell lines and clinical tissuesamples (Shi et al., 2007). Furthermore, from a large-scale miRnome analysis including lung, breast, stomach,prostate, colon and pancreatic tumors, several miRNAs,such as miR-218-2, have also shown inconsistentexpression in different human solid tumors from distinctorgans (Volinia et al., 2006). Recently, miR-663 wasreported to be a tumor suppressor, which was decreasedin human gastric cancer cell lines and acted to represscell proliferation and tumor growth by inducing amitotic catastrophe (Pan et al., 2010). In this study, wealso analyzed the function of miR-663 in two gastriccancer cell lines, MKN-45 and SCG-7901, and verifiedits role in repressing cell proliferation of the cell lineMKN-45 (Supplementary Figure S8). In contrast, wedemonstrated that miR-663 may act as an oncogene inNPC cells by promoting the G1/S transition via directlytargeting p21. The functions of some miRNAs, such asmiR-125b, miR-218-2 and miR-663, need to be deter-mined individually in each cancer type, as they may actas tumor suppressors or oncogenes in a cell-specificcontext.
As a critical cell cycle regulator, p21 was found to bedirectly regulated by miR-663 at the post-transcriptionallevel in the present study. It was recently reported thatp21 is directly regulated by multiple miRNAs. Membersof the miR-106b and miR-17-92 families modulate p21expression in different cell types and are involved inmultiple cellular processes, including cell cycle transi-tion, cell proliferation, stress-induced premature senes-cence and the self-renewal of stem cells (Fontana et al.,2008; Ivanovska et al., 2008; Petrocca et al., 2008;Inomata et al., 2009; Li et al., 2009; Chen et al., 2010;Wong et al., 2010). The post-transcriptional regulationof p21 by the embryonic stem cell-specific miRNAs,miR-290 cluster, was shown to promote the G1/Stransition and rapid proliferation of embryonic stemcells (Wang et al., 2008). The Kaposi’s sarcoma-associated herpes virus miR-K1 represses the expressionof p21 and attenuates p21-mediated cell cycle arrest(Gottwein and Cullen, 2010). A number of miRNAs,such as miR-302a, miR-146a and miR-146b, whichtarget p21 can rescue human mammary epithelial cellsfrom RasG12V-induced senescence by preventing theRasG12V-induced upregulation of p21 (Borgdorff et al.,2010). A screening strategy identified 28 miRNAs that
can suppress p21 expression by directly targeting its30UTRs and some of them were verified to promote cellproliferation and cell cycle progression (Wu et al., 2010).In the present study, we provide evidence that miR-663,by targeting p21 and causing the aberrant transitionfrom the G1 to the S phase, promoted cell proliferationand tumorigenicity in NPC cells.
Although we showed that miR-663 could function asoncogenic miRNA through the molecular axis miR-663/p21 in NPC cell lines in vitro and our animal modelin vivo, the inverse correlation between miR-663 andp21 expression was not detectable in the same NPC/nasopharingitis tissues. This inconsistence may be due tothe complicated regulation of p21. For instance, Leiet al. (1999) reported that the positive expression rates ofp21 were different between the NPC tissues and non-tumor nasopharyngeal tissue; (45.7 vs 80.0%), whereasWang et al. (2005) reported that the expression level ofp21 were indistinguishable between the NPC tissues andthe chronic inflammatory nasopharyngeal mucosa. Infact, p21 is tightly regulated at multiple levels. First,p21 is controlled by p53-dependent and -independentmechanisms at the transcriptional level (el-Deiry et al.,1994; Parker et al., 1995). Second, the p21 mRNAstability was regulated by miRNAs and the RNA-binding protein (Joseph et al., 1998; Filipowicz et al.,2008). Third, p21 is also regulated by phosphorylationand ubiquitination at the post-translational level (Liet al., 1996; Zhou et al., 2001; Child and Mann, 2006).For example, as mentioned above, there are multiplemiRNAs directly regulated p21 in the literature.Notably, various factors, such as the EBV nuclearantigen 2 and lactotransferrin, have been reported toregulate p21 in NPC (Lin et al., 2000; Zhou et al., 2008).
Chemically modified antisense oligonucleotides arewidely used as miRNA inhibitors, such as 20 O-methyl,LNA (Hutvagner et al., 2004; Meister et al., 2004; Oromet al., 2006), which only provide a correspondinglytransient derepression of miRNA targets in cells.Artificial miRNA decoys termed ‘miRNA sponges’ wererecently introduced as a long-term inhibitor that canstably block the function of miRNAs in cell lines andtransgenic organisms (Ebert et al., 2007). This approachhas proved to be a useful tool to probe miRNAfunctions in a variety of experimental systems, such asmodels of breast cancer and glioma (Valastyan et al.,2009; Mei et al., 2011). In the present experimentalmodel, we structured a miR-663 sponge to block miR-663 stably in NPC cells in a stronger way than withLNA, in accordance with the report by Ebert et al.(2007) Therefore, we speculate that ‘miRNA sponges’can be used as a powerful tool to investigate thefunctions of miRNAs and as a potential therapeuticstrategy by targeting miRNAs. However, it is worthnoting that the most appropriate dose of sponges thatshould be used is yet to be determined due to their highcytotoxicity (data not shown).
In summary, expression profiling and functionalstudies suggest an important role of miR-663 in themolecular etiology of oncology. For the first time, wehave revealed that miR-663 has oncogenic functions in
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NPC. The newly identified miR-663/p21 axis sheds lighton the molecular mechanism of NPC cell proliferation.It is logical to predict that the inhibition of miR-663may serve as a basis for the development of a potentiallynew therapeutic regimen against NPC.
Materials and methods
Cell cultureTwo human immortalized nasopharyngeal epithelial cell lines(NP69 and N5-Bmi-1) were grown in keratinocyte/serum-freemedium supplemented with growth factors (Gibco, GrandIsland, NY, USA; Li et al., 2004; Liao et al., 2005; Song et al.,2006, 2009). One EBV latently infected NPC cell line(HONE1), one well-differentiated NPC cell line (CNE1), threepoorly differentiated NPC cell lines (CNE2, C666 andSUNE1) and two SUNE1 subclones (6-10B and 5-8F) werecultured in RPMI-1640 supplemented with 10% fetal bovineserum (Glaser et al., 1989; Liao et al., 2005; Song et al., 2006,2009; Kong et al., 2010). Two human gastric cancer cell lines(MKN-45 and SCG-7901; provided by Jun Li, Department ofBiochemistry, Sun Yat-sen University School of Medicine,Guangzhou, China) were cultured in RPMI-1640 supplemen-ted with 10% fetal bovine serum. Cells were maintained in a5% CO2 incubator at 37 1C.
Tissue samplesA total of 12 samples of NPC tissues were biopsied frommasses in nasopharyngeal cavities of 12 patients with NPC anddiagnosed pathologically as undifferentiated non-keratinizingcarcinoma. Again, 12 samples of nasopharyngitis tissues werebiopsied from mucosa in nasopharyngeal cavities of 12patients with chronic nasopharyngitis. The 24 tissue sampleswere formalin-fixed and paraffin-embedded. The patients gaveinformed consent before the tissue sampling and the researchprotocols were approved by the Sun Yat-sen UniversityCancer Center Institute Research Ethics Committee.
Vector constructionpsiCHECK-2, a firefly luciferase-expressing vector, was used inthe luciferase reporter assay. To construct the psiCHECK2-p21-30UTR-wt plasmid, a wild-type 30UTR segment of p21mRNA that contained the putative miR-663-binding sites wasamplified and cloned into the PmeI and NotI sites downstreamof the luciferase reporter gene in psiCHECK-2. psiCHECK-2-p21UTR-mt1 and psiCHECK-2-p21UTR-mt2 each carriedthe mutated sequence in one of the two miR-663-binding sites,respectively, whereas psiCHECK-2-p21UTR-mt3 containedmutations of both the miR-663-binding sites.To construct a sponge-miR-663 plasmid vector, we inserted
tandemly arrayed miR-663-binding sites into a enhance greenfluorescent protein vector (pEGFP-C2). The sponge-C1plasmid vectors were constructed as the negative control, witha tandemly arrayed sequence of Caenorhabditis elegans gene(cel-miR-239b) instead of the miR-663-binding sites.P21 complementary DNA lacking its 30UTR (30UTR-less
p21) or carrying a wild-type 30UTR expression cassette at miR-663 response element were cloned into pcDNA 3.1 for ‘rescue’experiments.
miRNA microarrayTotal RNA samples were isolated from nasopharyngealepithelial cells (N5-Bmi-1) and NPC cells (HONE1 and 5-8F) using Trizol reagent (Invitrogen, Carlsbad, CA, USA).
Approximately, 5mg of total RNA from each sample werebiotin-labeled by reverse transcription using 50-biotin end-labeled random octomer oligo primers. Hybridization ofbiotin-labeled cDNA was carried out on a miRNA microarraychip, which contained 686 miRNA oligo probes printed induplicate. Hybridization signals were scanned with a Lux-Scan3.0 scanner (Capital Bio. Corporation, Beijing, China).The resultant images were digitized with Genepix Pro 6.0software (Axon Instruments, Foster City, CA, USA).
RT–PCR and quantitative real-time RT–PCRFor mRNA and miRNA analyses, total RNA was purifiedwith Trizol Reagent (Invitrogen). cDNA was synthesized usingMoloney murine leukemia virus reverse transcriptase (Prome-ga, Madison, WI, USA). The expression levels of maturemiRNAs were determined by SYBR Green quantitative PCRamplifications performed on the Stratagene Mx3000P Real-Time PCR system (Agilent Technologies, Inc., Santa Clara,CA, USA). The primers used for PCR amplification were asfollows: forward, 50-CTCGCTTCGGCAGCACA-30 and re-verse, 50-AACGCTTCACGAATTTGCGT-30 for U6; for-ward, 50-TGGCACCCAGCACAATGAA-30 and reverse, 50-CTAAGTCATAGTCCGCCTAGAAGCA-30 for b-actin;forward, 50-GCGGGGCGCCGCGGGACCG-30 and reverse,50-GTGCAGGGTCCGAGGT-30 for miR-663; and forward,50-GGGATGAGTTGGGAGGAGG-30 and reverse, 50-CGGCGTTTGGAGTGGTAG-30 for p21. RT–PCR was per-formed with the following cycling conditions: 95 1C for5min, 30 cycles of 95 1C for 30 s, 60 1C for 30 s, 72 1C for1min and a final extension of 10min at 72 1C. The thermalprofile for the real-time PCR was 95 1C for 10min, followed by40 cycles of 95 1C for 10 s, 60 1C for 20 s and 72 1C for 10 s.The fold-changes for miRNA expression levels were calculatedusing 2�DDCt.
Transient transfectionAsynchronously growing cells were seeded at 2� 105 cells/wellof a six-well plate format. Transfection of cells with 100 nMpre-miR-663, 100 nM LNA anti-miR or 50 nM p21 siRNA wasperformed using Lipofectamine RNAiMAX (Invitrogen).
Stable transfectionCNE1 and 5-8F cells were transfected with miR-663 sponge orcontrol sponge-C1 plasmid vectors to generate stable cells thatlacked miR-663 or control cells, respectively. TransfectedCNE1 and 5-8F cells were selected with G418 (Merck & Co.Inc., Whitehouse Station, NJ, USA) at 350 mg/ml for 4 weeks.The stable cell clones were obtained and maintained in G418 at150 mg/ml.
Cell proliferation assayCells were plated at 1500 cells/well in 96-well plates. Aftertransfection, the cells were cultured for 1, 2, 3, 4 and 5 days.On the indicated days, MTT reagent (AMRESCO, Solon, OH,USA) was added and incubated for 3 h at 37 1C. Thesupernatant was discarded and replaced with dimethylsulfoxide to dissolve the formazan product. Absorbance wasmeasured at an optical density of 490 nm in a spectro-photometric plate reader.
Analysis of cell clonogenicityAfter transfection, 500 cells were placed in six-well plates andmaintained in complete medium for 2 weeks. Colonies werefixed with methanol, stained with 0.1% crystal violet andcounted.
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Tumorigenicity assay in nude miceFive-week-old nude BALB/c athymic strain mice were subcuta-neously injected in the right flank with 4� 106 CNE1 cells thatstably expressed the miR-663 sponge or control sponge vector(CNE1-sponge-663 or CNE1-NC). Tumor growth was examinedevery 3 days for 6 weeks. Tumor volume (V) was monitored bymeasuring the length (L) and width (W) of the tumors andcalculated with the following equation: V¼ (L�W2)� 0.5.
SynchronizationCells were synchronized with serum deprivation for 48 h andthen released into the S-phase by the re-addition of serum.Cells were harvested at the indicated time points.
Cell cycle analysisAt 48 h post-transfection, cells were collected and fixed with70% ethanol at 4 1C overnight. Cells were washed and thenstained with propidium iodide for 30min at 37 1C. Cells wereanalyzed for DNA content by flow cytometry (fluorescence-activated cell sorting) using a Cytomics FC 500 (BeckmanCoulter, Brea, CA, USA). Data were analyzed with theMulticycle AV for Windows software (Beckman Coulter).
Luciferase reporter assayFor the luciferase reporter assay, 293T cells co-transfected with20mM of either pre-miR-663 or the negative control, and 500ngof psiCHECK2-p21-30UTR-wt, psiCHECK-2-p21UTR-mt1,psiCHECK-2-p21UTR-mt2 or psiCHECK-2-p21UTR-mt3,using Lipofectamine 2000 (Invitrogen). Cells were collected48h after transfection and analyzed using the Dual-LuciferaseReporter Assay System (Promega). Luciferase activity wasdetected by the GloMax fluorescence reader (Promega). ThepsiCHECK-2 vector that provided the constitutive expression ofRenilla luciferase was co-transfected as an internal control.
Western blotWhole cell proteins were separated in 12% SDS–PAGE gelsand blotted on nitrocellulose membranes. The filters werehybridized with polyclonal anti-p21, anti-ppRb, anti-pRb,anti-E2F1, anti-cyclin E, anti-cyclin A and anti-p27 (CellSignaling Technology, Danvers, MA, USA) at 4 1C overnight,followed by incubation with the secondary anti-rabbit or anti-mouse (Santa Cruz Biotechnology, CA, USA) for 1 h at roomtemperature. Anti-GAPDH (Santa Cruz Biotechnology) wasused as the loading control.
Confocol microscopyCells grown on coverslips were fixed using 4% paraformalde-hyde solution for 15min. Samples were blocked for 30minwith 5% bovine serum albumin/phosphate-buffered saline atroom temperature and then incubated with primary antibody(1:50 anti-p21; Cell Signaling Technology) at 4 1C overnight.The coverslips were incubated with the secondary anti-rabbitantibody (Invitrogen) for 1 h at room temperature in the darkand stained with 4-,6-diamidino-2-phenylindole (Invitrogen)for 5min to visualize the nuclei. The coverslips were mountedonto glass slides and visualized by confocal microscopy.
ImmunohistochemistryImmunohistochemistry was performed as described previously(Yun et al., 2007; Zhang et al., 2009). Briefly, after blocking, thesections were incubated with primary antibodies overnight (anti-p21 (Cell Signaling Technology), 1:25; anti-Ki67 (Santa CruzBiotechnology), 1:1), followed by incubation with secondaryantibodies, and further incubation with the Streptavidin–Biotincomplex (Dakopatts, Glostrup, Denmark). Reactivity was devel-oped in chromogen 3,30-diaminobenzidine (DAB) solution. Aftercontrast staining, the sections were dehydrated and mounted. Abrown particle in a cell was considered as positive labeling.
Statistical analysisData are presented as means±s.d. from at least three separateexperiments. Unless otherwise noted, the Student’s t-test wasused for comparisons between groups. Differences wereconsidered significant for P-values less than 0.05.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
We thank Jun Li for kindly providing the human gastriccancer cells MKN-45 and SCG-7901. This work wassupported by grants from the National Natural ScienceFoundation of China (30973506 and 81172345), the 863Project (2006AA02A404), the Science Foundation of KeyHospital Clinical Program of Ministry of Health, China (2010-178), and the Project of State Key Laboratory of Oncology inSouth China.
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MiR-663 promotes the proliferation and tumorigenesis of NPCC Yi et al
