NME2 Is a Master Suppressor of Apoptosis in Gastric Cancer Cells

Published OnlineFirst November 6, 2019; DOI: 10.1158/1541-7786.MCR-19-0612

MOLECULAR CANCER RESEARCH | CELL FATE DECISIONS

NME2 Is a Master Suppressor of Apoptosis in Gastric Cancer Cells via Transcriptional Regulation of miR-100 and Other Survival Factors Yi Gong1, Geng Yang1, Qizhi Wang2, Yumeng Wang2, and Xiaobo Zhang1

ABSTRACT ◥ Tumorigenesis is a result of uncontrollable cell proliferation antiapoptotic genes including miRNA (i.e., miR-100) and protein- which is regulated by a variety of complex factors including encoding genes (RIPK1, STARD5, and LIMS1) through interacting miRNAs. The initiation and progression of cancer are always with RNA polymerase II and RNA polymerase II–associated protein accompanied by the dysregulation of miRNAs. However, the 2 to mediate the phosphorylation of RNA polymerase II C-terminal underlying mechanism of miRNA dysregulation in cancers is still domain at the 5th serine, leading to the suppression of apoptosis of largely unknown. Herein we found that miR-100 was inordinately gastric cancer cells both in vitro and in vivo. In this context, our upregulated in the sera of patients with gastric cancer, indicating study revealed that the transcription factor NME2 is a master that miR-100 might emerge as a biomarker for the clinical diagnosis suppressor for apoptosis of gastric cancer cells. of cancer. The abnormal expression of miR-100 in gastric cancer cells was mediated by a novel transcription factor NME2 (NME/ Implications: Our study contributed novel insights into the mech- NM23 nucleoside diphosphate kinase 2). Further data revealed that anism involved in the expression regulation of apoptosis-associated the transcription factor NME2 could promote the transcriptions of genes and provided a potential biomarker of gastric cancer.

Introduction As one of the first identified transcription factors, Sp1 is a member of the zinc-finger Sp family of proteins and serves as a tumor Cancer is a major cause of human death all over the world (1). suppressor (14). The transcription factor p53, one of the most It appears often along with unregulated cell growth and metastasis famous transcription factors, can regulate the expression of genes to nearby parts of the body during its development (2). As well involved in cell-cycle arrest, cellular metabolism, senescence, and known, apoptosis plays an essential role in tumorigenesis. There- apoptosis (15–18). Up to date, the transcription factors responsible fore, the regulation of apoptosis in tumorigenesis by miRNAs (3) for the expression of protein-encoding genes have been well char- and protein-coding genes (4–7) has attracted extensive attentions. acterized. However, the roles of transcription factors in the miRNA In recent years, the extremely important roles of miRNAs in cancer gene expression are not extensively explored. At present, the reports pathogenesis have been revealed. miRNAs negatively regulate indicate that a transcription factor can act as a tumor promoter or a expressions of specific mRNAs by either translational inhibition tumor suppressor in tumorigenesis (19). But whether there is a or mRNA degradation via RNA-induced silencing complex (8). It is transcription factor mediating the transcriptions of miRNAs and found that all the examined cancers, such as colorectal neopla- protein-encoding genes, which are involved in tumorigenesis, has sia (9), lung cancer (10), glioblastoma (11) and B cell lympho- not been investigated. mas (12) are characterized with abnormal miRNA expression Ourpreviousstudiesrevealedthat miR-100 was upregulated in patterns (13). Despite the dysregulation of miRNAs during tumor- gastric and breast cancers (20, 21). The miR-100 antagonism igenesis, the underlying mechanisms are still largely unknown. It is specifically induced the apoptosis of poorly differentiated gastric believed that transcription factors are required for the miRNA cancer cells but not noncancerous gastric cells via the miR-100– transcription. RNF144B-pirh2-p53 signaling pathway, leading to the suppression Transcription factors are the adaptor molecules of DNA, which in the progression of gastric tumors (21). In this study, therefore, target the assembly of protein complexes to control gene expression. the mechanism of miR-100 dysregulation in cancers was investigated. The results showed that miR-100 was remarkably upregulated in sera of patients with gastric cancer, suggesting the bio- 1 College of Life Sciences and Laboratory for Marine Biology and Biotechnology marker potential of miR-100 in clinical diagnosis of gastric cancers. of Qingdao National Laboratory for Marine Science and Technology, Zhejiang Further data revealed that the transcription factor NME/NM23 University, Hangzhou, P.R. China. 2Department of Gastroenterology, The First Affiliated Hospital of Bengbu Medical College, Bengbu, P.R. China. nucleoside diphosphate kinase 2 (NME2) promoted the expression of apoptosis-associated miR-100 and protein-encoding genes Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). (RIPK1, STARD5,andLIMS1) in cancers. NME2, serving as a cancer metastasis–related protein (22), is involved in gene tran- Corresponding Author: Xiaobo Zhang, Zhejiang University, 388 Yuhangtang scription regulation (23). The results of this study showed that Road, Hangzhou, Zhejiang 310004, P.R. China. Phone: 8657-1889-81129; Fax: 8657-1889-81151; E-mail: [email protected] NME2 promoted the transcriptions of apoptosis-associated genes through mediating the phosphorylation of RNA polymerase Mol Cancer Res 2019;XX:XX–XX II C-terminal domain at the 5th serine. Further results indicated doi: 10.1158/1541-7786.MCR-19-0612 the important role of NME2 protein in antiapoptosis of gastric 2019 American Association for Cancer Research. cancer.

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Materials and Methods Results Cell culture Abnormal expression of miR-100 in sera of patients with gastric Gastric cancer cell lines (HGC-27 and MKN-45) and normal gastric cancer cell line GES-1 were purchased from the Cell Bank of the Chinese To characterize the role of miR-100 in human cancer, the miR-100 Academy of Sciences (Shanghai, China). HGC-27 and MKN-45 cells expression level in normal and cancerous gastric cell lines was detected. were cultured in RPMI1640 medium (Gibco) with 10% FBS (Gibco). The results showed that miR-100 was significantly upregulated in All cells were cultured at 37C in a humidified atmosphere with 5% gastric cancer cells, HGC-27 and MKN-45, compared with the normal fi CO2. The identities of the cell lines were con rmed by short tandem gastric cells, GES-1 (Fig. 1A). Our previous study revealed that miR- repeat testing in 2014. 100 was highly expressed in gastric cancer tissues compared with normal tissues (21). The differential expression of miR-100 in cancer- Tumorigenicity in nude mice ous and normal cells/tissues suggested that miR-100 was abnormally Gastric cancer cells, HGC-27, were collected and adjusted to expressed in patients with gastric cancer. 5 106 cells/mL with physiologic saline. Then, a 100 mL cell suspension To further evaluate the miR-100 expression in sera of patients with was subcutaneously injected into nude mice to induce tumor growth. gastric cancer and healthy persons, a total of 218 serum samples were One week later, when the tumor volume was around 30 mm3, the mice analyzed. qRT-PCR data showed the prominent upregulation of miR- were injected via the lateral tail vein with 80 mg/kg of NME2-siRNA or 100 in the sera of 90 patients with gastric cancer compared with that of NME2-siRNA-scrambled once every 3 days. Thirty days after the 128 normal sera (Fig. 1B), indicating that the abnormal expression of first injection, the mice were sacrificed and the solid tumors were miR-100 in patients with gastric cancer could be detected in sera. To collected. Animal experiments were approved by The Animal Exper- reveal the relationship between miR-100 expression level and gastric iment Centre of Zhejiang University. carcinogenesis, the sera of patients with various grades of the malig- nant tumor were subjected to the miR-100 detection using qRT-PCR. Apoptosis detection with annexin V The results presented that the content of miR-100 in patient sera was Apoptosis detection with Annexin V (Becton Dickinson) was significantly increased along with the higher grade of tumor malig- conducted according to the manufacturer's instructions. Cells were nancy (Fig. 1C). On the basis of the types of gastric cancer, the miR- collected and washed with cold PBS, followed by resuspension in 1 100 level in signet-ring cell carcinoma (poor differentiation and high annexin-binding buffer. Then the cells were added with Alexa Fluor malignancy) was remarkably higher than that in other gastric adeno- 488 annexin V and propidium iodide and incubated in the dark for 15 carcinoma (Fig. 1D), suggesting the connection between the miR-100 minutes at room temperature. To stop the reaction, 1 annexin- content and the tumor malignancy. Among the patients with gastric binding buffer was added. Apoptosis of cells was measured by flow cancer, the miR-100 content was the highest in the sera of patients with cytometry at 530 nm and 575 nm using an excitation wavelength of cancer metastasis (Fig. 1E), suggesting that the abnormal expression of 488 nm. miR-100 was related with cancer metastasis. Taken together, our study revealed that miR-100 could be a Coimmunoprecipitation assay biomarker for the clinical diagnosis of gastric cancer by detecting its Cells were harvested through centrifugation at 300 g for expression in serum. 10 minutes and lysed with ice-cold cell lysis buffer (Beyotime Biotechnology). An antibody was incubated with Protein G- Effects of miR-100 promoter on the inordinate expression of agarose Beads (Invitrogen) for 4 hours at 4 C, and then the lysate miR-100 in gastric cancer was added to the mixture. The mixture was incubated at 4 C To explore the mechanism of the abnormal expression of miR-100 overnight. After washing three times with ice-cold PBS, in human gastric cancer, the expression levels of primary and pre- the coimmunoprecipitation (Co-IP) product was collected for cursor miR-100 during posttranscription processing were examined. later use. The results indicated that the primary miR-100, the precursor miR- 100, and the mature miR-100 were significantly upregulated in gastric Chromatin immunoprecipitation analysis cancer cells (HGC-27 and MKN-45) compared with noncancerous HGC-27 cells were incubated with 1% formaldehyde for 10 minutes cells (GES-1; Fig. 2A). This indicates that the abnormal expressions of at 37 C, followed by the addition of glycine. After washing with cold miR-100 in cancer cells were independent of posttranscriptional PBS twice, the cells were lysed with cell lysis buffer (Beyotime processing of miR-100 and might rely on the miR-100 promoter. Biotechnology) containing 2 mmol/L of phenylmethylsulfonylfluor- To identify the RNA polymerase (RNA pol) involved in the miR- ide. Subsequently the cells were ultrasonicated for 1 minute. The anti- 100 transcription, the activity of RNA pol II or RNA pol III was NME2 IgG and dynabeads were added into the ultrasonicated cells and inhibited, followed by the miR-100 quantification. The results showed incubated at 4 C overnight. Rabbit IgG was used as a negative control. that the RNA pol II inhibitor caused a significant decrease of miR-100 The beads were washed with PBS for at least three times, and then expression level, while the RNA pol III inhibitor had no effect on the eluted with elution buffer (1% SDS, 0.1 mol/L NaHCO3). The mixture miR-100 expression (Fig. 2B), indicating that RNA pol II was required was centrifuged for 5 minutes at 15,000 g and the supernatant was for the transcription of miR-100. subjected to agarose gel electrophoresis. On the basis of 50 rapid amplification of cDNA ends (RACE) analysis, the transcription start site of primary miR-100 was identified. To Statistical analysis obtain the promoter of miR-100, the sequences containing the putative All experiments were biologically repeated three times. Numer- miR-100 promoter were cloned and then subjected to the promoter ical data were analyzed using a one-way ANOVA. The statistical activity assays. The results showed that a 50-bp fragment of the putative significance between treatments was assessed by one-tailed Student miR-100 promoter was the core promoter region for the transcription t test. of primary miR-100 (Fig. 2C). In this 50-bp fragment, a typical TATA

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Figure 1. A B Abnormal expression of miR-100 in 60 sera of patients with gastric cancer. A, The expression level of miR-100 6 in gastric cancer and noncancerous 40 cells. The expression level of miR- 4 100 in different cells including normal gastric epithelial cells (GES-1) 20 and gastric cancer cells (HGC-27 2 and MKN-45) was examined with of miR-100 Level qRT-PCR. B, The detection of 0 0 miR-100 expression in the sera of Relative miR-100 level patients with gastric cancer and GES-1 HGC-27 MKN-45 healthy donors (without cancer). persons C, The expression level of miR-100 Sera of healthy Sera of gastric in the sera of patients with gastric (n = 128) cancer(n patients = 90) cancer with different grades of cancer malignancy. D, The analysis of C Grade 1 (low malignancy) Grade 2 (medium malignancy) Grade 3 (high malignancy) miR-100 content in the sera of signet-ring cell carcinoma (n ¼ 25) and other gastric adenocarcinoma (n ¼ 65). E, The content of miR-100 in the sera of patients with cancer metastasis. Data presented are representatives of three independent experiments (, P < 0.05; , P < 0.01).

20 Level of miR-100 Level

Grade 1 Grade 2 Grade 3 (n = 40) (n = 35) (n = 15)

D 60 E 60

40 40

20 20 Level of miR-100 Level Level of miR-100 Level

0 0

carcinoma Other gastric metastasis Signet-ring cell Serawithout of patients cancer adenocarcinoma (n = 25) (n = 65) Sera cancerof patients metastasis with (n = 7) (n = 83) box was found (Fig. 2C). To examine whether this TATA box was cancerous cells (MKN-45 and HGC-27). The promoter activities in canonical, the six nucleotides of the TATA box were randomly mutated MKN-45 and HGC-27 cells were much higher than that in GES-1 cells (Fig. 2C). As controls, the sequences near the TATA box were mutated (Fig. 2E), which was consistent with the results of miR-100 expression (Fig. 2C). The results showed that the TATA box mutant (mutant-2) in gastric cancerous and noncancerous cells (Fig. 1A). The transfec- presented a remarkably decreased luciferase activity compared with tion of miR-100 promoter from gastric cancerous cells into noncan- the controls (mutant-1 and mutant-3; Fig. 2D), revealing that the cerous cells yielded the similar results (Fig. 2E), indicating that the TATA box was required for the transcription of primary miR-100. activity of miR-100 promoter was independent on its origin. These To evaluate the effects of miR-100 promoter on the abnormal data revealed that the mR-100 promoter activity was dependent on the expression of miR-100 in cancer cells, the miR-100 promoter cloned proteins bound with the promoter. It is well known that the gene from gastric noncancerous cells (GES-1) were transfected into gastric expression can be affected by DNA methylation, which generally

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A miR-100 Precursor miR-100 Primary miR-100 B 8 1.2

1.0 6 0.8

4 0.6

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Normalized fold 2 Normalized fold 0.2 expression of miR-100 expression expression of miR-100 expression 0 0.0

GES-1 GES-1 GES-1 HGC-27MKN-45 HGC-27MKN-45 HGC-27MKN-45 Nontreated

RNA pol II inhibitorRNA pol III inhibitor C 1 50

Mutant-1:GGACCC Mutant-2:TTGCGC Mutant-3:CCAACG

D 1.5 E Promoter from GES-1 Promoter from MKN-45 Promoter from HGC-27

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GES-1 GES-1 GES-1 Wild-type Mutant-1 Mutant-2 Mutant-3 MKN-45HGC-27 MKN-45HGC-27 MKN-45HGC-27

F 100% GC%

50%

Figure 2. Effects of miR-100 promoter on the inordinate expression of miR-100 in gastric cancer. A, The expression levels of primary miR-100, precursor miR-100, and mature miR-100 in gastric cancer cells (HGC-27 and MKN-45) and noncancerous cells (GES-1) by qRT-PCR. B, The requirement of RNA pol II for the miR-100 transcription. HGC-27 cells were used. C, The sequence of miR-100 promoter. The six nucleotides of the TATA box were randomly mutated (mutant-2). As controls, the sequences nearby the TATA box were also mutated (mutant-1 and mutant-3). D, The inﬂuence of TATA box on the miR-100 promoter activity. E, The effects of miR-100 promoter origin on the promoter activity in gastric cells. F, Bioinformatics analysis of methylation of miR-100 promoter sequence. Data presented are representatives of triplicate assays (, P < 0.01).

occurs on CpG islands. To explore whether the transcription of miR- Influence of transcription factor NME2 on the miR-100 100 was dependent on methylation, a bioinformatics approach was transcription used to analyze the promoter of miR-100. The results indicated that the To explore the transcription factors required for the miR-100 GC content of the miR-100 promoter was only 32.47% and no obvious transcription, the proteins bound to the miR-100 promoter were CpG enrichment area was found in the miR-100 promoter (Fig. 2F), screened. The biotin-labeled miR-100 promoter was incubated with showing that the transcription of miR-100 was not regulated by cell lysate. The results from mass spectrometry identification indicated methylation. that NME2 specifically interacted with the miR-100 promoter Taken together, these findings showed that the inordinate expres- (Fig. 3A). Western blots also confirmed the binding of NME2 to the sion of miR-100 in cancer relied on the transcription factors bound miR-100 promoter (Fig. 3B). To further confirm the interaction with the miR-100 promoter. between the NME2 protein and the miR-100 promoter,

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A B

Biotin-labeled Biotin-labeled

M (kDa) Biotin miR-100 promoter NME2 protein Biotin miR-100 promoter

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Mass (m/z)

C NME2 protein concentration BSA protein concentration ChIP product miR-100 promoter bound to NME2 protein

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M (bp) NME2 Rabbitantibody IgG BSA protein NME2 protein 500 400 300 200 100 miR-100 promoter E

NME2 binding region TATA box G GES-1 12

8 F NME2-D1-66 NME2-D66-118 NME2-D66-152 protein concentration protein concentration protein concentration 6

miR-100 promoter bound miR-100 promoter bound 4 to NME2-D66-118 protein to NME2-D118-152 protein 2 Relative promoter activity Relative promoter 0 Free miR-100 promoter Free miR-100 promoter Free miR-100 promoter

Control pEasy-M1 NME2-D1-66 protein NME2-D66-118 protein NME2-D118-152 protein pEasy-M1-NME2

pGL3-miR-100pGL3-miR-100 promoter+ promoter+

H GES-1 4 8 HGC-27 MKN-45 I HGC-27 MKN-45 3 6

2 4 Nontreated Nontreated NontreatedNME2 overexpression NME2-siRNA NME2-siRNA 1 2

NME2 NME2 Relative miR-100 level Relative miR-100 level 0 0 b-Tubulin b-Tubulin

Nontreated Nontreated Nontreated NME2-siRNA NME2-siRNA

NME2 overexpression

Figure 3. Influence of transcription factor NME2 on the miR-100 transcription. A, Identification of proteins bound to the miR-100 promoter. The lysate of HGC-27 cells was incubated with miR-100 promoter. The proteins bound to the miR-100 promoter were separated with SDS-PAGE (top) and then identified with mass spectrometry (bottom). The protein identified is indicated with an arrow. The matched peptides to the NME2 protein sequence are indicated with numbers, asterisks, and solid underlines. M, protein marker. B, Western blot of the protein bound to the miR-100 promoter. C, The binding of miR-100 promoter to NME2 protein by EMSA analysis. BSA was used as a control. D, The DNA bound to the NME2 protein. The DNA was analyzed with (Continued on the following page.)

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electrophoretic mobility shift assay (EMSA) was conducted. The miR- by NME2, ChIP assays were conducted in HGC-27 cells using the 100 promoter displayed concentration-dependent binding to the NME2-specific antibody (Fig. 3D). The sequencing data of ChIP NME2 protein and did not bind to the control protein (Fig. 3C). The products indicated that the promoter sequences bound to the NME2 DNAs bound to the NME2 protein were obtained with chromatin protein could be classified into 10 motifs (Fig. 4A). These promoters immunoprecipitation (ChIP) using the anti-NME2 IgG (Fig. 3D). The were involved in the transcriptions of 65 genes (motif 1: 26 genes; motif PCR data revealed that the miR-100 promoter was amplified from the 2: 28 genes; motif 3: 21 genes; motif 4: 32 genes; motif 5: 15 genes; motif ChIP products using the NME2 antibody but not the control (Fig. 3D), 6: 18 genes; motif 7: 19 genes; motif 8: 42 genes; motif 9: 16 genes; and which was further confirmed by DNA sequencing. These results motif 10: 17 genes). To further evaluate the effects of NME2 protein on indicated that the NME2 protein specifically interacted with the the 10 motif-mediated genes’ transcriptions, the expression of NME2 miR-100 promoter. gene was knocked down by the NME2-specific siRNA (NME2- To characterize the binding region of NME2 protein to the miR-100 siRNA), followed by the detection of 10 motif-mediated genes’ expres- promoter, DNase I footprinting assay was conducted. The sequencing sion. On the basis of the possibility of each base in a motif sequence, the data revealed that a 96-bp fragment of miR-100 promoter (including gene was selected for its mRNA detection (motif 1, RIPK1; motif 2, the core 50-bp fragment) was bound to the NME2 protein (Fig. 3E). NTF3; motif 3, ITGA9; motif 4, MAP3K19; motif 5, CD14; motif 6, The collective data presented that the NME2 protein specifically STARD5; motif 7, LIMS1; motif 8, POLR3B; motif 9, RPL23; and motif interacted with the miR-100 promoter. 10, ALDH2). The qRT-PCR results showed that the mRNA levels of To explore the binding of miR-100 promoter to three domains of RIPK1, STARD, LIMS1, RPL23, and ALDH2 were significantly NME2 protein including binding domain (amino acids 1–66), energy decreased along with the downregulation of NME2 (Fig. 4B), suggest- domain (amino acids 66–118), and catalytic domain (amino acids ing that the transcription factor NME2 was responsible for the 118–152), the purified proteins of the deletion mutants and miR-100 expression of these 5 genes. promoter were analyzed by EMSA. The results showed that there was a To explore whether the NME2 protein was involved in the tran- concentration-dependent binding of miR-100 promoter to the NME2 scriptions of RIPK1 (belonging to motif 1), STARD (motif 6), LIMS1 mutants NME2-D66-118 and NME2-D118-152, while the miR-100 (motif 7), RPL23 (motif 9), and ALDH2 (motif 10) genes, the expres- promoter did not bind to the NME2-D1-66 mutant (Fig. 3F). These sion of NME2 was knocked down in HGC-27 cells, and then the results indicate that the binding domain (amino acids 1–66) of NME2 promoter activities of the 5 genes were evaluated. The NME2 silencing protein interacted with the miR-100 promoter. led to significant decreases of the promoter activities of RIPK1, STARD, To investigate the effects of NME2 on the transcription of miRNA, and LIMS1 genes (Fig. 4C). On the other hand, the overexpression of the construct of miR-100 promoter and the plasmid expressing NME2 NME2 resulted in significant increases of the promoter activities of was cotransfected into GES-1 cells, in which the NME2 protein was not RIPK1, STARD5, and LIMS1 (Fig. 4D). These data showed that the detected by Western blot analysis. The results indicated that the NME2 protein could be a transcription factor of expression of motif 1-, activity of miR-100 promoter was significantly enhanced by the NME2 motif 6-, and motif 7–mediated genes. protein (Fig. 3G), showing the possible role as a miRNA transcription Asreported,RIPK1,LIMS1(alsoknownasPINCH1),and factor of NME2 protein. STARD5 are involved in the apoptosis signaling pathway (24–26). The above data revealed that miR-100 and NME2 were highly Our results showed that the knockdown of RIPK1, STARD5,or expressed in gastric cancer cells (HGC-27 and MKN-45). Therefore LIMS1 by gene-specificsiRNAsignificantly promoted apoptosis of the expression of NME2 gene was knocked down in HGC-27 and gastric cancer cells compared with the control (Fig. 4E). In this MKN-45 cells, followed by the evaluation of the miR-100 expression context, the NME2 protein might be a transcription factor for genes level. The results showed that the NME2 silencing led to a significant involved in apoptosis. decrease of the miR-100 expression level in gastric cancer cells To explore the role of NME2 in apoptosis, the NME2 expression was (Fig. 3H), indicating a very important role of NME2 protein in the knocked down in HGC-27 and MKN-45 cells, followed by the transcription of miR-100 in cells. To overexpress NME2, the plasmid examinations of cell viability and apoptosis. The results showed that expressing the MNE2 protein was transfected into gastric normal cells the cell viabilities of HGC-27 and MKN-45 cells were significantly (GES-1). The overexpression of NME2 protein significantly increased decreased when the NME2 expression was silenced (Fig. 4F). The the level of miR-100 in cells (Fig. 3I). percentage of apoptotic cells was remarkably increased after NME2 These findings revealed that the NME2 protein was a transcription knockdown (Fig. 4G). These data indicated that the NME2 protein factor required for the transcription of miR-100. took an inhibitory effect on apoptosis of gastric cancer cells. Further- more, Western blots demonstrated that the NME2 silencing led to Role of transcription factor NME2 in the transcriptions of significant increase of the expression levels of proapoptotic genes [p53, apoptosis-associated genes PUMA (p53 upregulated modulator of apoptosis), Bax, and cleaved As a transcription factor, the genes transcribed by NME2 need caspase 3] and a significant decrease of the antiapoptotic gene Bcl-2 (B- to be addressed except for miR-100. To explore the genes transcribed cell lymphoma 2) expression level (Fig. 4H). However, the

(Continued.) agarose gel electrophoresis (left). The rabbit IgG was used as a control. The miR-100 promoter was amplified using the ChIP product (right). M, DNA marker. E, The sequence of miR-100 promoter bound to the NME2 protein. F, The binding domain analysis of NME2 protein to the miR-100 promoter. The NME2 deletion–mutant proteins and the miR-100 promoter were analyzed by EMSA. G, The influence of NME2 protein on the miR-100 promoter activity. The construct of miR-100 promoter (pGL3-miR-100 promoter) and the plasmid expressing the NME2 protein (pEasy-M1-NME2) were cotransfected into GES-1 cells. The vector alone (pEasy-M1) was included in the cotransfection. Cells transfected with the control pRL-TK plasmid were used as controls. At 48 hours after transfection, the miR-100 promoter activity was examined. H, The effects of NME2 silencing on the expression of miR-100 in cells. The gastric cancer cells (HGC-27 and MKN-45) were treated with NME2-specific siRNA (NME2-siRNA). Forty-eight hours later, the NME2 protein was detected using Western blot analysis (left) and the expression level of miR- 100 was examined by qRT-PCR (right). I, The influence of NME2 overexpression on the miR-100 expression in normal gastric cells (GES-1). Cells were transfected with the plasmid expressing NME2 protein. At 48 hours after transfection, the NME2 protein and the miR-100 expression level were evaluated with Western blot analysis (left) and qRT-PCR (right), respectively. All the numeral data referes to the mean SD of triplicate assays (, P < 0.05; , P < 0.01).

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overexpression of the full-length or truncated NME2 protein had no targets (miR-100, RIPK1, STARD5, and LIMS1) did not rescue gastric effect on the cell viability of gastric cancer cells (HGC-27; Fig. 4I). Our cancer cells from the NME2 siRNA-induced cell death compared with results indicated that the overexpression of the NME2 downstream the controls (Fig. 4J).

A B Motif 1 Motif 2 Motif 3 Motif 4 Motif 5 Motif 6 Motif 7 Motif 8 Motif 9 Motif 10 D Motif 1 Motif 6 Motif 7 Motif 9 Motif 10 1.5 12 10 1.0 8

Motif 1 6 0.5 4 expression 2 Motif 2 Normalized gene 0.0 activity Relative promoter 0 -+ -+ -+ -+ -+ -+ -+ -+ -+ -+NME2-siRNA

Motif 3 Control Control Control Control Control NTF3 CD14 RIPK1 ITGA9 LIMS1 RPL23 ALDH2 STAED5 POLR3B MAP3K19 RIPK1 promoter LIMS1 promoter RPL23 promoter ALDH2 promoter RIPK1 promoter+STARD5 promoter LIMS1 promoter+ RPL23 promoter+ ALDH2 promoter+ Motif 4 STARD5 promoter+ NME2 overexpressionNME2 overexpressionNME2 overexpressionNME2 overexpressionNME2 overexpression Motif 5 C E Motif 1 Motif 6 Motif 7 Motif 9 Motif 10 Motif 6 8

6 60 Motif 7 40 4 Nontreated RIPK1-siRNA Motif 8 20 2 0

Motif 9 activity promoter Percentage 0 Percentage of apoptotic cells (%) Percentage Nontreated RIPK1-siRNA LIMS1-siRNA Control Control Control Control Control STARD5-siRNA Motif 10 promoter+ NME2-siRNA NME2-siRNA NME2-siRNA NME2-siRNA NME2-siRNA STARD5-siRNA LIMS1-siRNA RIPK1 promoter LIMS1 promoter RPL23 promoter ALDH2 promoter RIPK1 promoter+ STARD5 promoter LIMS1 RPL23 promoter+ ALDH2 promoter+ STARD5 promoter+

FG H

HGC-27 MKN-45 HGC-27 MKN-45 40 HGC-27 MKN-45 -+-+NME2 siRNA 1.5 30 NME2 HGC-27 MKN-45 p53 1.0 20 PUMA 10 0.5 Bcl-2 0 -+ -+NME2-siRNA Bax

Relative cell viability 0.0 -+ -+NME2-siRNA Cleaved caspase 3 Percentage of apoptotic cells (%) Percentage b-Tubulin HGC-27+NME2-siRNA MKN-45+NME2-siRNA

HGC-27 MKN-45 I J 1.5 1.5

1.0 1.0

0.5 0.5 Relative cell viability Relative cell viability 0.0 0.0

D66-118 Nontreated pEasy-NME2 Nontreated Nontreated NME2-siRNA NME2-siRNA pEasy-NME2-D1-66 pEasy-NME2- pEasy-NME2-D118-152 NME2-siRNA+RIPK1NME2-siRNA+LIMS1 NME2-siRNA+RIPK1NME2-siRNA+LIMS1 NME2-siRNA+miR-100NME2-siRNA+STARD5 NME2-siRNA+miR-100NME2-siRNA+STARD5

Figure 4. Role of transcription factor NME2 in the transcriptions of apoptosis-associated genes. A, The motifs of promoters bound to the NME2 protein. B, The effects of NME2 protein on the 10 motif-mediated genes’ transcriptions. The expression of NME2 gene was silenced by NME2-siRNA in HGC-27 cells. Then 10 motif-mediated genes’ expression at mRNA level were detected with qRT-PCR. b-actin was used as an internal reference. C, The influence of NME2 silencing on the gene promoter activity. The plasmid containing a gene promoter and NME2-siRNA were transfected into HGC-27 cells. Forty-eight hours later, the promoter activity was examined. The empty PGL3-basic vector was used as an internal control. D, The effects of NME2 overexpression on the gene promoter activity. The promoter and the plasmid expressing NME2 were transfected into HGC-27 cells. At 48 hours after transfection, the promoter activity was evaluated. E, The influence of RIPK1, STARD5, or LIMS1 silencing on apoptosis of gastric cancer cells. The expression of RIPK1, STARD5, or LIMS1 was knocked down in HGC-27 cells. At 48 hours after the siRNA transfection, the percentage of apoptotic cells was examined with annexin V. F, The influence of NME2 silencing on cell viability. G, The effects of NME2 silencing on apoptosis. H, The impact of NME2 silencing on the expression of apoptosis-associated genes by Western blot analysis. I, The effects of the overexpression of the full-length or truncated NME2 protein on cell viability. The full-length or truncated NME2 was overexpressed in HGC-27 cells. Forty-eight hours later, the cell viability was evaluated. J, The influence of overexpression of the NME2 downstream targets on apoptosis of NME2-silenced gastric cancer cells. Gastric cancer cells (HGC-27 and MKN-45) were cotransfected with NME2-siRNA and the plasmid expressing miR-100, RIPK1, STARD5, or LIMS1. At 48 hours after cotransfection, the viability of gastric cancer cells was examined. In all panels, data represent mean SD of triplicate assays (, P < 0.05; , P < 0.01).

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Taken the above data together, these findings presented that the Suppression of tumorigenesis of gastric cancer by NME2 transcription factor, NME2, was a master suppressor for apoptosis of antagonism in vivo gastric cancer. To further explore the antiapoptotic role of transcription factor NME2 in vivo, gastric cancer cells (HGC-27) were transplanted into Underlying mechanism of NME2 promoting the transcriptions of nude mice, followed by the injection of NME2-siRNA every 3 days for a apoptosis-associated genes month. The comparison analysis of gene silencing efficiency between To reveal the mechanism that NME2 promoted the transcriptions of NME2-siRNA and NME2-shRNA showed that NME2-siRNA and apoptosis-associated genes, the proteins interacting with NME2 were NME2-shRNA generated the same results of NME2 gene silencing in characterized. The results of Co-IP assays in HGC-27 cells showed that HGC-27 cells (Fig. 6A). In this study, NME2-siRNA was used in the three specific protein bands were found in the Co-IP products using in vivo experiments. The results indicated that the tumor sizes of the NME2-specific antibody compared with the control (Fig. 5A). MS NME2-siRNA–treated mice were much smaller than those in NME2- identification revealed that these proteins were RNA pol II subunit 1 siRNA-scrambled–injected mice (Fig. 6B). IHC analysis revealed that (RPB1), RNA pol II-associated protein 2 (RPAP2), and NME2, the NME2 knockdown resulted in a decreased expression of the tumor respectively (Fig. 5A). Western blot data confirmed the presence of proliferation marker Ki-67 and enhanced expression of apoptosis RPB1 and RPAP2 in the Co-IP products using the NME2-specific marker caspase 3 (Fig. 6C). At the same time, the expression levels antibody (Fig. 5B). These results indicate that NME2 interacted with of miR-100, RIPK1, STARD5, and LIMS1 were significantly down- PRB1 and RPAP2. regulated in tumors of mice treated with NME2-siRNA compared with To explore the interaction between NME2 and RPAP2 proteins the control (Fig. 6D). These findings revealed that the NME2 silencing in cells, the full-length NME2, deletion mutants of NME2, full- downregulated the expression of miR-100, RIPK1, STARD5, and length RPAP2, or deletion mutants of RPAP2 were transfected into LIMS1 and then triggered apoptosis of gastric cancer cells, thus HGC-27 cells, followed by immunoprecipitation (IP) analysis. suppressing tumor growth in vivo. Western blot results showed that the RPAP2 protein was not detected in the IP product of cells transfected with NME2-D1–66 Upregulation of transcription factor NME2 in patients with and RPAP2 (Fig. 5C), indicating that the binding domain (amino gastric cancer acids 1–66) of NME2 protein was bound to RPAP2 protein. When The findings of this study revealed that the NME2 protein could the cells were transfected with RPAP2-D1–77, NME2 did not exist promote the transcriptions of antiapoptotic genes including miR-100 in the IP product (Fig. 5C), showing that the binding domain and protein-encoding genes (RIPK1, STARD5, and LIMS1) in gastric (amino acids 1–77) of RPAP2 protein interacted with NME2. cancers. Thus, the expression of NME2 in gastric cancers was explored. Further data demonstrated the interaction between NME2 (amino Western blot analysis indicated that NME2 was significantly upregu- acids 1–66) and RPAP2 (amino acids 1–77) (Fig. 5D). To inves- lated in gastric cancer cells (HGC-27 and MKN-45) compared with tigate the interaction between NME2 and RPB1 proteins, the full- normal gastric epithelial cells (GES-1; Fig. 7A). The data from NCBI length NME2, deletion mutants of NME2, or the C-terminal showed a significantly increased level of NME2 mRNA in patients with domain (CTD) of RPB1 was transfected into HGC-27 cells. The gastric cancer compared with the healthy donors (Fig. 7B). results demonstrated that the catalytic domain (118–152 amino To evaluate the expression of NME2 protein in patients with gastric acid) of NME2 was bound to the CTD of RPB1 (Fig. 5E). It has been cancer, the gastric tissues of patients with cancer and healthy donors revealed that RNA pol II is composed of 12 subunits (RPB1–12; were analyzed. Western blot results demonstrated that the NME2 ref. 27) and RPAP2 can directly bind to RPB6 (28). In this context, protein was significantly upregulated in gastric cancer tissues com- NME2, RPB1, and RPAP2 formed a complex in gastric cancer cells. pared with the normal samples (Fig. 7C). The immunohistology As reported, NME2 (nucleoside diphosphate kinase 2) serves as a analysis also showed that the NME2-positive signals were remarkably kinase (29) and the phosphorylation of RNA pol II always occurs in the increased in gastric cancer tissues (Fig. 7D). To explore the impact of CTD of RPB1 (30). Thus the binding of the catalytic domain of NME2 NME2 on the therapy-induced cell death, NME2 was silenced in the to the CTD of RPB1 might activate the phosphorylation of RNA pol II. cisplatin-treated gastric cancer cells (HGC-27 and MKN-45) and then The results indicated that NME2 could effectively induce the phos- the cell viability was examined. The results showed that the NME2 phorylation of RNA pol II compared with the controls (Fig. 5F). It has silencing significantly promoted the cisplatin-induced cell death com- been reported that the transcriptional activity of RNA pol II depends pared with the control (Fig. 7E). on the phosphorylation level of RPB1 CTD at the 2nd serine and the Taken together, these findings presented that the upregulation of 5th serine (31). Our results revealed that NME2 mediated the phos- transcription factor NME2 promoted the expression of antiapoptotic phorylation of RPB1 CTD at the 5th serine but not the 2nd serine genes via mediating the phosphorylation of RNA pol II in gastric (Fig. 5F). cancers, leading to the suppression of apoptosis of gastric cancer cells To explore the effects of NME2 on the phosphorylation of RNA pol (Fig. 7F). II in cells, NME2 was silenced or overexpressed in cells, followed by the detection of the phosphorylation level of RNA pol II. The results indicated that the NME2 overexpression significantly increased the Discussion phosphorylation level of RNA pol II at the 5th serine, while the NME2 Tumorigenesis results from uncontrollable cell proliferation silencing had little influence on the phosphorylation level of RNA pol regulated by a variety of complex factors (32). During tumorigen- II (Fig. 5G). The data presented that NME2 was required for the esis, the inhibition of apoptosis plays an essential role, which is phosphorylation of RNA pol II in cells. controlled by oncogenes and tumor suppressors (33). As well The above findings demonstrated that NME2 could promote the known, transcription factors are central modulators of cellular phosphorylation of RNA pol II C-terminal domain at the 5th serine via events including cell-cycle progression and apoptosis through the NME2–RPAP2–RNA pol II protein complex in cells, leading to the transcriptional regulation of a series of related genes (34). Thus transcriptions of apoptosis-associated genes. far, many transcription factors are found to take great effects on

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IP product

A protein RPAP2 RPB1 protein

M (kDa) Rabbit IgGAnti-NME2 IgG 170 RPB1 130 100 70 RPAP2 55 40

35 25

NME2 % Intensity 15 % Intensity 0 500 1,000 1,500 0 500 1,000 1,500 2,000 1,000 1,500 2,000 2,500 800 1,000 1,200 1,400 1,600 1,800 2,000 Mass (m/z) Mass (m/z)

His IP

77-160 B C D D160-612 IP product His IP

His-RPAP2His-RPAP2-His-RPAP2-His-RPAP2-D1-77 D1-66 IP NME2 Rabbit Anti-NME2IgG IgG

RPB1 His-NME2His-NME2-His-NME2-His-NME2-D66-118D118-152 IP RPAP2 RPAP2 Input His

Input His

D E His IP RabbitAnti-His IgG IgG Rabbit Anti-HisIgG IgG His-CTD His-NME2-1-66 D1-66D66-118 Flag-NME2-118-152 Flag-RPAP2-1-77 RabbitAnti-His IgG IgG His-CTD His His IP IP His-NME2His-NME2-His-NME2-His-NME2-D118-152 His Flag IP Flag IP RPB1 NME2 His His His Input Input His Input Input Flag NME2 Flag

FGRNA Pol II IP

WCE InputCDK7NME2 NME2-siRNA IIO NME2 overexpression RNA Pol II IIA NME2 Ser5-P Ser2-P b Ser5-P -Tubulin

Figure 5. Underlying mechanism of NME2 promoting the transcriptions of apoptosis-associated genes. A, Identification of proteins bound to NME2. The nuclear extract of HGC-27 cells was subjected to Co-IP using the anti-NME2 IgG. The proteins of Co-IP assays were separated with SDS-PAGE (top) and then identified using mass spectrometry (bottom). The proteins identified are indicated with arrows. The matched peptides to the protein sequences are indicated with numbers, asterisks, and solid underlines. M, protein marker. B, Western blot analysis of the proteins bound to NME2. C, The interaction between NME2 and RPAP2 in gastric cancer cells. HGC- 27 cells were transfected with full-length NME2, deletion mutants of NME2, full-length RPAP2, or deletion mutants of RPAP2. At 48 hours after transfection, IP analysis was conducted using the antibody against His tag. The IP products were analyzed by Western blots using antibodies as shown on the right. D, The interaction between the binding domain of NME2 and the binding domain of RPAP2. The construct of His-NME2-1-66 or Flag-RPAP2-1-77 was transfected into HGC-27 cells. Forty-eight hours later, the cells were subjected to IP with anti-His IgG, followed by Western blot analysis with antibodies indicated on the right. E, The interaction between NME2 and RPB1 in HGC-27 cells. The full-length NME2, deletion mutants of NME2, or the RPB1 CTD was transfected into cells. At 48 hours after transfection, the cells were analyzed using IP analysis with anti-His or anti-Flag antibody. Subsequently, Western blot analysis was conducted with antibodies indicated on the right. F, The influence of NME2 on RNA pol II phosphorylation. The unphosphorylated RNA pol II was immunoprecipitated using an antibody recognizing unphosphorylated RNA pol II CTD in HGC-27 cells. The IP product was incubated with NME2 protein. As controls, CDK7 (RNA pol II CTD 5th serine phosphorylase) and WCE (whole-cell extract) were included in the assays. The phosphorylation of RNA pol II was examined by Western blot analysis using antibodies indicated on the right. IIO and IIA represented hyper-phosphorylated and hypo-phosphorylated RNA pol II, respectively. G, The impact of NME2 silencing or overexpression on the 5th serine phosphorylation of RNA pol II CTD in HGC-27 cells.

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Figure 6. A 1.5 Suppression of tumorigenesis of gastric cancer by NME2 antagonism in vivo. 1.0 A, The comparison of gene silencing NontreatedNME2-siRNANME2-shRNA efﬁciency between NME2-siRNA and NME2-shRNA. HGC-27 cells were trans- 0.5 NME2 fected with NME2-siRNA or NME2- shRNA. At 48 hours after transfection,

Relative NME2 level the gene silencing efﬁciency was eval- 0.0 b -Tubulin uated by qRT-PCR (left) and Western blot analysis (right). B, The effects of NME2 silencing on solid tumors in nude Nontreated mice. HGC-27 cells were injected into NME2-siRNANME2-shRNA nude mice, and then siRNAs were sub- 600 cutaneously and intravenously injected B into the mice. The tumor images were NME2-siRNA-scrambled-treated mice obtained (top) and the tumor weight 400 was evaluated (bottom; , P < 0.01) at 30 days after siRNAs injection. 200 C, IHC analysis of solid tumors. The positive signals are indicated with -

NME-siRNA treated mice weight (mg) Tumor arrows (top) and calculated (bottom; 0 , P < 0.01). Scale bar, 25 mm. D, Expres- sion levels of miR-100, RIPK1, STARD5, and LIMS1 in solid tumors. The data represent the mean SDs of triplicate scrambled NME2-siRNA NME2-siRNA- assays and asterisks indicate statisti- C NME2-siRNA- cally signiﬁcant differences between scrambled NME2-siRNA treatments (, P < 0.01). ki-67 Caspase 3

Ki-67 80 60 m m 25 m 25 m 40

20 Positive signal Positive 0 Caspase 3

25 mm25 mm NME-siRNA-scrambledNME2-siRNANME-siRNA-scrambledNME2-siRNA

D 1.5 NME2 miR-100 LIMS1 1.0 STARD5 RIPK1

0.5 Relative gene expression 0.0

NME2-siRNA-scrambled NME2-siRNA

apoptosis during tumorigenesis (5, 35–37). However, these tran- miR-100 antagonism triggers apoptosis of gastric cancer cells by scription factors are responsible for the transcriptional regulation inhibiting ubiquitination-mediated p53 degradation (21), showing of protein-encoding genes. In this study, the ﬁndings highlighted miR-100 is an antiapoptotic miRNA. It is well known that apoptosis NME2 as a novel antiapoptotic transcription factor which is triggered by a sophisticated gene network consisting of miRNAs could mediate the transcriptions of antiapoptotic miRNA (miR- and protein-encoding genes. In this context, our study provided a 100) and antiapoptotic protein-encoding genes (RIPK1, STARD5, more comprehensive understanding of the mechanism involved in and LIMS1).Inourpreviousinvestigation,theresultsindicatethat the regulation of apoptosis.

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A B 500 400 300 200 GES-1MKN-45HGC-27 100 NME2

mRNA expression 0

b-Tubulin of NME2 Relative level Normal samples samples (n = 10)Gastric cancer (n = 24) CD Normal tissues Gastric cancer tissues Normal tissue Gastric cancer tissue

NME2 b-Tubulin NME2

1.5 25 mm 25 mm

1.0 50 40 0.5 30 protein level protein 20 Normalized NME2 10

0.0 signal Positive 0

Normal tissues tissues Normaltissue tissue (n = 10)Gastric (cancern = 10) Gastric cancer EF HGC-27 MKN-45

1.5 Gastric cancer cell RNA pol II RPAP2 RPB6 1.0 P53 NME2 RPB1 0.5 P27 0.0 RNA pol II HS3ST2

Relative cell viability RPAP2 RPB6 Apoptosis NF-κB NME2 Nontreated Nontreated RPB1 miR-100 P RIPK1 FXR STARD5 ERK-Bim Cisplatin+NME2-siRNA Cisplatin+NME2-siRNA LIMS1

Gene expression Bcl-2

Cisplatin+NME2-siRNA-scrambledCisplatin+NME2-siRNA-scrambled

Figure 7. Upregulation of NME2 in patients with gastric cancer. A, The differential expression of NME2 protein in gastric cancerous and noncancerous cells by Western blot analysis. B, The NME2 mRNA level in healthy donors and patients with gastric cancer. The data were collected from Gene Expression Omnibus (GEO) proﬁles in NCBI database (the GEO identities 105752367 and 19878818). C, The NME2 protein levels in gastric cancer tissues and normal tissues. D, Immunohistology analysis of NME2 protein in gastric cancer tissues and normal tissues. The positive signals of the representative images are indicated with arrows (top) and calculated (bottom; , P < 0.01). Scale bar, 25 mm. E, The inﬂuence of NME2 on therapy-induced cell death. NME2 was silenced in gastric cancer cells (HGC-27 and MKN-45), which were simultaneously treated with cisplatin at 50 mmol/L. Forty-eight hours later, the cell viability was evaluated (, P < 0.01). F, Model for the NME2-mediated transcriptional promotion of antiapoptotic genes in gastric cancer cells. All the numeral data represent the mean SDs of triplicate assays (, P < 0.01).

As reported, NME2 is associated with telomere ends and telomerase polymerase II-associated protein; ref. 40), to mediate the phosphor- to reduce telomerase activity (38). NME2 can also interact with G- ylation of RNA polymerase II C-terminal domain at the 5th serine. It quadruplex DNA of c-MYC promoter to induce c-MYC expression (23) has been revealed that RPAP2 speciﬁcally binds to the small GTPase and suppress the metastasis of lung cancer via transcriptional regu- GPN1 (GPN-loop GTPase 1) and promotes the RNA pol II nuclear lation of the key cell adhesion factor vinculin (39). In this study, the import (41). Moreover, RPAP2 can directly interact with the RNA pol results showed that NME2 served as a transcription factor responsible II subunit Rpb6 and participates in pre-mRNA 30-end formation in for the transcriptions of miR-100 and protein-encoding genes by HEK-293T cells (28). Our investigations indicated that RPAP2 could interacting with both RNA pol II and RPAP2 (the major RNA interact with NME2 and functioned as an accessory factor during the

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NME2-mediated RNA pol II phosphorylation process in gastric cancer Administrative, technical, or material support (i.e., reporting or organizing data, cells. In this context, our findings presented novel insights into the constructing databases): X. Zhang underlying mechanism of NME2, a master suppressor for apoptosis of Study supervision: X. Zhang gastric cancer. Acknowledgments Disclosure of Potential Conflicts of Interest This study was financially supported by the National Key Research and No potential conflicts of interest were disclosed. Development Program of China (2018YFC0310703) and China Ocean Mineral Resources R & D Association (DY135-B-04). Authors’ Contributions Conception and design: X. Zhang The costs of publication of this article were defrayed in part by the payment of page Development of methodology: Y. Gong, X. Zhang charges. This article must therefore be hereby marked advertisement in accordance Acquisition of data (provided animals, acquired and managed patients, provided with 18 U.S.C. Section 1734 solely to indicate this fact. facilities, etc.): Y. Gong, G. Yang Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y. Gong, G. Yang, Q. Wang, Y. Wang Received June 9, 2019; revised September 8, 2019; accepted November 1, 2019; Writing, review, and/or revision of the manuscript: Y. Gong, X. Zhang published first November 6, 2019.

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NME2 Is a Master Suppressor of Apoptosis in Gastric Cancer Cells via Transcriptional Regulation of miR-100 and Other Survival Factors

Yi Gong, Geng Yang, Qizhi Wang, et al.

Mol Cancer Res Published OnlineFirst November 6, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-19-0612

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