Oncogene (2014), 1–13 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

ORIGINAL ARTICLE

Selective killing of lung cancer cells by miRNA-506 molecule through inhibiting NF-kB p65 to evoke reactive oxygen species generation and p53 activation M Yin1,2,6, X Ren1,2,6, X Zhang3, Y Luo4, G Wang1, K Huang4, S Feng4, X Bao1, K Huang5, X He2, P Liang1,5, Z Wang1, H Tang3, J He3 and B Zhang1,2 The tumor suppressor p53, nuclear factor-kB (NF-kB) and reactive oxygen species (ROS) have crucial roles in tumorigenesis, although the mechanisms of cross talk between these factors remain largely unknown. Here we report that miR-506 upregulation occurs in 83% of lung cancer patients (156 cases), and its expression highly correlates with ROS. Ectopic expression of miR-506 inhibits NF-kB p65 expression, induces ROS accumulation and then activates p53 to suppress lung cancer cell viability, but not in normal cells. Interestingly, p53 promotes miR-506 expression level, indicating that miR-506 mediates cross talk between p53, NF-kB p65 and ROS. Furthermore, we demonstrated that miR-506 mimics inhibited tumorigenesis in vivo, implicating that miR-506 might be a potential therapeutic molecule for selective killing of lung cancer cells. Oncogene advance online publication, 27 January 2014; doi:10.1038/onc.2013.597 Keywords: miR-506; NF-kB p65; ROS; p53; lung cancer; selective killing

INTRODUCTION MicroRNAs (miRNAs) are short noncoding RNAs of 19–25 nucleotides that are thought to modulate the expression of at least 30% of all human genes, with marked effects on fundamental biological processes such as development, cell proliferation, apoptosis, differentiation and metabolism. miRNAs exert their effects through base-pairing interactions between the seed region of the miRNA (nucleotides 2–8 from its 50 -end) and complementary sequences that usually reside in the 30 -untranslated regions (UTRs) of target mRNAs.1,2 miRNAs appear to be involved in signal transduction pathways in most diseases, including cancer. Hence, not only protein-encoding genes but also noncoding RNAs especially miRNAs, should be considered as key factors in signaling cascades. p53, a DNA-binding transcription factor identified in 1979,3 has been one of the most intensively studied tumor suppressors in the past three decades. Expression of p53 is usually below detectable level in resting cells, but is activated by cellular stresses such as DNA damage, oncogene activation, hypoxia, ribosomal stress and chemotherapeutic drugs.4 Upon stimulation, p53 enters the nucleus and induces transcription of target genes, including miRNAs.5,6 Loss of p53 function is associated with most human cancers.7 Consequently, modulating the stability and/or activation of p53 represents a promising anticancer strategy.8,9 Pro-inflammatory transcription factors such as nuclear factor-kB (NF-kB) are central mediators of immune responses in mammals. Activation of the classical NF-kB pathway and the resulting chronic inflammation10,11 contribute to cancer development

and pathology. Activation of NF-kB has been observed in human lymphomas and many types of human carcinomas.12,13 NF-kB can be activated by many divergent stimuli, including pro-inflammatory cytokines and ionizing radiation, and chemotherapeutic agents such as tumor necrosis factor a (TNFa) and doxorubicin. Activated NF-kB binds to the NF-kB-binding elements of its target genes, including inhibition of apoptosis proteins (IAPs) and bcl-2, which mediate anti-apoptosis and chemoresistance in tumor cells.14,15 Lung cancer is the leading cause of cancer deaths worldwide. Approximately 50% of lung cancer patients are already at advanced stages of diagnosis,16 making their treatment poor. Thus, it is crucial to identify better targets for lung cancer diagnosis and therapy. In advanced stages, tumors generally exhibit multiple genetic mutations and higher levels of oxidative stress than at earlier stages.17 Persistent oxidative stress occurs in many types of cancers,18 and while moderate levels are thought to be beneficial for tumor cells, excessive levels are anti-tumorigenic.19 For this reason, inducing reactive oxygen species (ROS) accumulation in cancer cells is considered to be a novel therapeutic approach for preferential stimulation of apoptosis.17,20 The key genes involved in regulation of ROS have been extensively studied; however, the role of noncoding RNAs, including miRNAs, in modulation of ROS has only recently emerged. miR-21 protects against H2O2-induced cell apoptosis by targeting programed cell death protein 4 (PDCD4) in cardiomyocytes.21 miR-17–92 overexpression modulates ROS generation and reduces excessive DNA damage to a tolerable level.22 Here, we report that human miR-506, a member of the

1 The State Key Laboratory of Respiratory Diseases, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China; 2School of Life Science, University of Science and Technology of China, Hefei, China; 3Department of Cardiothoracic Surgery, The First Affiliated Hospital of Guangzhou Medical College, Guangzhou, China; 4Guangzhou RiboBio Co., Ltd, Guangzhou, China and 5University of Chinese Academy of Sciences, Beijing, China. Correspondence: Dr B Zhang or Dr J He, The State Key Laboratory of Respiratory Diseases, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kai Yuan Avenue, Science Park, Guangzhou 510530, China. E-mail: [email protected] or [email protected] 6 These two authors contributed equally to this work. Received 3 June 2013; revised 10 November 2013; accepted 16 December 2013

miR-506 mediates cross talk between p53 and NF-kB in lung cancer M Yin et al

2 X-linked miRNA cluster in primates,23 is upregulated in 83% lung cancer patients and strongly correlates with ROS levels. miR-506 mimics inhibit cell viability in lung cancer cells, but not in normal cells. Furthermore, we found that miR-506 mediates the cross talk between p53 and NF-kB signal pathways. RESULTS miR-506 is upregulated in lung cancer and functions as a pro-apoptotic factor To identify miRNAs associated with lung cancer, we profiled the expression of 500 human miRNAs in non-small-cell lung cancer (NSCLC) patient samples by quantitative real-time reverse transcription–PCR (qPCR). Expression levels of 51 miRNAs were altered in cancerous lung tissues when compared with normal lung tissues. We explored the biological relevance of these deregulated miRNAs by manipulating their expression in lung cancer cells. Seventeen miRNA mimics were synthesized and transfected into the NSCLC cell line 95D, and the cells were then assayed for caspase-3/7 activity (Figure 1a). Among the analyzed miRNAs, miR-506 mimics induced the most robust increase in caspase-3/7 activity (B2.5-fold compared with the mock or notarget control miRNA; Figure 1a). The pro-apoptotic activity of miR-506 mimics was confirmed by fluorescence-activated cell sorting (FACS) in 95D cells and three other lung cancer cell lines (Figure 1b). The percentage of apoptotic 95D cells increased by 180% (from 9.38 to 26.4%; Figure 1b). However, less-pronounced increases were observed in H1299 and H661 human lung

carcinoma cells (57% and 74%, respectively, Figure 1b). Taken together, these results suggest that miR-506 has a role in stimulating apoptosis of lung cancer cells. We next assayed expression levels of miR-506 in lung cancer cell lines as well as in samples from 156 NSCLC patients by using qPCR. miR-506 expression levels were higher in most cancer cells when compared with normal cell lines (Figure 2a). In NSCLC patients, miR-506 expression level was upregulated in lung cancer tissues compared with the adjacent non-cancer tissues in 83% (129/156) of cases (Figure 2b). To assess the expression level of miR-506, in situ hybridization experiments were carried out in lung samples of NSCLC patients (Figure 2c). The association between miR-506 level and clinicopathlogical variables showed the upregulated miR-506 levels in men to be significantly higher than those in women (Supplementary Table S1). The other variables yielded no significant differences in miR-506 levels. Tumor samples with higher miR-506 expression level displayed increased cleavage of the pro-apoptotic protein poly ADP-ribose polymerase (PARP) (Figure 2d). This was consistent with the increased apoptosis seen in lung cancer cell lines with higher miR-506 expression level (95D, A549 and H1299) (Figure 1b). Thus, higher expression of miR-506 correlates with increased apoptosis in lung cancer samples. miR-506 directly targets NF-kB p65 to induce apoptosis of lung cancer cells To elucidate the miR-506 regulatory mechanism, it is important to identify this miRNA’s target(s). Using TargetScan 5.2, we identified

Figure 1. miR-506 mimics induced apoptosis in lung cancer cells. (a) Caspase-3/7 activity of 95D cells treated with 17 miRNA mimics. Cells without any treatment (Mock), with adriamycin (positive control) and with no-target control miRNA (NC) were used as experimental controls. The data are normalized to NC. Error bars indicated s.d. (n ¼ 3); *Po0.05; **Po0.001 relative to NC. (b) A FACS analysis of cell apoptosis of A549, H1299, 95D and H661 lung cancer cells treated with miR-506 mimics. Oncogene (2014) 1 – 13

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Figure 2. Upregulated miR-506 levels in lung cancer patients’ samples and in lung cancer cell lines. (a) miR-506 expression levels in lung cancer cells and normal cell lines. The abundance of miR-506 was normalized to U6 rRNA. (b) The miR-506 expression levels of 156 lung cancer patients’ samples by comparing the lung cancerous tissues and adjacent non-cancerous tissues. The P-value was calculated by paired Student’s t-tests. (c) A representative image of a sample section with notable miR-506 staining (dark blue) in the normal lung tissue. In situ hybridization of miR-506 was used a miR-506-LNA probe in lung cancerous tissues and adjacent non-cancerous tissues. (d) Relationship between miR-506 expression level and PARP activation in lung cancer patients. The western blot was performed to assess PARP activation. The numbers of 1, 61, 65, 83 and 89 s are represented as the patient numbers of 1, 61, 65, 83 and 89, respectively; N represents the adjacent non-cancerous tissue; and T represents the tumor tissue.

putative miR-506 targets, and 12 out of these are associated with cell proliferation, cell cycle or survival/apoptosis. From these, we identified CCND2, MAPK1 and NF-kB p65 that show higher scores compared with others (Supplementary Figure S1a). NF-kB p65 is a subunit of the NF-kB dimer.24 To determine whether these genes are regulated by miR-506 in NSCLC, we used a 30 -UTR reporter assay. The reporter plasmid and the miR-506 mimics were cotransfected into A549 cells and the activity of the luciferase reporter was monitored. Although the Renilla luciferase activity of NF-kB p65 30 -UTR reporter plasmid was downregulated upon cotransfection with miR-506 mimics, but not in response to control oligonucleotides, the CCND2 and MAPK1 30 -UTR reporters were not regulated either (Supplementary Figure S1a). The miR-506 mimics also induced downregulation of luciferase activity in 95D cells, where reporter activity was reduced by 43.4%. Mutation of the putative miR-506-binding site in the 30 -UTR of the reporter abolished this regulation (Figure 3a), indicating that suppression is specifically mediated through miR-506-dependent base-pairing interactions. Similarly, miR-506 significantly reduced NF-kB p65 mRNA (Figure 3b) and protein (Figure 3c) levels in 95D cells. Furthermore, NF-kB p65 protein levels were also underexpressed in 95D cells as well as in three other NSCLC cell lines after transfection with miR-506 mimics (Figure 3c). In addition, p65 protein expression was negatively correlated with miR-506 levels in lung cancer samples (Figure 3d). Next, we investigated whether induction of apoptosis in NSCLC cells treated with miR-506 mimics resulted from deregulation of NF-kB target genes. For this, we examined relative mRNA levels of apoptosis-related genes by qPCR (Figure 3e). Expression of multiple anti-apoptotic genes known to be activated by NF-kB, including A1/A20, c-IAP1/2 and TRAF2, was inhibited in the presence of miR-506 mimics. As these genes displayed no & 2014 Macmillan Publishers Limited

significant miR-506 target sequences within their 30 -UTRs, these findings suggest downstream effects resulting from miR-506mediated NF-kB p65 downregulation. Overall, our results suggest that NF-kB p65 is a downstream target of miR-506 in lung cancer cells. To further examine the relevance of NF-kB p65 regulation of apoptosis in lung cancer cells, we knocked down NF-kB p65 using a p65-specific small-interferring RNA (siRNA) (si-p65) and observed canonical cell apoptosis phenotypes, including altered cell morphology and blebbing (Supplementary Figure S1b). Expression of genes downstream of NF-kB p65, including c-FLIPs, c-IAP1/2 and TRAF2, reported before to function in suppressing caspase-8 activation,25 was decreased in cells transfected with miR-506 mimics (Figure 3e). We also detected significant accumulation of activated caspase-8 and enhanced cleavage of PARP, both hallmarks of apoptosis, in response to miR-506 mimics in 95D cells (Figure 3g). Si-p65 and miR-506 mimics both induced apoptosis in 95D cells (Figures 3e and f and Supplementary Figure S1c). Moreover, the apoptosis-inducing effect could be repressed in cells transfected with an antisense oligonucleotide (antagomir) complementary to miR-506, which also enhanced NF-kB p65 protein levels in lung cancer cells (Figures 3f and g). We then constructed an EGFP-tagged NF-kB p65-encoding gene lacking miR-506 target sequences, and expressed it in 95D cells. This modified gene could induce activation of NF-kB p65, but was not inhibited by co-transfection with miR-506 mimics (Figure 3h). Furthermore, expression of this transgene caused attenuation of PARP cleavage, even when co-expressed with miR-506 mimics, suggesting that the NF-kB p65 transgene reversed the effects of miR-506 and prevented apoptosis (Figure 3h). TNFa-induced apoptosis is mediated in part by transcriptional activation of caspase-8 and is antagonized by NF-kB activation.25 Oncogene (2014) 1 – 13

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4 We predicted that sensitivity of A549 cells, which contain low endogenous levels of miR-506, to TNFa-induced apoptosis would be enhanced after treatment with miR-506 mimics. Consistent

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with this idea, cleaved PARP accumulated in cells treated simultaneously with TNFa and miR-506 mimics (Figure 3j). Moreover, this treatment increased the percentage of apoptotic cells

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5 from 9.86 to 13.36%, while a non-specific control miRNA mimetic administered along with TNFa treatment caused no significant change over TNFa treatment alone (Figure 3h). The increase in PARP activation and apoptosis induced by miR-506 mimics was comparable to that induced by exposure to si-p65 (Figure 3j), indicating that suppression of NF-kB p65, and hence of NF-kB activity, underlies the increased sensitivity to TNFa-induced apoptosis. Expression of miR-506 positively correlates with oxidative stress and participates in oxidative stress-induced apoptosis Previous research reported that many types of cancer cells have higher ROS levels than their normal counterparts.17,26,27 Hence, we investigated whether high miR-506 levels could be due to ROS accumulation by measuring SOD2 protein expression in the samples of lung cancer patients. The results showed that SOD2 levels strongly correlate with the expression of miR-506 in these tested samples (Figures 4a and b). Next, we examined miR-506 expression level after treating lung cancer cells with serum-free medium, oxidants and antioxidants. miR-506 levels were increased in serum-free medium and after H2O2 treatment, but were decreased after exposure to antioxidants, such as butyl-hydroxy anisd (BHA) and N-acetyl-l-cysteine (NAC) (Figures 4c and d). Expression levels of miR-506 correlated inversely with NF-kB p65 protein levels in lung cancer cells treated with oxidants or antioxidants (Figure 4e). Moreover, H2O2 treatment induced p53 activation in A549 cells (Figure 4e). We further found that expressions of miR-506 and hemeoxygenase-1, a classic RedOx marker,28 were reduced with NAC treatment in A549 cells (Figure 4f). However, miR-506 and hemeoxygenase-1 expressions were upregulated after treated with phenethyl isothiocyanate (PEITC), but the affect was abolished by combined treatment of PEITC with NAC (Figure 4f). In the non-cancer lung cells (HLF1), miR-506 levels were also increased by exogenous H2O2 treatment in a concentration-dependent manner (Figure 4g); p53 was elevated but NF-kB p65 was decreased (Figure 4g). In addition, PEITC elevated ROS levels, and enhanced p53 and miR-506 expression level (Supplementary Figures S2a–c). Upregulation of ROS, p53 and miR-506 expression level could be returned to basal by NAC treatment in HLF1 cells (Supplementary Figures S2a–c). Moreover, miR-506 and SOD2 protein levels positively correlated with ROS levels in HFL-1 cells (Supplementary Figures S2b and c). There is growing consensus that oxidative stress has pivotal role in regulating apoptosis. As described above, lung tumor samples show increasing cell apoptosis reflected by increasing PARPcleaved levels (Figure 2d). Interestingly, we found that miR-506 participated in inducing cell apoptosis (Figures 4e and j) and inhibiting the viability (Figure 4i). However, although NF-kB p65 level was decreased and miR-506 level was increased with elevating ROS levels, PARP activation failed to occur in H1299 (p53 null) cells treated in the same conditions (Figure 4e). These

results suggest that activation of p53 has a key role in PARP activation induced by ROS. AntagomiR-506 could restore inhibition of H2O2-induced cell viability in A549 and 95D lung cancer cells (Figure 4i), and suppressed H2O2-mediated induction of apoptosis in A549 cells (Figure 4j). These results demonstrated that miR-506 is an important factor in the ROS-induced cell apoptosis cascade. Expression of miR-506 is regulated by p53 We next explored the mechanism underlying ROS-related miR-506 regulation in lung cancer cells. ROS stress is thought to activate p53 expression,4,29 and we found that exogenous ROS can activate p53 and promote miR-506 expression level (Figure 4e). Therefore, we examined genome sequences upstream of miR-506 and found a putative p53-response element (p53 RE) B782-bp upstream of MIR506 (Figure 5f), suggesting that miR-506 can potentially be regulated by p53. We assayed p53 and miR-506 levels in lung cancer patients using qPCR and demonstrated significant correlation (Pearson correlation index, R ¼ 0.73) between MIR506 and TP53 mRNAs levels in stage I lung cancer patients (Figure 5a). We then examined the effect of adriamycin as p53 activator on miR-506 expression level in a series of lung cancer cell lines. Consistent with the regulation of p53, miR-506 was markedly induced by the treatment of adriamycin in A549, H460 and 95D cell lines, which express functional p53. In H661 and H1299 cells, which lack and lost functional p53, respectively (http://p53.free.fr/ Database/Cancer_cell_lines/NSCLC.html), miR-506 level was extremely low and not inducible by adriamycin (Figure 5b). Moreover, A549 cells treated with p53-specific siRNA abolished adriamycininduced expression of miR-506 (Figure 5c). Conversely, when the expression of wild-type p53 was restored in H1299 cells by transfection of exogenous expression vectors, all expressions of the primary (pri-), precursor and mature forms of miR-506 were increased relative to control cells transfected with empty vectors (Figure 5d). In addition, expression of wild-type p53 caused 460% reduction in NF-kB p65 protein levels (Figure 5e). Notably, the mutant p53 (R273H), which lacks DNA-binding activity, failed to induce pri-miR-506 expression level upon adriamycin stimulation. Under these conditions, pri-miR-506 expression level was similar to that of control cells, suggesting that p53 may directly regulate the pri-miR-506 promoter. Chromatin immunoprecipitation using p53-specific antibodies revealed that this promoter region is bound by p53 in A549 cells treated with adriamycin (Figure 5f), implicating that miR-506 transcription might be induced directly by p53. Although induction of miR-506 expression level by R273H mutant p53 was not as prominent as that by functional p53, it still caused an approximately twofold increase in mature miR-506 levels compared with empty vector. We speculate that miR-506 expression level were regulated by other mechanisms, such as that DNA-binding-defective mutant p53 may affect processing

Figure 3. NF-kB p65 is a direct target of miR-506 and involved in miR-506-induced apoptosis in lung cancer cells. (a) The miR-506-binding region at NF-kB p65 3’-UTR and the luciferase reporter assay. The seed region of miR-506 is underlined and the mutations are shown in bold italicized letters. (b) qPCR analysis of NF-kB p65 mRNA levels in 95D cells treated with miR-506 mimics. NF-kB p65 mRNA levels were normalized to b-actin by using the 2  DDCt method. (c) Western blot analysis of NF-kB p65 protein levels in the different lung cancer cell lines treated with miR-506. (d) Relationship between miR-506 and NF-kB p65 expression levels in lung cancer patients. Total proteins from lung tumor and normal tissues were extracted by RIPA lysis and assayed by western blot to assess NF-kB p65 protein levels. The numbers of 17, 18, 38, 43 and 45 s are represented as the patient numbers of 17, 18, 38, 43 and 45, respectively. (e) Expression of NF-kB downstream genes in 95D cells treated with miR-506 by qPCR assay. (f ) Cell apoptosis assay of 95D cells treated with NC, miR-506 mimics, antagomiR-506 or si-p65 by FACS. (g) Western blot analysis of caspase-8 and PARP cleavage treated with miR-506 or si-p65. The PARP protein was normalized to b-actin. (h) Western blot analysis of PARP activation induced by miR-506 being reverted by NF-kB p65 overexpression in 95D cell. NC, no-target control miRNA; pEGFP-c1, empty vector; Ex-p65, exogenous p65 expression vector pEGFP-p65 with no miR-506-binding site. (i) miR-506 affects on TNFa-induced apoptosis in A549 cells by FACS analysis. FACS was performed in A549 cells treated TNFa (final concentration of 15 ng/ml) at 48 h after miRNA/siRNA transfection. Cells were harvested and stained for Annexin-V and 7-AAD 24 h after TNFa addition. (j) PARP activation in A549 cells upon TNFa-treatment (15 ng/ml) treated with miR-506 or si-p65. Cells were harvested for western blot analysis at 24 h after TNFa addition. Error bars indicate s.d. (n ¼ 3); *Po0.05, **Po0.001. & 2014 Macmillan Publishers Limited

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6 and maturation of miR-506 through other mechanisms as previously reported.30 A luciferase reporter vector was constructed by inserting a 1047-bp genome fragment, which located upstream of MIR506 into PGL3 vector. Then the reporter

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vector was co-transfected with wild-type P53, mutant P53 (R273H) or empty vector. It showed that only the wild-type P53 can activate the luciferase activity. In addition, exogenous H2O2 treatments enhanced more the activity than that of the wild-

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miR-506 mediates cross talk between p53 and NF-kB in lung cancer M Yin et al

7 type P53 (Figure 5g). Taken together, our findings revealed that p53 directly regulates miR-506 expression level, which in turn directs inhibition of NF-kB p65 accumulation. Downregulation of NF-kB p65 by miR-506 leads to ROS-induced p53 activation in a cancer cell-specific manner NF-kB activity can inhibit ROS by promoting antioxidant enzymes.31 Therefore, we next evaluated whether NF-kB p65 inhibition by miR-506 leads to ROS activation by measuring ROS levels in cancer cells transfected with miR-506 and si-p65. We observed that treatment of miR-506 and si-p65 increased ROS levels in A549 and 95D cells, but failed to induce ROS in non-cancer HLF1 cells (Figures 6a and e). ROS stress is thought to activate p53 expression.4,29 We examined expression level of p53 after ROS enhancement induced by NF-kB p65 inhibition, and found that expression of p53 mRNA and protein increased upon NF-kB p65 downregulation after cell transfection with either miR-506 mimics or si-p65 (Figures 6b–d). On the other hand, antagomiR-506 enhanced NF-kB p65 protein levels and inhibited p53 levels in A549 cells (Figures 6b–d) and 95D cells (Supplementary Figures S3a and b). In A549 cells, p53 expresses at a relatively low level in untreated lung cancer cells, where the majority of p53 protein was localized in the cytoplasm (Figure 6b). Suppression of NF-kB p65 by miR506 mimics or si-p65 led to nuclear translocation of p53, an important indicator of p53 activation (Figure 6b). Nuclear translocation of p53 was also observed in 95D cells upon NF-kB p65 downregulation by treatment with miR-506 mimics or si-p65 (Supplementary Figure S3a). Consistent with activation of p53, we found that p53-targeted BBC3 gene expression was significantly induced in cells treated with either miR-506 mimics or p65 siRNA (Figure 6d). AntagomiR-506 slightly decreased ROS level in A549 cells, as that is of low abundance of miR-506 (Figure 6a). Intriguingly, both miR-506 mimics and p65 siRNA failed to activate p53 expression in the non-cancer cells HLF1 (Figures 6b and c) and MRC-5 (Supplementary Figures S3a and b). At the same time, after treatment with miR-506 mimics or si-p65, p53, p53-targeted BBC3 gene and miR-506 levels showed no activation in normal lung HLF1 cells (Figure 6b–d). This might be partially attributed to the low basal ROS levels found in the normal cell lines (Supplementary Figure S3c), which were not affected by miR-506 mimics or p65 siRNA (Figure 6a). In addition, the antioxidants BHA and NAC blocked NF-kB p65 suppression-induced p53 activation by abrogating ROS generation (Figure 6f). The similar ROS-droved p53-miR-506 circuits were found to exist in normal lung HLF1 cells by exogenous ROS treatment (Figure 4g; Supplementary Figure S2a–c). These results demonstrated that ROS, p53 and miR-506 constitute a loop that negatively regulates NF-kB p65 expression in cancer cells (Figure 6g). Upregulated miR-506 serves as a selective tumor suppressor and inhibits lung cancer tumorigenesis in vivo The qPCR studies of 156 NSCLC patients revealed increased expression of miR-506 relative to cancer-stage progression (Figure 7a). Although miR-506 is upregulated in lung cancer,

survival analysis revealed that advanced-stage patients with elevated miR-506 levels survived better than those with lower miR-506 levels in stages II plus III, the results showed significance (P-value ¼ 0.0216) (Figure 7b). The early-stage patients showed upregulated miR-506 levels, suggesting worse survival than those down-regulated and the results showed no significance (P-value ¼ 0.174) (Supplementary Figures S4b–d). The total patients and showed no significant advantage in survival analysis (Supplementary Figure S4a). We have performed a dosedependent cell viability study of miR-506 molecules with six NSCLC cell lines and three normal cell lines. Our data showed that cell viability was affected in a dose-dependent manner after treatment with miR-506 mimics for NSCLC cells. Significantly, miR506 selectively inhibited cell viability in lung cancer cells and not in normal cells (Figure 7c). A possible mechanism by which miR506 selectively kills lung cancer cells might be ‘oncogene addiction’- NF-kB p65 addiction in lung cancer cells. Previous works have reported that the NF-kB activity is required in lung cancer.32,33 The expression of NF-kB p65 downstream genes including c-IAP2,c-FLIPs and TRAF2, which antagonize cell apoptosis and promote cell proliferation, are much higher than in normal cells (Figure 7d). In addition, the transfection of miR-506 mimics or siRNAs of NF-kB p65/NF-kB p50 (si-p65, si-p50) significantly inhibited the cell viability of A549 and H358 lung cancer cells, but with only minor suppression in normal lung cells HLF1 (Figure 7e). So, we postulated that miR-506 selectively killing lung cancer cells might be through addicted NF-kB p65 inhibition. To confirm the tumor suppressor function of miR-506, we established a BALB/c nude mouse xenograft model by using human lung cancer 95D cells. These cells were pre-treated with miR-506 mimics or non-targeting control and were then injected into the right forelimb flank of nude mice. The tumor volume in animals that received cells pre-treated with miR-506 mimics was significantly (Po0.05) smaller than that in animals that received administered cells treated with an identically formulated nontargeting control (Figure 7f). This difference in volume was observed from day 20 and was confirmed in tumors harvested from animals killed at day 48 (Figure 7f). These results demonstrated that miR-506 can significantly inhibit tumor growth in a nude mouse xenograft model.

DISCUSSION Typically, miRNAs are considered as tumor suppressors when downregulated in tumors, or oncogenes when upregulated in tumors. However, similar to p53, p16, p21 and other classic tumor suppressors, miR-506 is a tumor suppressor but upregulated in tumors.34 Such tumor suppressors are thought to act as sensors and defenders against abnormal oncogene activation and abnormal cell stress. It was also reported that the tumor suppressor miR-34a was upregulated in 460% colon cancer tissues compared with normal colon mucosa.35 miR-34a was also increased in NSCLC.5 Moreover, high-grade human ovarian adenocarcinomas have high miR-200a levels, and the miR-200adependent stress signature correlates with good survival of

Figure 4. The expression levels of miR-506 were regulated by ROS levels and induced cell apoptosis. (a) miR-506 expression levels in lung cancer patients’ tissue samples. (b) The protein levels of SOD2 in lung cancer patients. The numbers of 17, 18, 34, 43, and 45 s are represented as the patient numbers of 17, 18, 34, 43 and 45, respectively. (c–e) A549 and H1299 cells were plated at 30% confluence. After 48 h culture, cells were treated with serum-free medium, H2O2 (1 mM in A549; 100 mM in H1299), BHA (50 mM) or NAC (5 mM) for 24 h. Cells were then harvested for ROS FACS analysis (c), miR-506 qPCR assay (d) and western blot (e). (f ) NAC restored ROS and miR-506 levels in A549 cells. A549 cells were subcultured at 30% confluence for 24 h culture, and then treated with mock, NAC (5 mM), PEITC (50 mM) or PEITC þ NAC (50 mM þ 5 mM) for another 24 h. Cells were harvested to assess hemeoxygenase-1 and miR-506 levels by qPCR. (g) miR-506 expression level in different concentrations of H2O2 and NF-kB p65 protein levels with 100 mM H2O2 in HLF1 cells. (h) Suppression of cell viability by miR-506 mimics rescued by antagomiR-506 in 95D cells. (i) AntagomiR-506 rescued H2O2-induced cell viability suppression in A549 and 95D cells. Cells were treated with H2O2 (500 mM in A549 and 100 mM in 95D) for another 24 h. Cell viability was examined by CCk-8 assay. (j) Western blot assay of PARP activation in A549 cells treated same as in i. Data are presented as mean±s.d. (n ¼ 3); *Po0.05, **Po0.001. & 2014 Macmillan Publishers Limited

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Figure 5. miR-506 expression level is regulated by p53 in lung cancer cells. (a) The expression relationship between p53 and miR-506 in lung cancer patients analyzed by Pearson’s correlation test. LN means that the data of relative expression levels are changed through natural logarithms. (b) miR-506 abundance is dependence with p53 in various lung cancer cell lines. U6 rRNA was used as internal control. (c) Downregulation of p53 led to decreased miR-506 expression level. The A549 cells were treated with p53-specific siRNA (si-p53), mock (adriamycin  ) and 100 nM adriamycin (adriamycin þ ). The cells were harvested for miR-506 expression level test by qPCR. U6 rRNA was used as internal control. (d) miR-506 expression level in H1299 cells transfected with 1 mg/ml empty vector (M04), wild-type p53 vector (M04-p53WT) or mutant p53 vector (M04-p53 R273H). The expression levels of mature miR-506, primary-miR506 and precursor-miR-506 were normalized to U6 rRNA. (e) Western blot analysis of exogenous p53 (Ex-p53) and endogenous NF-kB p65 in H1299 cells treated same as described in d. (f ) Enriched p53-binding region in the upstream of MIR506 transcription starting site. The putative p53 RE is shown as a box, and the DNA sequences are aligned with the canonical p53 RE. The forward primer (FP) and reverse primer (RP) used for qPCR and semiqRT–PCR are indicted as small blue arrows. Chromatin immunoprecipitation assays were carried out in A549 cells treated with 100 nM adriamycin for 24 h. Cell lysates were aliquoted into input, IgG pull-down (IgG) and p53 pull-down (anti-p53). Equal amounts of PCR products were subjected to agarose electrophoresis. (g) miR-506 promoter reporter assay. The 1047-bp genome fragment contains the p53 RE, which is located in the upstream of MIR506, were clone to the pGL3-promoter vector. The constructed reporter vectors were co-transfected with an expression construct for wild-type p53, mutant p53 or empty vector in H1299 cells. After 24 h transfection, the cells were treated with mock or 0.5 mM H2O2 for another 24 h. Then the reporter assay was conducted. Data are presented as mean±s.d. (n ¼ 3); *Po0.05, **Po0.001.

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9 patients in response to treatment.36 In this study, we found that miR-506 is over-expressed in 83% lung cancer tissues compared with lung non-cancer tissues. The advanced-stage lung cancer patients with upregulated miR-506 had better survival rates, although elevated miR-506 did not seem sufficient to completely inhibit lung cancer formation. It is necessary to identify the mechanisms that allow cancer cells to cope with elevated levels of tumor suppressor miRNAs in order to develop more effective miRNA-dependent therapies. In addition, pre-treatment of lung cancer cells with miR-506 mimics effectively inhibited tumorigenesis in a mouse xenograft model. Besides the lung cancer cells, the miR-506 can inhibit p65 and activated the p53 in the cervical carcinoma cell HeLa (Supplementary Figures S5b–e). The miR-506 mimics also suppressed the cell viability in the HeLa cell, the prostate cancer cell DU145 and the renal carcinoma cell RCC4 (Supplementary Figure S5a). Consistent with this, the recent reports demonstrated that miR-506 functions as a tumor suppressor in breast cancer and ovarian cancer through inhibition of cancer cell metastasis.37,38 Altogether, these results suggest that miR-506 functions as a tumor suppressor rather than an oncogene in cancer cells. Our studies provide first evidence that a p65 subunit of NF-kB is a direct downstream target of miR-506. In the lung cancer samples, the upregulation of miR-506 correlated with the downregulation of NF-kB p65 in lung cancer tissue samples (Figure 3d). And miR-506 mimics inhibited NF-kB p65 protein levels and induced cell apoptosis and reduced cell viability in lung cancer cell lines, but not in the normal cells (Figures 7c and d). It was reported that NF-kB is constitutively expressed in various cancers including lung cancer,39 and NF-kB activation is required for lung cancer tumorigenesis.32,33 Inhibition of NF-kB activation induces apoptosis in a mouse lung cancer model and in human NSCLC.32,40 Interestingly, the cancer cells seems to be more dependent on or ‘addicted’ to one or a few genes for both maintenance of the malignant proliferation and cell survival, which is called the ‘oncogene addiction’.41,42 Our results also indicate that the lung cancer is more addicted to NF-kB p65 than normal lung cells, therefore, the lung cancer cells were more vulnerable to miR-506-induced NF-kB p65 inhibition. In addition, the NF-kB p65 have crucial roles in normal cell function. However, the miR-506 transfections of miR-506 in lung normal cells have minor influence on cell viability and cell apoptosis than lung cancer cells. The reason is probable because of the fact that the miR-506-induced NF-kB p65 inhibition is in the ‘tuning’ way.43 Our results showed the NF-kB p65 protein levels were higher in the miR-506 mimics treatment than the sip-65 transfection (Figures 3g, 6b and c).43 Furthermore, cross talk between the p53 and NF-kB pathways has been studied in various physiological contexts. Activation of p53 antagonizes NF-kB transcriptional activation and induces apoptosis in a mouse model.32 Moreover, p53 can suppress lipopolysaccharide-induced inflammatory response by inhibiting activation of NF-kB.44,45 In this study, our results revealed that downregulation of NF-kB p65 by miR-506 transfection leads to an increase in ROS levels and stimulates p53 expression in a ROS-dependent manner, which, in turn, induces miR-506 expression level in lung cancer cells. Furthermore, activation of p53 by exogenous oxidative stress enhanced miR506 expression level and suppressed NF-kB p65 protein levels. The end result of this process is a decrease in cell survival signaling and increased apoptosis of lung cancer cells (Figure 6g). Many studies have focused on identifying naturally occurring and synthetic compounds that simultaneously target both the p53 and NF-kB pathways.15,46–50 The miR-506 transfection have better results in cell viability inhibition (Figure 7c) and apoptosis assay, and (Figure 1b) in the lung cancer cells containing functional p53 protein than the mutant or null p53 protein. Our findings suggest that administration of miR-506 mimics could provide a potent therapeutic approach for the induction of p53-dependent & 2014 Macmillan Publishers Limited

apoptosis in cancer cells, which have functional p53 protein and over-activated NF-kB p65 activity. It has been suggested that transformed cells possessing the potential to elevate ROS generation are likely to be more vulnerable to damage by further oxidative stress induced by exogenous agents.17,51–54 Oxidative stress is considered to be a target for selectively eliminating tumor cells without affecting normal cells.17 Our results showed that upexpression of miR-506 contributes to accumulation of ROS, and miR-506 expression level highly correlates with ROS levels both in vivo and in vitro. Interestingly, the lung cancer cells contain higher level of ROS than normal lung cells (Supplementary Figure S3c), and miR-506 specifically enhanced ROS levels by inhibiting NF-kB p65 expression in lung cancer cells but not in normal cells. Hence, the activation of p53 via miR-506 mimics appeared to be specific to lung cancer A549 cells (Figures 6b–d) and 95D cells (Supplementary Figures S3a and b). We found that in the noncancer cell lines, such as MRC-5 and HLF1, while miR-506 reduced NF-kB p65 expression, it failed to activate p53 (Figures 6b–d; Supplementary Figures S3a and b). In cells lacking function of p53, miR-506 seems to be regulated by other pathways, which are also regulated via ROS and are interesting for future studies (Figure 6g). In the p53 null lung cancer cell H1299 the exogenous ROS also can enhance miR-506 level and inhibit NF-kB p65 (Figure 4c–e). So miR-506 would be a remedy similar to other p53-related miRNAs, such as miR-34, miR-192 and miR-107.6,55,56 We found that ectopic expression of miR-506 significantly reduced cell viability in p53 null lung cancer line H358 and H1299, with no significant inhibition of the viability of normal cells (Figure 7c), supporting our concept that miR-506 molecules as a promising therapeutic agent can selectively kill lung cancer cells. Therefore, miR-506 molecule can selectively kill lung cancer cells, further implicating as a promising therapeutic agent. In conclusion, our results suggest that miR-506 inhibits NF-kB pathway, evokes ROS generation and activates p53 to promote miR-506 expression level in lung cancer cells (Figure 6g). Furthermore, exogenous miR-506 molecules elevate ROS generation and p53 activation to trigger apoptosis and suppress lung cancer cell viability, but has no affect for normal cells (Figure 7c). These observations demonstrate that miR-506 molecule might provide a potential therapy for successful selective killing of lung cancer cells.

MATERIALS AND METHODS Cell culture and tissue sample collection A549 and H1299 cells (human lung adenocarcinoma cell lines), H460, 95D, and H661 cells (human large-cell lung cancer cell line), and HCC827 cells (human small-cell lung cancer cell line) were maintained in Prime-1640 medium (Gibco, Invitrogen, Shanghai). MRC-5 cells (normal lung cell line) were maintained in Ham’s F12K (Gibco). HeLa, HEK293, DU145 and HLF-1cells (human normal lung cell line) were maintained in modified Eagle’s medium (Gibco). All cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), except 95D, which was a kind gift from Dr Duanqing Pei at the Guangzhou Institute of Biomedicine and Health, Guangzhou, China. One hundred fifty-six lung cancer tissues and respective non-cancer lung tissues were obtained from the Guangzhou Institute of Respiratory Diseases (Guangzhou, China) in 2008 with informed consent and Institutional Review Board permission. All clinical and biological data were available for the samples. Samples were immediately snap-frozen in liquid nitrogen and stored in liquid nitrogen until use.

Flow cytometric apoptosis analysis After transfection, cells were trypsinized and washed twice in cold phosphate-buffered saline. Cells were stained using the Annexin-V-PE apoptosis detection kit (BD, San Diego, CA, USA) according to the manufacturer’s protocol and were analyzed by FACS (BD). Oncogene (2014) 1 – 13

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10

Figure 6. Repression of NF-kB p65 increased ROS levels and selectively induced p53 activation in lung cancer cells but not in normal cells. (a) The intensity of DCF fluorescence of A549 and HLF1 cells treated with NC miRNA, miR-506 mimics, antagomiR-506 or si-p65 measured by FACS. The purple shading indicates the treatment cell population and the green line represents cell population treated with NC. (b) Cell imaging of A549 and HLF1 cells treated with NC miRNA, miR-506 mimics, antagomiR-506 or si-p65. Immunostaining was performed 72 h after transfection. The scale bar at the bottom right corner indicates 100 mm. (c) Western blot assay of NF-kB p65 and p53 protein levels in A549 and HLF1 cells treated same as described in a. (d) Expression of p53, BBC3 and miR-506 in A549 cells treated same as described in a. Data represent mean±s.d.; *Po0.05, **Po0.001 compared with NC. (e) The intensity of DCF fluorescence in A549 and 95D cells treated with NC miRNA, miR506 mimics or si-p65 measured by FACS. Data represent mean±s.d.; *Po0.05, **Po0.001 compared with NC. (f ) Western blot analysis of endogenous p53 and NF-kB p65 proteins in A549 cells treated with the antioxidants BHA (50 nM) and NAC (5 mM). (g) Postulated miRNA-506 regulation pathway. miR-506 mediates the cross talk between the p53 and p65/NF-kB pathways. The downregulation of NF-kB p65 by miR506 caused the activation of miR-506 and contributed to the ‘vicious cycle’ of ROS.

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11

Figure 7. miR-506 functions as a tumor suppressor and selective killing of lung cancer cells through inhibiting NF-kB. (a) miR-506 was upregulated in cancer tissues along with lung cancer progression. miR-506 levels were normalized to U6 rRNA. LN means the data of relative expression levels were changed through natural logarithms. The P-value was calculated by paired Student’s t-tests; *Po0.05, **Po0.001. (b) Kaplan–Meier survival curves for stages II þ III lung cancer patients. The red curve shows the patients whose miR-506 levels were upregulated, and the black curve shows the patients whose miR-506 levels were downregulated in the tumor tissues. (c) Cell viability assay of NSCLC and normal cell lines treated miR-506 mimics. Cell viability was determined 72 h after transfection with different concentrations of miR-506 mimics by the CellTiter96 assay. Error bars indicate s.d. (n ¼ 3). (d) The downstream genes of NF-kB p65 were tested by qPCR. The expression level of c-IAP2,c-FLIPs and the TRAF2 in lung cancer cells and normal cells were measured and normalized to HLF1 cell. (e) The cell viability was measured after inhibiting the NF-kB activity in lung cancer cell and normal cells. The lung cancer cells A549 and the normal cells HLF1 were transfected with NC miRNA, miR-506 mimics, antagomiR-506, si-p50 and sip-65. Seventy hours after transfection the cell viability were tested by CellTiter96 assay. (f) Tumor growth plots of BALB/c nude mice treated with miR-506. 95D cells were treated with NC miRNA, miR-506 mimics or Lipofectamine 2000 (Lipo) only. Cells were then implanted into BALB/c nude mice. Tumor volumes of mice treated miR-506 (n ¼ 6), NC (n ¼ 3) and Lipo (n ¼ 3) were measured at the indicated days after implantation. Tumor volume (V) was monitored by measuring the length (L) and width (W) with calipers and calculated with the formula V ¼ (L  W2)  0.5. Data represent mean±s.d.; *Po0.05, **Po0.001 compared with NC. Images of mice and tumors (right) were taken at day 48 after implantation. & 2014 Macmillan Publishers Limited

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12 Caspase-3/7 activity assay

Kaplan–Meier survival analysis and statistical analysis

Cell culture and transfections were carried out in 96-well plates, and the final concentration of miRNA mimics used for transfection was 50 nM. Caspase-3/7 activity was analyzed at 72-h post transfection according to the manufacturer’s protocol (Promega, Madison, WI, USA).

The analysis was conducted using R software (www.R-project.org) with Bioconductor packages (www.bioconductor.org) named ‘EMA’. The ‘EMA’ was used to compute and draw survival curves for censored data using Kaplan–Meier’s method. The P-values were tested by using log-rank test to assess the difference between the survival curves. The expression level of miR-506 was measured by qRT–PCR. The downregulated/upregulated means the miRNA level were decreased/increased in lung cancer tumors compared with normal counterparts. Statistical significance was determined by paired or unpaired Student’s t-tests in cases of standardized expression data. Multiple group comparisons were performed using analysis of variance. Significance was determined by Po0.05.

miRNA expression analysis Real-time qPCR was performed to examine the expression level of mature miRNAs, as described previously.23

Locked nucleic acid—in situ hybridization The in situ hybridization kit (no. 10000-89999-15) was purchased from Exiqon (Vedbaek, Denmark). In situ hybridization of mR-506 was performed as previously described.57

CONFLICT OF INTEREST Luciferase reporter assay

The authors declare no conflict of interest.

Luciferase reporter assays were performed as previously described.58 The luciferase activity was assessed using the Dual-Glo luciferase assay kit (Promega), and luminescence intensity was read with a microplate luminometer following the manufacturer’s protocol. Transfections were performed in duplicate and repeated three times.

ACKNOWLEDGEMENTS

Chromatin immunoprecipitation assay Adriamycin was added to A549 cells (final concentration: 100 nM) for 24 h. Cells were fixed in 1% formaldehyde for 15 min at room temperature, followed by incubation with glycine (final concentration: 125 nM) for 15 min. Cells were then washed twice with cold phosphate-buffered saline and harvested in lysis buffer (20 mM Tris–HCl, pH 8.0, 85 mM KCl, 1.0 mM EDTA, 0.5 mM EGTA, 0.5% Nonidet-P40, protease inhibitors). After centrifugation, cell pellets were dissolved in nuclear lysate buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS and protease inhibitors) and subjected to sonication. Nuclear extracts were pre-cleaned with 50 ml of 50% Protein A–Sepharose slurry at 4 1C for 30 min. p53 antibody (DO-1, Santa Cruz Biotechnology, Dallas, TX, USA) was added to the extract, and the associated DNAs were recovered from Protein A–Sepharose beads by phenol/chloroform extraction. Enrichment across the putative p53-binding site at the miR-506 locus was tested using real-time PCR analysis. Primers used are as follows: FP: 50 -GCATTGCCCTATTTTGTGAGC-30 RP: 50 -GGTCTAAACACTGGTTTGCAAGTG-30 .

Western blot and immunohistochemistry Antibodies against NF-kB p65 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53, caspase-8 (R&D Systems, Minneapolis, MN, USA), PARP (Cell Signaling, No.9542, Danvers, MA, USA) and actin (Sigma-Aldrich, St Louis, MO, USA) were used in standard western blot and immunostaining experiments, according to manufacturer’s instructions.

Measurement of ROS levels Cells were plated in six-well plates and transfected with no-target control miRNA, miR-506 mimics, antagomir-506 or si-p65. After 72 h, the cells were incubated with 5 mM dichlorodihydrofluorescein diacetate (Sigma-Aldrich) for 30 min at 37 1C. The intensity of dichlorofluorescein (DCF) fluorescence was tested by flow cytometric analysis by using the FACS Calibur (BD) using the CellQuest program (BD).

Cell viability assay Cell viability was determined by using the CellTiter96 assay kit (Promega). Cell Counting Kit-8 assay kit (Dojindo, Kumamoto, Japan) was used to evaluate the viability of lung cancer cells and normal cell lines after treatment with miR-506 mimics and siRNAs. Cells were transfected with miRNA mimics or siRNAs in 96-well plates. At 72 h after transfection, cell viability was measured according to manufacturer’s instructions.

Tumor formation in BALB/c nude mice All experimental protocol involving animals were performed according to the institutional ethical guidelines for animal experiments at the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences. Oncogene (2014) 1 – 13

We would like to thank Professor Craig Mello for valuable advice throughout this research, Professor Miguel Esteban for manuscripts revision and renal cell line RCC4, Professor Duanqing Pei for the lung cancer cell line 95D. This research was supported by Introduced Innovative R&D Team Program of Guangdong Province (no. 201001Y0104789252), 863 Program of China (no. 2012AA022501), Strategic Emerging Industry Key Technology Project of Guangdong Province (no. 2012A080800006), the National Natural Science Foundation of China (nos. 30870535 and 90913017), the "Hundred Talents Plan" of Guangzhou Municipality and Combination Project of Guangdong Province and the Ministry of Education (no. 2011B090400478).

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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

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