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 investi- gated. The results showed that miR-100 was remarkably upregu- lated 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 Ó2019 American Association for Cancer Research. cancer. AACRJournals.org | OF1 Downloaded from mcr.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst November 6, 2019; DOI: 10.1158/1541-7786.MCR-19-0612 Gong et al. 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 Â 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 Â 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 Â 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 Â 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.