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A Novel Micropeptide Encoded by Y-Linked LINC00278 Links

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A Novel Micropeptide Encoded by Y-Linked LINC00278 Links Author Manuscript Published OnlineFirst on March 13, 2020; DOI: 10.1158/0008-5472.CAN-19-3440 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 A Novel Micropeptide Encoded by Y-Linked LINC00278 Links 2 Cigarette Smoking and AR Signaling in Male Esophageal Squamous 3 Cell Carcinoma 4 Running title: Role of micropeptide encoded by lncRNA in male ESCC. 5 6 Siqi Wu 1*, Liyuan Zhang2*, Jieqiong Deng1, Binbin Guo1, Fang Li1, Yirong Wang1, Rui Wu1, 7 Shenghua Zhang1, Jiachun Lu3, Yifeng Zhou1† 8 *Siqi Wu and Liyuan Zhang contributed equally to this work. 9 10 Author affiliations 11 1Department of Genetics, Medical College of Soochow University, Suzhou 215123, China; 12 2Department of Radiotherapy & Oncology, The Second Affiliated Hospital of Soochow 13 University, San Xiang Road No. 1055, Suzhou 215004, China 14 3The Institute for Chemical Carcinogenesis, The First Affiliated Hospital, The School of Public 15 Health, Guangzhou Medical University, Guangzhou 510182, China 16 17 Correspondence to: †Dr. Yifeng Zhou, Medical College of Soochow University, Suzhou 215123, 18 China. Tel: 86-512-65884720; Fax: 86-512-65884720; E-mail: [email protected] 19 20 Competing interests None. 21 Keywords: lncRNAs, micropeptide, m6A, male ESCC, cigarette smoking 1 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 13, 2020; DOI: 10.1158/0008-5472.CAN-19-3440 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 22 ABSTRACT 23 Long non-coding RNAs (lncRNA) have been shown to play critical roles in many diseases, 24 including esophageal squamous cell carcinoma (ESCC). Recent studies have reported that some 25 lncRNA encode functional micropeptides. However, the association between ESCC and 26 micropeptides encoded by lncRNA remains largely unknown. In this study, we characterized a 27 Y-linked lncRNA, LINC00278, which was downregulated in male ESCC. LINC00278 encoded a 28 Yin Yang 1 (YY1)-binding micropeptide, designated YY1BM. YY1BM was involved in the 29 ESCC progression and inhibited the interaction between YY1 and androgen receptor (AR), 30 which in turn decreased expression of eEF2K through the AR signaling pathway. 31 Downregulation of YY1BM significantly upregulated eEF2K expression and inhibited apoptosis, 32 thus conferring ESCC cells more adaptive to nutrient deprivation. Cigarette smoking decreased 33 m6A modification of LINC00278 and YY1BM translation. In conclusion, these results provide a 34 novel mechanistic link between cigarette smoking and AR signaling in male ESCC progression. 35 36 SIGNIFICANCE 37 Post-transcriptional modification of a micropeptide-encoding lncRNA is negatively impacted by 38 cigarette smoking, disrupting negative regulation of the AR signaling pathway in male ESCC. 39 2 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 13, 2020; DOI: 10.1158/0008-5472.CAN-19-3440 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 40 INTRODUCTION 41 Esophageal squamous cell carcinoma (ESCC) is two to four times more common in men 42 than in women worldwide(1). Previous studies suggest that several male-specific factors 43 contribute to such gender disparity, including cigarette smoking and sexual hormone. A survey 44 in 2010 indicated that 52.9% of Chinese men while only 2.4% of Chinese women were current 45 smokers(2,3). Expression of androgen receptors has been reported in ESCC as well as 46 association with prognosis(4,5). However, the exact underlying molecular mechanisms in male 47 ESCC progression remain largely unknown. 48 A recent study identified a tumor suppressor gene on Y chromosome for male breast 49 cancer(6), suggesting that genetic material encoded by Y chromosome could be involved in 50 male-dominant tumors. Long non-coding RNAs (lncRNAs) are defined as RNA transcripts 51 longer than 200nt that lack protein-coding potential(7,8). LncRNAs act as master regulators for 52 gene expression, thus play an important role in many biological functions and diseases, including 53 cancer(9). However, no study so far has reported on the involvement of Y-linked lncRNAs in 54 ESCC. 55 Recent computational and genome-wide studies have demonstrated that hundreds of 56 functional micropeptides (less than 100 amino acids) are embedded in lncRNAs. For example, 57 myomixer is an 84-amino acid muscle-specific micropeptide encoded by a lncRNA that controls 58 the critical steps in myofiber formation during muscle development(10); myoregulin is identified 59 as a skeletal muscle-specific lncRNA, which regulates muscle performance by impeding Ca2+ 60 uptake into the SR(11). It is still unclear whether micropeptides play a key role in tumor 3 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 13, 2020; DOI: 10.1158/0008-5472.CAN-19-3440 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 61 development, although a recent study has identified a micropeptide encoded by HOXB-AS3 62 lncRNA that suppresses colon cancer growth(12). 63 N6-methyladenosine (m6A) is the most abundant post-transcription modification on 64 eukaryotic mRNAs and lncRNAs(13). Recent studies show that m6A modification is dynamic 65 and reversible in cells, whose level is regulated by m6A methyltransferases (also called “writers”: 66 METTL3, METTL14, etc.) and m6A demethylases (also called “erasers”: FTO, ALKBH5, etc.). 67 m6A regulates gene expression through m6A binding proteins (also called “readers”: YTHDF1, 68 YTHDF2, YTHDF3, etc.)(14,15). These m6A-associated proteins play critical roles to regulate 69 the metabolism and functions of m6A-modified mRNAs and lncRNAs(15). 70 In this work, we identified a micropeptide encoded by a Y-linked lncRNA, LINC00278, 71 which is downregulated in male ESCC. The expression of this micropeptide was downregulated 72 by cigarette smoking in ESCC through erasing m6A modification. It specifically bound to Yin 73 Yang 1 (YY1) and blocked the interaction between YY1 and AR, therefore named YY1- 74 blocking micropeptide (YY1BM). YY1BM downregulated eEF2K expression through AR 75 signaling pathway and induced apoptosis in ESCC under nutrient deprivation (ND). 76 Furthermore, YY1BM also acts as a potential anticancer micropeptide for ESCC. 77 78 MATERIALS AND METHODS 79 Human study subjects 80 A total of 281 pairs of fresh frozen ESCC and adjacent non-cancerous tissue samples were 81 obtained from patients in eastern China who underwent tylectomies at the Affiliate Hospitals of 4 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 13, 2020; DOI: 10.1158/0008-5472.CAN-19-3440 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 82 Soochow University (Suzhou cohort). Another 288 pairs of fresh frozen ESCC tissues were 83 collected from patients in southern China at the Cancer Hospitals affiliated with Guangzhou 84 Medical University (Guangzhou cohort). None of the patients received anti-cancer treatment 85 before surgery, including chemotherapy or radiotherapy. The Medical Ethics Committees of 86 Soochow University and Guangzhou Medical College approved this study. The clinical 87 characteristics of patients in this study are listed in Table. S1. 88 Statistical Analysis 89 The data analysis was performed using the SPSS 19.0 software for Windows. The statistical 90 significance between data sets was expressed as P values, and P<0.05 was considered 91 statistically significant. Survival curves were obtained using the Kaplan-Meier method and 92 compared using the log-rank test. Multivariable Cox regression analysis was performed using the 93 R package “survival”. Paired or unpaired Student’s t-test, Pearson correlation coefficients were 94 used for various types of data comparison. Mediation analysis was conducted using the 95 procedure described by Baron and Kenny (16) and a P<0.05 was considered significant. 96 Animals and Cell cultures 97 Male nude mice of 6-8 weeks of age were purchased from the Shanghai Laboratory Animal 98 Center at the Chinese Academy of Sciences (Shanghai, China). All animal studies were 99 conducted with the approval of Soochow University Institutional Animal Care and Use 100 Committee and were performed in accordance with established guidelines 101 All cell lines were purchased from Procell Life Science&Technology Co.,Ltd. These cell 102 lines were all characterized by DNA finger printing analysis and passaged less than 6 months in 5 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 13, 2020; DOI: 10.1158/0008-5472.CAN-19-3440 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 103 this study. DMEM, RPMI-1640 and fetal bovine serum (FBS) were purchased from Invitrogen. 104 Eca-109, TE-1, KYSE-30 cells were grown in RPMI-1640 with 10% FBS; Het-1A, 293T cells 105 were grown in DMEM with 10% FBS. All cell lines were grown in penicillin/streptomycin 106 containing
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