Cancer Medicine Open Access ORIGINAL RESEARCH Cancer-­testis gene PIWIL1 promotes cell proliferation, migration, and invasion in lung adenocarcinoma Kaipeng Xie1,2,3,a, Kai Zhang1,2,3,a, Jing Kong1,2,3,a, Cheng Wang1,2,3, Yayun Gu1,2,3, Cheng Liang1,2,3, Tingting Jiang1,2,3, Na Qin1,2,3, Jibin Liu4, Xuejiang Guo1, Ran Huo1, Mingxi Liu1, Hongxia Ma1,2,3 , Juncheng Dai1,2,3 & Zhibin Hu1,2,3 1State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 211166, China 2Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing 211166, China 3Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center For Cancer Personalized Medicine, Nanjing Medical University, Nanjing 211166, China 4Tumor Biobank, Nantong Tumor Hospital, Nantong 226000, China Keywords Abstract Cancer-testis genes, DNA methylation, Epi-driver genes, Lung adenocarcinoma, Piwi-­like RNA-­mediated gene silencing 1 (PIWIL1) has been identified as a PIWIL1 novel extremely highly expressed cancer-­testis (CT) gene in lung adenocarci- noma. However, the exact function and mechanism of PIWIL1 in lung adeno- Correspondence carcinoma remains unclear. Herein, we sought to investigate the role of PIWIL1 Zhibin Hu, State Key Laboratory of in the occurrence and development of lung adenocarcinoma. We examined Reproductive Medicine, Nanjing Medical the expression pattern of PIWIL1 in The Cancer Genome Atlas (TCGA) lung University, Nanjing, China. Tel: 86-25-8686-8440; adenocarcinoma samples, and validated it by Real-­Time PCR (RT-­PCR) in Fax: 86-25-8686-8437; additional 21 paired lung adenocarcinoma tissues and 16 normal tissues. Sub- E-mail: [email protected] sequently, we explored the biological function of PIWIL1 in A549 and H1299 cell lines by gain and loss-­of-­function analyses. Using TCGA lung adenocar- Funding Information cinoma data, we further performed coexpression and Gene Ontology (GO) This research was supported by the National analyses, and analyzed the association of DNA methylation levels in PIWIL1 Key Basic Research Program Grant promoter region with its expression. Finally, we evaluated its expression in (2013CB911400), Science Fund for Creative Research Groups of the National Natural different mutation status of significantly mutated genes (SMGs) in TCGA lung Science Foundation of China (81521004), adenocarcinoma data. We observed that PIWIL1 was expressed in testis and National Natural Science of China (81230067, lung adenocarcinoma but not in other normal tissues, and its high expression 81225020), Jiangsu Specially-Appointed was associated with shortened survival of lung cancer patients. Overexpression Professor project, Natural Science Foundation of PIWIL1 could facilitate the proliferation, invasion and migration of lung of Jiangsu Province (BK20160046), the adenocarcinoma cells and vice versa. GO analysis revealed that PIWIL1 up- Priority Academic Program for the regulated genes were enriched in embryonic development, cell proliferation Development of Jiangsu Higher Education Institutions (Public Health and Preventive and regulation of transcription. Moreover, promoter DNA hypomethylation of Medicine) and Top-notch Academic Programs PIWIL1 could contribute to its aberrant expression in tumors. Interestingly, Project of Jiangsu Higher Education PIWIL1 expression was significantly higher in patients without hepatocyte growth Institutions (PPZY2015A067). factor (HGF) or serine/threonine kinase 11 (STK11) mutation (P = 0.006 and 0.005, respectively). PIWIL1 is an epidriver gene in lung adenocarcinoma, Received: 29 August 2017; Revised: 7 ­indicating a potential target for further therapy. October 2017; Accepted: 8 October 2017 doi: 10.1002/cam4.1248 aThese authors contributed equally to this work. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. 1 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. PIWIL1 drives lung cancer genesis and development K. Xie et al. Introduction Materials and Methods Cancer- testis (CT) genes are a group with limited expres- Patients and specimens sion in normal tissues except testis but frequently expressed in various types of cancers, the existence of This study was approved by the institutional review board which indicates the similarities between the processes of Nanjing Medical University and informed consent was of gametogenesis and tumorigenesis [1]. Owing to the obtained from all patients included in this study. A total special expression patterns and antigenic properties of of 21 lung adenocarcinoma patients with pairs of the CT genes, emerging studies have showed that these genes tumor and the adjacent normal tissues were recruited may be targeted as therapeutic cancer vaccines and bio- from the Nantong Cancer Hospital (Nantong City, Jiangsu markers for early clinical diagnosis and prognosis judg- Province, China), which were histologically or cytologically ment [2–5]. For example the peptides derived from CT confirmed by at least two local pathologists. Tissues were antigens have been used in the clinical trials for several frozen in liquid nitrogen after the surgery and stored at types of cancer, including head and neck cancer and −80°C. lung cancer [6, 7]. To date, more than 200 known CT genes have been Cell culture identified in the CT database (http://www.cta.lncc.br) [8]. Human lung cancer cell lines (A549 and H1299) were Using publically available databases, such as The Cancer obtained from the Shanghai Institute of Biochemistry and Genome Atlas (TCGA), The Encyclopedia of DNA Elements Cell Biology, Chinese Academy of Sciences (Shanghai, (ENCODE), and The Functional Annotation of The China). Cells were both cultured in RPMI- 1640 medium Mammalian Genome (FANTOM), we previously performed (Gibco, Carlsbad, MA) and supplemented with 100 U/ a comprehensive analysis to describe the expression char- mL penicillin, 100 μg/mL streptomycin and 10% fetal acteristics of CT genes and define the extremely highly bovine serum (Gibco). These cells were grown at 37°C expressed CT genes (EECTGs) that may be potential epi- with 5% CO in a humidified incubator. A549 and H1299 driver genes in 19 cancer types [9]. Epi- driver genes are 2 cell lines were certified by STR genotyping (HKgene, Beijing expressed aberrantly in tumors but not frequently mutated and MicroRead, Beijing, respectively). and regulated by DNA methylation or chromatin modi- fication according to the criteria demonstrated by Vogelstein [10]. In lung adenocarcinoma, we identified 327 potential RNA extraction and Real- time polymerase EECTGs with testis- specific proteins expression [9]. chain reaction (PCR) analysis However, limited studies have explored the function of Total RNA was extracted from the tissues and cells using these EECTGs in cancer. Trizol reagent (Invitrogen). Approximately 500 ng of RNA Piwi like RNA- mediated gene silencing 1 (PIWIL1), was used for the reverse transcription reaction with a lung cancer EECTG in our previous study, is the PrimeScript RT Master Mix (TaKaRa, Dalian, China). member of PIWI family and can bind to PIWIL- Human Multiple Tissue cDNA (MTC) Panels I and II interacting RNAs (piRNAs) during spermatogenesis [11]. (cat# 636742, 636743; Clontech Laboratories, Palo Alto, MIWI, a murine homolog of PIWIL1, encodes a cyto- CA) were used to validate the PIWIL1 expression in nor- plasmic protein specifically expressed in spermatocytes mal tissues. Using TaqMan Universal PCR Master Mix and spermatids, and controls translation in pachytene (Applied Biosystems, Inc.), the cDNA was amplified with spermatocytes and spermatids [12]. Accumulating evi- probes specific for PIWIL1 and ACTB (Cat. # 4331182, dence has demonstrated that PIWIL1 was frequently 4331182, Applied Biosystems, Inc., Marsiling, Singapore). expressed in various cancers including lung cancer, sug- gesting the potentially oncogenic roles of PIWIL1 in Protein isolation and western blot the formation or progression of cancer [13–16]. Existing studies mainly focused on the aberrant expression of Total cell lysates were prepared with RIPA mixed with PIWIL1 in tumors; however, the biological role of PIWIL1 1 mM Phenylmethanesulfonyl fluoride (PMSF). Protein in lung cancer has never been elucidated. Thus, in this samples (50 μg each) were separated on 10% Tris- study, we aimed to examine the expression pattern of polyacrylagel (Invitrogen) by electrophoresis and blotted PIWIL1, and further characterized the biological func- onto Polyvinylidene Fluoride (PVDF) membranes. Blots tion and potential regulatory mechanism of PIWIL1 in on the membranes were stained with primary antibodies lung cancer. (1:1000, abcam, ab12337 for PIWIL1 and 1:2000, Beyotime, 2 © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. K. Xie et al. PIWIL1 drives lung cancer genesis and development for Tubulin) overnight at 4°C and secondary antibody at a density of 2 × 104 cells per 200 μL culture solu- (Beyotime, Shanghai, China) for 1 h at room temperature. tion and cultured 24 h for migration and 48 h for Protein bands were visualized using the ECL Plus western invasion. Then cells were fixed with methanol and colo- blotting detection reagents (Millipore). nies were stained with 0.5% crystal violet for 30 min. The membranes were then dried, inverted, and mounted on microscope slides for analysis. Images