Zinc-Finger Protein 331, a Novel Putative Tumor Suppressor

ORIGINAL ARTICLE Zinc-ﬁnger protein 331, a novel putative tumor suppressor, suppresses growth and invasiveness of gastric cancer

JYu1,4, QY Liang1,4, J Wang1,4, Y Cheng2, S Wang1, TCW Poon1,MYYGo1,QTao2, Z Chang3 and JJY Sung1

Zinc-finger protein 331 (ZNF331), a Kruppel-associated box zinc-finger protein gene, was identified as a putative tumor suppressor in our previous study. However, the role of ZNF331 in tumorigenesis remains elusive. We aimed to clarify its epigenetic regulation and biological functions in gastric cancer. ZNF331 was silenced or downregulated in 71% (12/17) gastric cancer cell lines. A significant downregulation was also detected in paired gastric tumors compared with adjacent non-cancer tissues. In contrast, ZNF331 was readily expressed in various normal adult tissues. The downregulation of ZNF331 was closely linked to the promoter hypermethylation as evidenced by methylation-specific PCR, bisulfite genomic sequencing and reexpression by demethylation agent treatment. DNA sequencing showed no genetic mutation/deletion of ZNF331 in gastric cancer cell lines. Ectopic expression of ZNF331 in the silenced cancer cell lines MKN28 and HCT116 significantly reduced colony formation and cell viability, induced cell cycle arrests and repressed cell migration and invasive ability. Concordantly, knockdown of ZNF331 increased cell viability and colony formation ability of gastric cancer cell line MKN45. Two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomic approach were applied to analyze the molecular basis of the biological functions of ZNF331. In all, 10 downstream targets of ZNF331 were identified to be associated with regulation of cell growth and metastasis. The tumor-suppressive effect of ZNF331 is mediated at least by downregulation of genes involved in cell growth promotion (DSTN, EIF5A, GARS, DDX5, STAM, UQCRFS1 and SET) and migration/invasion (DSTN and ACTR3), and upregulation of genome-stability gene (SSBP1) and cellular senescence gene (PNPT1). A novel target of ZNF331 (DSTN) was functionally validated. Overexpression of DSTN in BGC-823 cells increased colony formation and migration ability. In conclusion, our results suggest that ZNF331 possesses important functions for the suppression of gastric carcinogenesis as a novel functional tumor-suppressor gene.

Oncogene (2013) 32, 307--317; doi:10.1038/onc.2012.54; published online 27 February 2012 Keywords: Zinc-ﬁnger protein 331 (ZNF331); gastric cancer; tumor suppressor; methylation; invasion; 2-D gel electrophoresis

INTRODUCTION associated box-A box and a zinc-finger domain with 12 Cys2His2 Gastric cancer is one of the leading causes of cancer-related death zinc fingers and locates on chromosome 19q13.4,5 The role of in China. Although the molecular mechanisms of gastric carcino- ZNF331 in tumor development remains largely unknown. The genesis remain unclear, epigenetic silencing of tumor-related functions of zinc-finger proteins are extraordinarily diverse, genes by promoter hypermethylation has recently emerged as an including DNA recognition, RNA packaging, transcriptional activa- important mechanism of tumorigenesis. The promoter hyper- tion, regulation of apoptosis, and so on. Structural studies of zinc- methylation profile differs in each cancer type and within each finger proteins have shed new insights into their extraordinary gene, providing tumor type- and gene-specific hypermethylation diversity of structure and function.6 Proteins containing the profiles that may involve in the corresponding molecular mechan- classical Cys2His2 zinc finger are among the most abundant in ism of tumorigenesis. The identification of a novel gene targeted eukaryotic genomes. Many of these proteins function by by promoter hypermethylation may provide insights into the recognition of specific DNA sequences. Because all the Kruppel- mechanisms for the inactivation of the tumor-suppressive path- associated box zinc-finger genes so far have been shown to act ways and is important for the identification of tumor markers in only as transcriptional repressors,7--9 ZNF331 is considered to be gastric cancer.1,2 Recently, using suppression subtraction hybridi- a transcriptional repressor.10 The engineered chimeric fusion zation,3 we have identified zinc-finger protein 331 (ZNF331)asa proteins of Kruppel-associated box domains combined with candidate tumor-suppressor gene. We aimed to clarify the different DNA-binding domains of oncogenic transcription factors epigenetic regulation and biological functions of ZNF331 in gastric have been shown to target their respective oncogenes and cancer in this study. specifically suppress malignant growth.11 The application of ZNF331, also known as ZNF361, ZNF463 and Rita, is a Kruppel- artificial zinc-finger protein transcription factors to repress the associated box zinc-finger protein gene consisting of a Kruppel- expression of cancer-related genes, such as human telomerase

1Institute of Digestive Disease and Department of Medicine and Therapeutics, Li Ka Shing Institute of Health Sciences, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China; 2Cancer Epigenetics Laboratory, Department of Clinical Oncology, The Chinese University of Hong Kong, Hong Kong, China and 3State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Medicine, National Engineering Laboratory for Anti-tumor Therapeutics, Tsinghua University, Beijing, China. 4These authors contributed equally to this work. Correspondence: Professor J Yu, Institute of Digestive Disease, Department of Medicine and Therapeutics, Li Ka Shing Institute of Health Sciences, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, NT, Hong Kong. E-mail: [email protected] or Professor JJY Sung, Institute of Digestive Disease, Department of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, NT, Hong Kong. E-mail: [email protected] Received 15 July 2011; revised 25 November 2011; accepted 10 January 2012; published online 27 February 2012 Zinc-finger protein 331 in gastric cancer JYuet al 308 reverse transcriptase, has presented a new promising strategy for silenced or downregulated in the tested gastric cancer cell lines inhibiting the growth of human cancer cells.12 These findings (12/17, 71%). Expression of variant 2 was detected only in 1 out demonstrate important tumor-suppressor functions of zinc-finger of the 17 cell lines (SNU719) by RT--PCR (Figure 1c). Real-time proteins and their promising application in cancer therapy. In this quantitative RT--PCR (qRT--PCR) indicated that expression of study, we investigated the gene-expression pattern of ZNF331, variant 2 in SNU719 was over 200 times higher than in the other molecular characterization of the promoter of ZNF331 and its cells. Dissimilarly, ZNF331 waslessfrequentlysilencedor tumor-suppressor function in gastric cancer. downregulated in colon, esophageal, liver and other human cancer cell lines (data not shown). These results implied that loss of ZNF331 expression, especially variant 2, may be mainly RESULTS involved in gastric carcinogenesis. Correlation of ZNF331 mRNA expression level with methylation Sequence analysis using both CpG island searcher (http:// status in gastric cancer cell lines cpgislands.usc.edu/) and CPGPLOT (http://www.cbib.u-bordeaux2. Three transcription variants of ZNF331 have been identified in fr/pise/cpgplot.html) revealed CpG islands around the transcrip- human tissues previously.4 The three variants are transcribed from tion start sites of both variants 1 and 2, indicating potential different loci and share the same open reading frame (ORF) epigenetic regulation of ZNF331 expression by DNA methylation. (Figure 1a), suggesting that they possess the same function and To evaluate the association of silencing or downregulation of might be transcriptionally regulated in a tissue-specific manner. ZNF331 variants 1 and 2 with methylation status, methylation- RT--PCR results indicated that variants 1 and 2 of ZNF331 were specific PCR was carried out. Heavy methylation of ZNF331 variant ubiquitously expressed in various normal human tissues, including 2 was observed in all silenced cell lines, whereas partial or full gastric tissue, whereas variant 3 was found only in the methylation of variant 1 was detected in cancer cells with reduced reproductive tissue, testis (Figure 1b). These results demonstrated or silenced expression (Figure 1d). These results indicated a that constitutive expression of ZNF331 in normal tissues mainly correlation between ZNF331 mRNA expression and its methylation involves variants 1 and 2 but not variant 3. So we further status in gastric cancer cell lines. analyzed the transcription of variants 1 and 2 in a series of cancer To further confirm that DNA methylation is indeed responsible cell lines. RT--PCR results indicated that variant 1 was frequently for the silencing or downregulation of ZNF331, six gastric cancer

Figure 1. Transcriptional silencing/downregulation of ZNF331 is associated with DNA methylation. (a) Schematic for the three transcription variants of ZNF331, which are transcribed from different transcription start sites while containing the same ORF. (b) Ubiquitous mRNA expression of ZNF331 variants 1 and 2 in normal human adult tissues. (c) ZNF331 variant 1 and variant 2 expression were silenced or reduced in many gastric cancer cell lines. (d) By methylation-specific PCR, full methylation of ZNF331 variant 2 was observed in most silenced cell lines, and partial or full methylation of ZNF331 variant 1 was detected in cell lines with reduced or silenced expression. MSP, methylation-specific PCR; M, methylated; U, unmethylated. (e) The mRNA expression of ZNF331 was restored after treatment with demethylation agent 5-Aza as evidenced by RT--PCR (left) and real-time qRT--PCR (right). Bisulfite genomic sequencing results indicated that methylation level of ZNF331 was reduced after 5-Aza treatment, in concordance with its restored expression.

Oncogene (2013) 307 --317 & 2013 Macmillan Publishers Limited Zinc-finger protein 331 in gastric cancer JYuet al 309 cell lines and one colon cancer cell line (HCT116) were treated ZNF331 nuclear expression is associated with male gender with the demethylation agent 5-Aza-20-deoxycytidine (5-Aza). (Po0.05), late TNM staging (Po0.05) and diffuse type of gastric Expression of ZNF331 variants 1 and 2 was dramatically restored cancer (Po0.001). There was no correlation between the or upregulated after treatment in all these cancer cell lines. expression of ZNF331 and other clinicopathological features such Bisulfite genomic sequencing indicated lower level of methylation as age and Helicobacter pylori status (Supplementary Table 2). in 5-Aza-treated cells as compared with untreated cells (Figure 1e). Moreover, as shown in the Kaplan--Meier survival curves These results confirmed that DNA methylation mediated the (Supplementary Figure 1), gastric cancer patients with low transcriptional silencing or downregulation of ZNF331. ZNF331 expression had significantly shorter survival than those with high ZNF331 expression (Po0.05, log-rank test) However, ZNF331 expression was not associated with the outcome in gastric Genetic deletion or mutation of ZNF331 was not detected in cancer patients by multivariate Cox regression analysis (Supple- gastric cancer cell lines mentary Table 3). To find out whether genetic alteration also contribute to the silencing of ZNF331, we performed sequence screening on the exons, exon--intron junctions and 42-kb upstream of the Expression of ZNF331 suppressed cell growth and induced cell cycle arrest transcription start sites of all transcripts of ZNF331 using PCR and direct sequencing. Results showed no genetic mutation/deletion The frequent silencing or downregulation of ZNF331 in gastric of ZNF331 in all the seven gastric cancer cell lines, including cancer cell lines suggests that it is likely a tumor-suppressor gene. MKN28, MKN45, AGS, Kato III, SNU16, SNU719 and NCI87, In order to test this point, we constructed and transfected a suggesting that silencing or downregulation of ZNF331 might pcDNA3.1--ZNF331 expression vector into the gastric cancer cell not be regulated by genetic alteration. line MKN28 and the colon cancer cell line HCT116, which shows no or low level of ZNF331 expression. Expression of ZNF331 after stable transfection was evidenced using RT--PCR and Western blot Downregulation of ZNF331 in primary gastric cancer tissues (Figure 3a). Expression of ZNF331 suppressed the growth of both ZNF331 was downregulated in gastric tumor samples as compared MKN28 (Po0.01) and HCT116 cells (Po0.001) by MTT assay with their adjacent normal samples as determined by immuno- (Figure 3b). This was confirmed by colony formation assay. The histochemistry (Figure 2a). ZNF331 protein was located mainly in colonies formed by ZNF331-transfected cells were significantly less the nuclei of normal cells, but predominantly in the cytoplasm of and smaller in MKN28 (Po0.05) and HCT116 (Po0.001) than cancer cells, suggesting that the subcellular localization of ZNF331 empty vector-transfected cells (Figure 3c). may also be regulated distinctly between cancerous and normal To determine the molecular mechanism by which ZNF331 cells as a transcription repressor. We counted the cells with inhibits cell growth, we analyzed the effect of ZNF331 on cell cycle positive nuclear staining and found that ZNF331 was significantly distribution by flow cytometry after propidium iodide staining. downregulated in gastric tumor samples as compared with their Ectopic expression of ZNF331 led to a significant decrease in the adjacent normal samples (5.8% vs 39.6%, Po0.001) (Figure 2b). number of the S-phase cells of MKN28 (Po0.001) and HCT116 The association between clinicopathological features and the (Po0.05) (Figure 3d). Western blot analysis of protein extracts expression of ZNF331 was evaluated in 224 primary gastric derived from ZNF331-transfected cells showed a clear decrease in cancers by immunohistochemistry on tissue array slides. Low proliferating cell nuclear antigen expression (Figure 3e) as compared with vector-transfected cells, confirming the inhibitory effect of ZNF331 on cell cycle progression. Concomitant with this inhibition, there was a significant increase in the number of cells accumulating in the G1 (Po0.05) and G2 phases (Po0.01) in MKN28 and in the G1 phase only in HCT116 (Po0.05) (Figure 3d). We also examined the contribution of apoptosis to the observed growth inhibition of ZNF331-transfected cells using flow cytometry with Annexin V:FITC and propidium iodide staining. However, no significant induction of apoptosis by ZNF331 was observed in both cell lines. These results indicated that expression of ZNF331 inhibited cancer cell growth by suppressing cell proliferation and inducing cell cycle arrest.

ZNF331 reduced migration and invasion ability of cancer cells To investigate the effects of ZNF331 on cancer cell migration and invasive ability, the monolayer scratch healing assay and Martrigel invasion assay were performed. Ectopic expression of ZNF331 markedly slowed cell migration at the edges of scratch wound of MKN28 and HCT116 (Figure 4a). Quantitative analyses at 48 h confirmed a significant reduction in wound closure in ZNF331- transfected cells compared with empty vector-transfected control cells (Figure 4a). In addition, ZNF331 also significantly impaired the invasiveness of both MKN28 and HCT116 cells (Figure 4b). These results suggest that ZNF331 has an important inhibitory role in the invasiveness of cancer cells.

Figure 2. Immunohistochemistry examination of ZNF331 expression in primary gastric tumor and adjacent normal samples. (a) Re- Knockdown of ZNF331 increased cell proliferation in MKN45 presentative results of immunohistochemical staining for ZNF331 in gastric cancer cells gastric tumor and adjacent normal tissues. (b) Quantitation of In order to further validate the inhibitory effect of ZNF331 on ZNF331 expression by counting cells with positive nuclear staining. cancer cell growth, we knocked down ZNF331 expression in the

& 2013 Macmillan Publishers Limited Oncogene (2013) 307 --317 Zinc-ﬁnger protein 331 in gastric cancer JYuet al 310

Figure 3. Effect of ZNF331 expression on cell growth and cell-cycle phase distribution. (a) Expression of ZNF331 in MKN28 and HCT116 cells after transfection with ZNF331-expressing vectors was conﬁrmed using RT--PCR and Western blot. (b) Representative results of MTT viability assay from empty vector- and ZNF331-transfected MKN28 and HCT116 cells. (c) Representative results of colony-formation assay from MKN28 and HCT116 cells. (d) Representative cell cycle analysis results from empty vector- and ZNF331-transfected MKN28 and HCT116 cells. (e) Western blot examination of proliferating cell nuclear antigen in control and ZNF331-transfected MKN28 and HCT116 cells.

ZNF331-expressing gastric cancer cell line MKN45 by short hairpin Identification of genes modulated by ZNF331 RNA vector transfection. Knockdown efficiency was evaluated by To elucidate the molecular mechanism underlying the inhibitory both real-time qRT--PCR and Western blot (Figure 5a). Knockdown effect of ZNF331 on cell growth and invasiveness, protein- of ZNF331 increased cell growth in MKN45 cells by MTT cell expression profiles in ZNF331-transfected MKN28 cells and viability assay (Figure 5b). In keeping with this, knockdown of vector-transfected MKN28 cells were analyzed using 2D gel assay. ZNF331 significantly increased colony formation ability of MKN45 In all, 33 spots were found differentially expressed with fold cells (Figure 5c). These results further indicated that ZNF331 acts changes more than two and they were subjected to further with a tumor-suppressor function in cancer cells. analysis by the combined mass spectrometry (MS) and MS/MS

Oncogene (2013) 307 --317 & 2013 Macmillan Publishers Limited Zinc-ﬁnger protein 331 in gastric cancer JYuet al 311

Figure 4. Effects of ZNF331 on MKN28 and HCT116 cell migration and invasion. (a) Representative result of scratch-healing assay. (b) Representative result of invasion assay. analysis (Figure 6). Ten spots were well recognized (Figure 7a) expression proﬁling were validated using Western blot (Figure 7b) (Table 1). When compared with control vector-transfected cells, and real-time qRT--PCR (Figure 7c) in MKN28 and HCT116 cells. In ZNF331 exerted its anti-growth effect by downregulation of six addition, ZNF331 downregulated the expression of DDX17 (the genes involved in cell growth and proliferation, including DEAD gene accompanying DDX5 in cell growth regulation) and c-Myc (Asp-Glu-Ala-Asp) box proteins (DDX5, À4.6-fold), eukaryotic (a downstream target of both PNPT1 and STAM) as demonstrated translation initiation factor 5A (EIF5A, À3.5-fold), glycyl-tRNA by real-time qRT--PCR in both cell lines. synthetase (GARS, À3.5-fold), signal-transducing adaptor molecule The correlations between ZNF331 and its downstream targets, (STAM, À3.3-fold), ubiquinol-cytochrome-c reductase 1 (UQCRFS1, as well as their association with the inhibition of gastric cancer À3.1-fold) and SET nuclear oncogene (SET, À4.8-fold). The anti- growth, were shown in Figure 7d. These results unveiled the invasive activity of ZNF331 was mediated by downregulation of molecular mechanism by which ZNF331 inhibited the growth and pro-metastatic genes, destrin (DSTN, À6-fold) and ARP3 actin- invasiveness of cancer cells. related protein 3 homolog (ACTR3, À2.8-fold). Further, ZNF331 induced the human PNPase gene (PNPT1) expression by 4.3-fold, a pro-cellular senescence regulator and single-stranded DNA-binding Functional validation of a novel ZNF331 downstream target DSTN protein 1 (SSBP1) by 4.6-fold, which has an important role in the We further selected DSTN, a novel downstream target of ZNF331, maintenance of genome stability (Table 1). These changes on for further functional validation. DSTN is required for cell

& 2013 Macmillan Publishers Limited Oncogene (2013) 307 --317 Zinc-finger protein 331 in gastric cancer JYuet al 312 migration and invasion in colon cancer cells (Estornes et al.13). scratch-healing assay indicated that DSTN significantly increased Ectopic expression of DSTN in BGC-823 cells (Figure 8a) sig- migration ability of BGC-823 cells (Figure 8c). These results nificantly increased colony formation ability as compared with indicated that ZNF331 inhibited gastric cancer growth, at least empty vector-transfected BGC-823 cells (Figure 8b). In addition, in part, by downregulation of DSTN.

DISCUSSION In this study, we identiﬁed and characterized a putative tumor- suppressor gene, ZNF331, in gastric cancer. The three transcript variants of ZNF331 share the same ORF, which suggests they exert the same function. Transcript variants 1 and 2 were readily expressed in normal human tissues but frequently silenced or downregulated in gastric cancer cell lines, whereas variant 3 was not found in normal tissue except the reproductive tissue (testis) (Figure 1b) and thus may not be involved in tumorigenesis. In addition, ZNF331 was also downregulated in gastric tumor samples compared with their adjacent normal samples by immunohistochemistry. Low ZNF331 expression was further revealed to be associated with advanced stage (Po0.05) and diffuse type (Po0.001) of gastric cancers. Moreover, low ZNF331 expression was correlated with poor survival of gastric cancer patients by Kaplan--Meier survival analysis (Po0.05). These results suggested that the downregulation of ZNF331 may be involved in tumor progression during gastric cancer development. Promoter methylation was demonstrated to mediate the transcriptional Figure 5. Knockdown of ZNF331 increased cell proliferation. silence of cancer cell lines at variants 1 and 2 of ZNF331. (a) Evaluation of knockdown of ZNF331 in MKN45 cells after trans- Demethylation treatment by 5-Aza successfully restored the fection with sh-ZNF331 (knockdown) and sh-Scrambled (control) expression of ZNF331, further conﬁrming its regulation by DNA using qRT--PCR and Western blot. (b) Representative results of MTT methylation. In addition, no genetic mutation or deletion of viability assay. (c) Representative results of colony-formation assay. ZNF331 was found in all the seven gastric cancer cell lines tested.

Figure 6. Identification of differentially expressed proteins modulated by ZNF331 expression. (a) 2D gel electrophoresis images of detergent extracts from vector- and ZNF331-transfected MKN28 cells. The first dimension was run on linear 13 cm IPG strip, pH 3--10. The second dimension was run on 10% SDS--PAGE. The major spots identified by MS and MS/MS analysis were labeled. (b) Histogram of fold-changes of the differentially expressed genes in ZNF331-transfected cells vs vector-transfected cells.

Oncogene (2013) 307 --317 & 2013 Macmillan Publishers Limited Zinc-finger protein 331 in gastric cancer JYuet al 313 and HCT116 significantly inhibited colony formation and cell proliferation. Concordantly, knockdown of ZNF331 increased proliferation of MKN45 cells as evidenced by cell viability and colony-formation assays. Flow cytometry analysis indicated that the suppression of cell growth by ZNF331 was mediated by cell- cycle phase arrest, without significant induction of apoptosis. Expression of ZNF331 decreased the number of both MKN28 and HCT116 cells in S phase (Figure 3d), inferring that ZNF331 blocked cell proliferation. There was an increase in the number of MKN28 cells in the G2/M phase, suggesting that G2 arrest was also involved. Furthermore, ectopic expression of ZNF331 reduced the migration and invasion ability of these cancer cells. These results indicate for the first time that ZNF331 functions as a tumor suppressor in gastric cancer. The molecular mechanisms by which ZNF331 exerts its anti- growth and anti-invasive functions in cancer cells were defined using two-dimensional gel electrophoresis protein profiling assay. Genes that were significantly altered in protein levels were then validated using RT--PCR and immunoblotting. We demonstrated that the suppression of cell growth and proliferation by ZNF331 was mediated by downregulation of genes involved in cell growth, including DDX5, DDX17, EIF5A, GARS, STAM, UQCRFS1 and SET oncogene (Figure 7d). The DEAD (Asp-Glu-Ala-Asp) box proteins DDX5 and DDX17 have important roles in ribosome biogenesis and cell proliferation. Overexpression of DDX5 and DDX17 has been found in colon, breast and prostate cancers, suggesting that DDX5/DDX17 promotes tumorigenesis as proto- oncoproteins.14 Co-silencing of both DDX5 and DDX17 causes perturbation of nucleolar structure and cell proliferation, whereas knocking down of either gene does not affect cell proliferation.15 Thus, downregulation of both DDX5 and DDX17 by ZNF331 contributes to its anti-proliferation effect. In addition, inhibition of EIF5A hypusination has been reported to impair melanoma growth;16 GARS, the glycyl-tRNA synthetase gene, has an important role in the synthesis of proteins;17 STAM, the signal- transducing adaptor molecule, is involved in signaling for cell growth through induction of the oncogene c-Myc;18 UQCRFS1 is known as the complex III of the mitochondrial respiratory chain passing electrons from reduced ubiquinol to cytochrome-c during the process of synthesis of ATP.19 The amplification of UQCRFS1 has been reported in breast, ovarian and gastric cancers,20 --22 which has an important role for dividing and proliferating cancer cells. The SET nuclear oncogene is involved in the regulation of cell growth23 and functions as a specific inhibitor of a gene suppressing tumor metastasis, non-metastatic cell 1.24 Collectively, suppression of DDX5/DDX17, EIF5A, GARS and STAM (followed by c-Myc downregulation), UQCRFS1 and SET may mediate the inhibitory activity by ZNF331 as a repressor on cancer cell growth. Figure 7. Molecular mechanism of the inhibitory activity of ZNF331 Cell cycle analysis indicated an increase in G2-phase cells in on gastric cancer growth. (a) Enlarged images of 10 differentially ZNF331-transfected MKN28 (Figure 3d). Members of the DEAD box expressed protein spots of interest. Selected regions of Two- proteins have been reported to be involved in the regulation of dimensional gel electrophoresis (2-DE) gels illustrate differentially cell division.25 A recent study has indicated that the proliferation- expressed proteins between vector- and ZNF331-transfected MKN28 associated nuclear antigen DDX5 has an important role in cells. Spots of interest are shown in circles. SET has two isoforms and mediating the G2-/M-phase arrest.26 We found that DDX5, a the spots 1 and 2 were both identified to be SET proteins by MS/MS ion search. (b) Validation of differentially expressed genes using member of the DEAD box family proteins, was significantly Western blot. (c) Validation of differentially expressed genes by real- downregulated by ZNF331 in MKN28 (À4.6-fold change) (Table 1). time qRT--PCR. (d) Schematic diagram for the molecular mechanism Thus, the G2-phase block by ZNF331 may be regulated through of the anti-tumorigenesis functions of ZNF331 deriving from 2-DE, the downregulation of DDX5. Western blot and real-time qRT--PCR. The anti-invasion function of ZNF331 appeared to be associated with the downregulation of DSTN and ACTR3 (Figure 7d). It has been reported that DSTN is required for cell migration and Collectively, these results revealed the epigenetic regulation invasion.13 ACTR3 is known to be a major constituent of the ARP2/3 mechanism on the expression of ZNF331 in gastric cancer cells. complex. The frequency of expression of ARP2/3 by the stromal In cell biology experiments, the full-length ORF was cloned into cells increased with the atypia of the colorectal neoplasms. It the expression vector pcDNA3.1 for gain-of-function study, contributes to the increased motility of both neoplastic and whereas in the knockdown experiment mediated by short hairpin stromal cells, and thus provides suitable conditions for invasion.27 RNA transfection, the ORF was targeted. Functional studies Thus, downregulation of DSTN and ACTR3 contributes to the revealed that restoration of ZNF331 in cancer cell lines MNK28 reduced migration and invasion ability induced by ZNF331.

& 2013 Macmillan Publishers Limited Oncogene (2013) 307 --317 Zinc-ﬁnger protein 331 in gastric cancer JYuet al 314 Table 1. Differentially expressed proteins in ZNF331-transfected MKN28 cells vs vector-transfected MKN28 cells

Spot Protein Fold Mass Protein Expectation Matched Sequence Function no. name change score value peptides coverage (%)

1, 2 SET À4.8 33 469 333 1.00E-29 10 34 Nuclear oncogene 3STAM À3.3 59 541 203 1.00E-16 9 22 Signaling for cell growth 5 EIF5A À3.5 17 049 103 1.00E-06 3 32 Cell proliferation 9 ACTR3 À2.8 47 797 156 5.10E-12 13 39 Cell motility and invasion 16 GARS À3.5 83 828 114 8.10E-08 4 8 Protein synthesis 17 UQCRFS1 À3.1 29 934 165 6.50E-13 4 12 Cell proliferation 25 PNPT1 4.3 86 524 195 6.50E-16 12 18 Cellular senescence 27 SSBP1 4.6 17 249 171 1.60E-13 8 56 Maintenance of genome stability 28 DDX5 À4.6 69 618 167 4.10E-13 13 25 Cellular growth and division 32 DSTN À6 18 950 181 1.60E-14 5 41 Cell migration and invasion

genome stability,32 inferring that ZNF331 can increase genomic stability by upregulating SSBP1. In conclusion, we characterized a novel tumor suppressor ZNF331 regulated epigenetically by DNA methylation in gastric cancer. ZNF331 suppressed growth and invasiveness of cancer cells through repressing genes involved in cell proliferation (DSTN, DDX5/DDX17, EIF5A, GARS, STAM, UQCRFS1 and SET) and migration/ invasion (DSTN and ACTR3), as well as inducing a cellular senescence gene (PNPT1) and a genome-stability gene (SSBP1) (Figure 7d). ZNF331 possesses important functions for the suppression of gastric cancer.

MATERIALS AND METHODS Cancer cell lines Seventeen gastric cancer cell lines (MKN28, MKN45, NCI87, BGC-823, AGS, Kato III, YCC1, YCC 2, YCC 3, YCC 6, YCC 7, YCC 9, YCC 10, YCC 11, YCC 16, SNU1, SNU16 and SNU719) and one colon cancer cell line (HCT116) were obtained from American Type Culture Collection (Manassas, VA, USA). The immortalized normal human gastric epithelial cell line GES-1 was provided by School of Oncology, Beijing University. Cells were cultured in RPMI 1640, DMEM or McCoy medium (Gibco BRL, Rockville, MD, USA) supplemented with 10% fetal bovine serum (Gibco BRL).

Semi qRT--PCR and real-time qRT--PCR analyses Total RNA was extracted from cell pellets using QiaZol reagent (Qiagen, Valencia, CA, USA). cDNA was synthesized from 2 mg of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Semi-qRT--PCR and real-time qRT--PCR analyses were Figure 8. Functional validation on DSTN, a novel downstream target performed using primers listed in Supplementary Table 1. of ZNF331.(a) Expression of DSTN in BGC-823 cells after transfection with DSTN-expressing vectors was confirmed by RT--PCR and Western blot. (b) Reexpression of DSTN reduced the colony-forma- Demethylation treatment tion ability in BGC-823 cells. (c) Reexpression of DSTN inhibited cell- Cells were seeded at a density of 1 Â 105 cells/ml in 100-mm dishes and migration ability in BGC-823 cells as evidenced by scratch-healing grew for 24 h. Cells were then treated with 2 mM 5-Aza (Sigma-Aldrich, assay. St Louis, MO, USA) for 48 h, with culture medium containing 5-Aza replenished every day. Then cells were harvested and gene expression was analyzed using RT--PCR. Two genes (PNPT1 and SSBP1) were found to be upregulated following ZNF331 reexpression. PNPT1, the gene encoding human DNA extraction, methylation-specific PCR and bisulfite genomic PNPase, is thought to contribute to cellular senescence through its sequencing RNA-degrading activity in the cytosol.28,29 It was also found to Genomic DNA from cancer cells and tissue samples were isolated using the induce growth arrest by downregulating expression of c-Myc in QIAamp DNA mini kit (Qiagen, Hilden, Germany). A total of 1 mg of DNA human melanoma cells.30 Therefore, upregulation of PNPT1, was modified by sodium metabisulfite as previously described.33 The followed by the downregulation of c-Myc, may contribute to the bisulfite-modified DNA was then amplified by methylation-specific PCR growth-inhibitory activity induced by ZNF331. SSBP1 is a house- using primer pairs that specifically amplify either methylated or keeping gene involved in mitochondrial biogenesis.31 It has an unmethylated sequences of the ZNF331 gene. Bisulfite genomic sequen- important role in the DNA-damage response and maintenance of cing was performed to assess the methylation levels of nine CpG sites

Oncogene (2013) 307 --317 & 2013 Macmillan Publishers Limited Zinc-finger protein 331 in gastric cancer JYuet al 315 spanning þ 92 to þ 285 of variant 1 of ZNF331 in cells with or without 5- manufacturer’s instructions. The experiment was conducted in three Aza treatment. Nucleotide sequences of the primers used in methylation- independent triplicates and results were shown as the means±s.d. specific PCR and bisulfite genomic sequencing were listed in Supplemen- tary Table 1. Flow cytometry The stably transfected MKN28 and HCT116 cells with pcDNA3.1--ZNF331- Immunohistochemistry and tissue array analysis expressing or pcDNA3.1 empty vector were fixed in 70% ethanol and Paired primary tumor and adjacent non-tumor samples were obtained stained with 50 mg/ml propidium iodide (BD Pharmingen, San Jose, CA, from 10 gastric cancer patients after surgical resection and from 224 USA). The cells were then sorted by FACSCalibur (BD Biosciences, Franklin gastric tumors in tissue array slides. Tissue types (tumor or normal) were Lakes, NJ, USA), and cell-cycle profiles were analyzed by ModFit 3.0 assessed by histological staining. Immunohistochemistry was performed software (Verity Software House, Topsham, ME, USA). Apoptosis was on 5-mm paraffin sections using anti-ZNF331 antibodies (Abcam, determined by dual staining with Annexin V:FITC and propidium iodide Cambridge, UK).34 ZNF331 staining in the nucleus and cytoplasm was (Invitrogen). The Annexin V-positive cells were counted as apoptotic cells. evaluated by scanning of the whole section and counting 41000 representative cells. The percentages of cells with positive nuclear staining Scratch-healing assay were used as immunohistochemical scores. The signal intensities and the Cell migration was assessed using scratch-healing assays. Briefly, MKN28 percentages of positive staining were both graded as 0, 1, 2 and 3 and HCT116 cells stably transfected with pcDNA3.1--ZNF331. BGC-823 cells independently. Then the score of a sample was defined as ‘high’ with stably transfected with pcDNA3.1--DSTN or empty vectors were cultured in ‘intensity þ percentage’ X3, otherwise as ‘low’ (‘intensity þ percentage’ o3). six-well plates. When the cells grew up to 90% confluence, three scratch The ethics committee of the Chinese University of Hong Kong approved of wounds across each well were made using a P-200 pipette tip. Fresh this study, and written consents were obtained from all patients involved. medium supplemented with reduced (5%) fetal bovine serum was added, and the wound-closing procedure was observed for 48 h. Photographs DNA mutation analysis were taken at 0, 24 and 48 h, respectively. Mutation of ZNF331-coding sequences were determined by direct DNA sequencing using an ABI prism 3100 genetic analyzer (Applied Biosystems). Invasion assay Sequence homology to published database was analyzed with the BLAST Invasion assay was performed using BD BioCoat Growth Factor Reduced program at the internet site of National Centre for Biological Information. MATRIGEL Invasion Chamber (BD Biosciences) according to the manufacturer’s instructions. Complete culture medium (supplemented with 10% Construction of expression vector for ZNF331 and DSTN fetal bovine serum) was used as chemoattractant. The insert membranes The expression vectors (pcDNA3.1--ZNF331 and pcDNA3.1--DSTN) encod- were stained with crystal violet and the permeating cells were counted ing the full-length ORFs of human ZNF331 and DSTN genes were under inverted microscope and photographed. Three independent ± constructed. Briefly, DNA sequences corresponding to the ORFs of experiments were performed and results were shown as the means s.d. ZNF331 or DSTN were generated by RT--PCR. The PCR products were confirmed by direct DNA sequencing and cloned into the mammalian Two-dimensional gel electrophoresis and image analysis expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA, USA). The sequences Samples containing 150 mg protein were dissolved in 250 ml rehydration of the constructs were further confirmed by sequencing. The ZNF331 solution (8 M urea, 2% CHAPS, 0.4%. DTT, 0.5% IPG buffer, 0.002% silencing cancer cell lines MKN28 and HCT116 were transfected with bromophenol blue) and subjected to two-dimensional gel electrophoresis pcDNA3.1--ZNF331 or empty vector, whereas DSTN-downregulated BGC- as previously described by us.35 The isoelectric focussing was carried out 823 cells were transfected with pcDNA3.1--DSTN or empty vector, using using IPG-PhorII apparatus (Amersham, Piscataway, NJ, USA). All Immobi- Lipofectamine 2000 (Invitrogen). Stably transfected cells were established line DryStrip gels strips (13 cm, pH 3--10, nonlinear) were used according to under selection with neomycin (G418) (Invitrogen). the manufacturer’s instructions. Two-dimensional SDS--PAGE was performed in tris-glycine buffer at a constant current setting of 15 mA/gel for Knockdown of ZNF331 30 min and 30 mA/gel thereafter. Gels were stained by a modified silver- staining method, which was compatible with the MS analysis, as previously A set of vectors carrying short hairpin RNAs against ZNF331 was purchased 35 from Origene (Rockville, MD, USA). The ZNF331-expressing gastric cancer described. Gel images were digitalized using a scanner (GS-800 calibrated densitometer, Bio-Rad, Hercules, CA, USA) and analyzed using cell line MKN45 was transfected, with vectors carrying scrambled sequence (sh-scrambled) as negative control. Knockdown efficiency was evaluated the software PDQuest (version 8.0, Bio-Rad) with manual editing. Spots on 3 days after transfection by real-time qRT--PCR. The one with the highest different gels were matched and analyzed. Statistical analysis was knockdown efficiency was further used to establish stable knockdown cells performed using the independent-samples t-test. under selection with puromycin (Invitrogen) for colony-formation assay and cell-viability assay. Protein identification using MS Differential protein spots were excised from stained gels, and subjected to Colony-formation assay protein identification by undertaking the in-gel digestion approach and using MALDI TOF/TOF MS. Briefly, the gel pieces were destained, reduced For overexpression assay, MKN28 and HCT116 cells (2 Â 105 cells/well) with 1.75% DTT, alkylated with 350 mM iodoacetamide and digested with were plated in a 12-well plate and transfected with pcDNA3.1--ZNF331 or modified porcine trypsin overnight (sequencing grade; Promega, Madison, empty vector. BGC-823 cells were transfected with pcDNA3.1--DSTN or WI, USA). The tryptic peptides were harvested, cleaned up with C18 ZipTips empty vector. For knockdown assay, MKN45 cells were transfected with (Millipore Corp., Billerica, MA, USA) and subjected to MALDI-TOF/TOF MS sh-ZNF331 or sh-scrambled. Two days after transfection, cells were (Ultraflex-III, Bruker Daltonics, Bremen, Germany) with a-Cyano 4-hydroxy subsequently split at 1:20 ratio on six-well plates with G418 or puromycin. cinnamic acid as the matrix. The MS and MS/MS spectra were automatically After culturing for 14--21 days, cells were fixed with 70% ethanol and processed with the FlexAnalysis program (version 3.0, Bruker Daltonics) stained with crystal violet solution. Colonies with 450 cells per colony with the default parameters. The MS spectrum data were searched via the were counted. All experiments were conducted three times in triplicates. MASCOT search engine to obtain the protein identity by undertaking the peptide mass fingerprinting approach and the MS/MS ion search Cell-viability assay approach. For the search parameters, one missed cleavage in trypsin Cell proliferation of stably transfected cells was examined using the digestion was allowed; partial oxidation of methionine, phosphorylation of Vybrant MTT Cell Proliferation Assay Kit (Invitrogen) according to the serine/threonine/tyrosine and iodoacetamide modification of cysteine

& 2013 Macmillan Publishers Limited Oncogene (2013) 307 --317 Zinc-finger protein 331 in gastric cancer JYuet al 316 residues were selected. The error-tolerance values of the parent peptides 4 Rippe V, Belge G, Meiboom M, Kazmierczak B, Fusco A, Bullerdiek J. A KRAB zinc and the MS/MS ion masses were 50 ppm and 0.1 Da, respectively. A search finger protein gene is the potential target of 19q13 translocation in benign result having an expectation value o0.05 was considered statistically thyroid tumors. Genes Chromosomes Cancer 1999; 26: 229 --236. significant. For a gel spot, an identification result was considered valid 5 Wu H, Zhang S, Qiu W, Zhang G, Xia Q, Xiao C et al. Isolation, characterization, and when both peptide mass fingerprinting and MS/MS ion search identified mapping of a novel human KRAB zinc finger protein encoding gene ZNF463. the same protein as the statistically significant hit from the Swiss-Prot Biochim Biophys Acta 2001; 1518: 190 --193. database, or MS/MS ion search identified at least two tryptic peptides with 6 Laity JH, Lee BM, Wright PE. Zinc finger proteins: new insights into structural and functional diversity. Curr Opin Struct Biol 2001; 11:39--46. sequences from the same protein as the statistically significant hits. 7 Margolin JF, Friedman JR, Meyer WK, Vissing H, Thiesen HJ, Rauscher 3rd FJ. Kruppel-associated boxes are potent transcriptional repression domains. Proc Natl Western blot analysis Acad Sci USA 1994; 91: 4509 --4513. Total protein was extracted and protein concentration was then measured 8 Vissing H, Meyer WK, Aagaard L, Tommerup N, Thiesen HJ. Repression of by the DC protein-assay method of Bradford (Bio-Rad). A total of 40 mgof transcriptional activity by heterologous KRAB domains present in zinc finger proteins. FEBS Lett 1995; 369: 153 --157. protein from each sample were separated by 10% SDS--PAGE gel and 9 Witzgall R, O’Leary E, Leaf A, Onaldi D, Bonventre JV. The Kruppel-associated box- transferred to an equilibrated polyvinylidene difluoride membrane A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression. (Amersham Biosciences, Buckinghamshire, UK). After incubation with Proc Natl Acad Sci USA 1994; 91: 4514 --4518. specific primary antibody at 4 1C overnight and then the secondary 10 Meiboom M, Murua Escobar H, Pentimalli F, Fusco A, Belge G, Bullerdiek J. A antibody, the proteins were detected by enhanced chemiluminescence 3.4-kbp transcript of ZNF331 is solely expressed in follicular thyroid adenomas. (Amersham Corporation, Arlington Heights, IL, USA). The primary Cytogenet Genome Res 2003; 101: 113 --117. antibodies anti-ZNF331, anti-DSTN and anti-EIF5A were purchased from 11 Fredericks WJ, Ayyanathan K, Herlyn M, Friedman JR, Rauscher 3rd FJ. An Abcam. Antibodies against PNPT1 and DDX5 were purchased from Santa engineered PAX3-KRAB transcriptional repressor inhibits the malignant pheno- Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against proliferating type of alveolar rhabdomyosarcoma cells harboring the endogenous PAX3-FKHR oncogene. Mol Cell Biol 2000; 20: 5019 --5031. cell nuclear antigen and GAPDH were purchased from Cell Signaling 12 Sohn JH, Yeh BI, Choi JW, Yoon J, Namkung J, Park KK et al. Repression of human Technology (Danvers, MA, USA). telomerase reverse transcriptase using artificial zinc finger transcription factors. Mol Cancer Res 2010; 8: 246 --253. Statistical analysis 13 Estornes Y, Gay F, Gevrey JC, Navoizat S, Nejjari M, Scoazec JY et al. Differential All measurements or variables were shown as means±s.d. Results of involvement of destrin and cofilin-1 in the control of invasive properties of Isreco1 colony-formation assays, flow-cytometry analyses, cell growth, migration human colon cancer cells. Int J Cancer 2007; 121: 2162 --2171. 14 Janknecht R. Multi-talented DEAD-box proteins and potential tumor promoters: and invasion assays were analyzed by analysis of variance. Survival p68 RNA helicase (DDX5) and its paralog, p72 RNA helicase (DDX17). Am J Transl analyses were assessed using the Kaplan--Meier method and a Cox Res 2010; 2: 223 --234. proportional hazards regression model. A difference was considered 15 Jalal C, Uhlmann-Schiffler H, Stahl H. Redundant role of DEAD box proteins p68 statistically significant when P-value is o0.05. All statistical analyses were (Ddx5) and p72/p82 (Ddx17) in ribosome biogenesis and cell proliferation. Nucleic conducted using the SPSS program (version 17.0; SPSS, Chicago, IL, USA). Acids Res 2007; 35: 3590 --3601. 16 Jasiulionis MG, Luchessi AD, Moreira AG, Souza PP, Suenaga AP, Correa M et al. Inhibition of eukaryotic translation initiation factor 5A (eIF5A) hypusination impairs melanoma growth. Cell Biochem Funct 2007; 25: 109 --114. 17 Freist W, Logan DT, Gauss DH. Glycyl-tRNA synthetase. Biol Chem Hoppe Seyler ABBREVIATIONS 1996; 377: 343 --356. 2-DE, two-dimensional gel electrophoresis; 5-Aza, 5-Aza-20-deox- 18 Takeshita T, Arita T, Higuchi M, Asao H, Endo K, Kuroda H et al. STAM, ycytidine; ACTR3, ARP3 actin-related protein 3 homolog; DDX5, signal transducing adaptor molecule, is associated with Janus kinases DEAD (Asp-Glu-Ala-Asp) box proteins 5; DSTN, destrin; EIF5A, and involved in signaling for cell growth and c-myc induction. Immunity 1997; ukaryotic translation initiation factor 5A; GARS, glycyl-tRNA 6: 449 --457. synthetase; MSP, methylation specific PCR; PNPT, the human 19 Pennacchio LA, Bergmann A, Fukushima A, Okubo K, Salemi A, Lennon GG. Structure, sequence and location of the UQCRFS1 gene for the human Rieske Fe-S PNPase gene; qRT--PCR, quantitative RT--PCR; RT--PCR, reverse protein. Gene 1995; 155: 207 --211. transcript PCR; SSBP, single-stranded DNA binding protein; STAM, 20 Ohashi Y, Kaneko SJ, Cupples TE, Young SR. Ubiquinol cytochrome c reductase signal transducing adaptor molecule; UQCRFS, Ubiquinol-cyto- (UQCRFS1) gene amplification in primary breast cancer core biopsy samples. chrome-c reductase, Rieske iron-sulfur polypeptide; ZNF, zinc- Gynecol Oncol 2004; 93: 54 --58. finger protein 21 Kaneko SJ, Gerasimova T, Smith ST, Lloyd KO, Suzumori K, Young SR. CA125 and UQCRFS1 FISH studies of ovarian carcinoma. Gynecol Oncol 2003; 90:29--36. 22 Leung SY, Ho C, Tu IP, Li R, So S, Chu KM et al. Comprehensive analysis of 19q12 CONFLICT OF INTEREST amplicon in human gastric cancers. Mod Pathol 2006; 19: 854 --863. 23 Anazawa Y, Nakagawa H, Furihara M, Ashida S, Tamura K, Yoshioka H et al. PCOTH, The authors declare no conflict of interest. a novel gene overexpressed in prostate cancers, promotes prostate cancer cell growth through phosphorylation of oncoprotein TAF-Ibeta/SET. Cancer Res 2005; ACKNOWLEDGEMENTS 65: 4578 --4586. 24 Fan Z, Beresford PJ, Oh DY, Zhang D, Lieberman J. Tumor suppressor NM23-H1 is This project was supported by the National Basic Research Program of China (973 a granzyme A-activated DNase during CTL-mediated apoptosis, and the Program, 2010CB529305), Research Grants Council RGC CERG CUHK (473008), CUHK nucleosome assembly protein SET is its inhibitor. Cell 2003; 112: 659 --672. Focused Investment Grant (1903026) and RFCID (10090942, 11100022). 25 Wassarman DA, Steitz JA. RNA splicing. Alive with DEAD proteins. Nature 1991; 349: 463 --464. 26 Choi YJ, Lee SG. The DEAD-box RNA helicase DDX3 interacts with DDX5, REFERENCES co-localizes with it in the cytoplasm during the G2/M phase of the cycle, and 1 Kang GH, Lee S, Kim JS, Jung HY. Profile of aberrant CpG island methylation along affects its shuttling during mRNP export. J Cell Biochem 2011; 113: 985 --996; multistep gastric carcinogenesis. Lab Invest 2003; 83: 519 --526. e-pub ahead of print 27 October 2011; doi: 10.1002/jcb.23428. 2 Lee JH, Park SJ, Abraham SC, Seo JS, Nam JH, Choi C et al. Frequent CpG island 27 Otsubo T, Iwaya K, Mukai Y, Mizokami Y, Serizawa H, Matsuoka T et al. methylation in precursor lesions and early gastric adenocarcinomas. Oncogene Involvement of Arp2/3 complex in the process of colorectal carcinogenesis. Mod 2004; 23: 4646 --4654. Pathol 2004; 17: 461 --467. 3 Li L, Tao Q, Jin H, van Hasselt A, Poon FF, Wang X et al. The tumor suppressor 28 Leszczyniecka M, Kang DC, Sarkar D, Su ZZ, Holmes M, Valerie K et al. UCHL1 forms a complex with p53/MDM2/ARF to promote p53 signaling and is Identification and cloning of human polynucleotide phosphorylase, hPNPase frequently silenced in nasopharyngeal carcinoma. Clin Cancer Res 2010; 16: old-35, in the context of terminal differentiation and cellular senescence. Proc Natl 2949 --2958. Acad Sci USA 2002; 99: 16636 --16641.

Oncogene (2013) 307 --317 & 2013 Macmillan Publishers Limited Zinc-finger protein 331 in gastric cancer JYuet al 317 29 Sarkar D, Park ES, Emdad L, Randolph A, Valerie K, Fisher PB. Defining the domains 32 Huang J, Gong Z, Ghosal G, Chen J. SOSS complexes participate in the of human polynucleotide phosphorylase (hPNPaseOLD-35) mediating cellular maintenance of genomic stability. Mol Cell 2009; 35: 384 --393. senescence. Mol Cell Biol 2005; 25: 7333 --7343. 33 Pollard KS, Serre D, Wang X, Tao H, Grundberg E, Hudson TJ et al. A genome- 30 Sarkar D, Leszczyniecka M, Kang DC, Lebedeva IV, Valerie K, Dhar S et al. Down- wide approach to identifying novel-imprinted genes. Hum Genet 2008; 122: regulation of Myc as a potential target for growth arrest induced by human 625 --634. polynucleotide phosphorylase (hPNPaseold-35) in human melanoma cells. J Biol 34 Yu J, Leung WK, Ebert MP, Ng EK, Go MY, Wang HB et al. Increased expression of Chem 2003; 278: 24542 --24551. survivin in gastric cancer patients and in first degree relatives. Br J Cancer 2002; 31 Tiranti V, Rossi E, Ruiz-Carrillo A, Rossi G, Rocchi M, DiDonato S et al. Chromosomal 87:91--97. localization of mitochondrial transcription factor A (TCF6), single-stranded DNA- 35 Li G, Zhang XA, Wang H, Wang X, Meng CL, Chan CY et al. Comparative proteomic binding protein (SSBP), and endonuclease G (ENDOG), three human housekeeping analysis of mesenchymal stem cells derived from human bone marrow, umbilical genes involved in mitochondrial biogenesis. Genomics 1995; 25: 559--564. cord, and placenta: implication in the migration. Proteomics 2009; 9: 20 --30.

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& 2013 Macmillan Publishers Limited Oncogene (2013) 307 --317