TMEFF2 is a novel prognosis signature and target for endometrial carcinoma

Journal Pre-proof TMEFF2 is a novel prognosis signature and target for endometrial carcinoma Lingling Gao, Xin Nie, Mingjun Zheng, Xiao Li, Qian Guo, Juanjuan Liu, Qing Liu, Yingying Hao, Bei Lin PII:

S0024-3205(19)30837-9

DOI:

https://doi.org/10.1016/j.lfs.2019.116910

Reference:

LFS 116910

To appear in:

Life Sciences

Received Date: 12 June 2019 Revised Date:

4 September 2019

Accepted Date: 23 September 2019

Please cite this article as: L. Gao, X. Nie, M. Zheng, X. Li, Q. Guo, J. Liu, Q. Liu, Y. Hao, B. Lin, TMEFF2 is a novel prognosis signature and target for endometrial carcinoma, Life Sciences (2019), doi: https://doi.org/10.1016/j.lfs.2019.116910. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

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TMEFF2 is a novel prognosis signature and target for

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endometrial carcinoma

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Lingling Gao1,2, Xin Nie1,2, Mingjun Zheng1,2, Xiao Li1,2, Qian Guo1,2, Juanjuan Liu1,2,

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Qing Liu1,2, Yingying Hao1,2, Bei Lin1,2*

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Heping District, Shenyang, Liaoning, China;

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Gynecology of Higher Education of Liaoning Province, Liaoning, China

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*

Department of Obstetrics and Gynaecology, Shengjing Hospital Affiliated to China Medical University,

Key Laboratory of Maternal-Fetal Medicine of Liaoning Province, Key Laboratory of Obstetrics and

Corresponding author at: Department of Obstetrics and Gynecology, Shengjing Hospital Affiliated to

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China Medical University, 36 Sanhao Street, Heping District, Shenyang, Liaoning, 110004, China.

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E-mail address: [email protected] (B Lin).

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Abstract

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Aims: Tomoregulin-2 (TMEFF2) is a single-pass transmembrane protein whose specific functions and

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mechanisms in endometrial carcinoma (EC) remain unclear. The aim of this study was to investigate the

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expression, prognostic role, and potential regulatory mechanisms of TMEFF2 in EC. Materials and

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Methods: The expression and prognosis of TMEFF2 in EC were analyzed via bioinformatics and verified

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by immunohistochemistry and survival analysis. Proliferation, invasion, and migration of EC cells in vitro

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were assessed by cell functional assays, while epithelial–mesenchymal transition (EMT) markers and key

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signaling pathway proteins were evaluated by western blotting. Key findings: The expression of

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TMEFF2 in EC was significantly higher than that in atypical hyperplasia and normal endometrium, the

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high expression of TMEFF2 was correlated with advanced stage, poor differentiation, and lymph node

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metastasis, and also predicted a poor prognosis of EC. Gene ontology (GO) and Kyoto Encyclopedia of

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Genes and Genomes (KEGG) analysis showed that TMEFF2 and its related genes were enriched in the

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central nervous system, cell adhesion, signal transduction, and several critical signaling pathways. We also

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elucidated TMEFF2 networks of kinase, microRNA, and transcription factor targets. In vitro, the

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proliferation, invasion, and migration abilities of EC cells decreased after TMEFF2 downregulation.

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Downregulation of TMEFF2 reduced the activation of MAPK and PI3K signaling pathways, and inhibited 1

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EMT. Significance: TMEFF2 plays an important role in the initiation, development, and malignant

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behavior of EC and can be a potential target for early diagnosis and treatment in EC.

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Keywords: endometrial carcinoma, TMEFF2, bioinformatics, prognosis, proliferation and invasion

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1. Introduction

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Endometrial carcinoma (EC) is one of the most common malignant cancers of the female

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reproductive system, accounting for 20–30 % of gynecological cancers and 7 % of all cancers in women

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[1]. EC is classified as type I (estrogen-dependent) or type II (non-estrogen-dependent), depending on the

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pathogenesis and biological behavior of the tumor. Currently, most patients with early EC prefer surgical

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treatment with 5-year survival rate as high as 95%. However, patients with advanced EC are prone to

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cancer recurrence after surgery, with vaginal, pelvic, and distant metastases, resulting in a 5-year survival

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rate reduction to 16–45 % [2-3]. Therefore, finding molecular markers of endometrial cancer for an early

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diagnosis and targeted therapy is crucial to increase survival rate.

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The TMEFF2 (transmembrane protein with epidermal growth factor (EGF)-like and two

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follistatin-like domains) gene, also known as tomoregulin-2 (TR-2), transmembrane protein containing

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EGF and follistatin domains (TPEF), hyperplastic polyposis protein 1 (HPP1), or transmembrane protein

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TENB2, is located on chromosome 2q32.3 and contains 11 exons. TMEFF2, which is widely expressed in

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brain tissue and regulate central nervous system (CNS) development [4], was first isolated from human

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gastric fibroblasts in 1999 by Uchida et al. [5]. TMEFF2 encodes a single-pass transmembrane protein

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consisting of 374 amino acids, and includes three isoforms, each of which has an extracellular domain, a

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transmembrane domain, and an intracellular domain. The extracellular domain includes two follistatin-like

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functional domains, which can bind to transforming growth factor beta (TGFβ) family, vascular endothelial

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growth factor (VEGF), as well as inhibit activation of their receptors, and an EGF-like functional domain,

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which may regulate EGF/neuregulin family and bind Ca2+. The intracellular domain contains a potential

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G-protein activation motif, involved in intracellular signal transduction [5,6,7]. TMEFF2 gene knockout

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mice showed growth retardation and a severe loss of white adipose tissue, suggesting that TMEFF2 may be

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involved in the growth and formation of white adipose tissue [8]. In adults, TMEFF2 expression is

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tissue-specific, with selectively high expression in brain and prostate tissues [4,9,10]. This gene is also

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expressed in tissues such as the normal colonic mucosa, myofibroblasts and primordial oocytes [11,12], 2

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and can maintain normal function of CNS [13,14]. In recent years, Studies have shown that TMEFF2 is

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downregulated in colorectal, gastric, and gallbladder cancers [15,16,17], playing a role in tumor

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suppression. However, TMEFF2 expression is significantly increased in androgen-dependent prostate

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cancer, its extracellular domain activates extracellular signal-regulated kinases (ERK1/2) and promotes

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proliferation of prostate cancer [9,18], indicating that TMEFF2 can promote cancer progression.

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There have been few studies on TMEFF2 in gynecological cancers, only limited to TMEFF2 DNA

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methylation in cervical intraepithelial neoplasia grade 3, cervical cancer and endometrial cancer [19-20].

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However, the expression and specific mechanism of TMEFF2 in endometrial cancer have not been

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reported. In this study, the expression of TMEFF2 in EC and functional regulatory networks were

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objectively analyzed using several bioinformatics databases. We also investigated the effects of TMEFF2

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on the biological behaviors and related signaling pathways and mechanisms in vitro.

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2. Materials and methods

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2.1. Oncomine database analysis

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The Oncomine database (http://www.oncomine.org) [21] was used in this study to analyze differential

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gene expression in cancer tissues and their corresponding normal tissues. The screening conditions were

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set as follows: cancer type: other; TCGA Endometrium; gene: TMEFF2; analysis type: cancer vs. normal;

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critical value settings: P-value <0.05, fold change >2, gene rank = top 10%.

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2.2. UALCAN database analysis

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UALCAN (http://ualcan.path.uab.edu) [22] is a reliable cancer database for online analysis and

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mining. The relative expression of query genes in tumor and normal samples can also be analyzed, as well

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as the relative expression based on the cancer stage, tumor grade, or other clinicopathological features in

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different tumor subgroups. The screening conditions were set as follows: Gene symbol: TMEFF2; TCGA

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dataset: Uterus corpus endometrial carcinoma; Expression based on: ①Sample types, ②Historical

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subtypes; Methylation profile based on: ①Sample types, ②Individual cancer stages, ③Patient’s ethnicity,

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④Patient’s age, ⑤Patient’s weight, ⑥Tumor grade.

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2.3. Survival analysis

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The Kaplan–Meier (KM) Plotter (http://kmplot.com) [23] is an online tool for analyzing prognosis of 3

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patients with tumors. The effects of 54,675 genes on survival were evaluated. Based on the expression of

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the TMEFF2 gene, patients with EC were divided into two groups: a high expression group and a low

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expression group. The hazard ratio (HR) at a 95% confidence interval and log-rank P-values were

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displayed online. The online survival analysis was carried out using the dataset of endometrial cancer in

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the KM Plotter database. The filter conditions were as follows: cancer: pan-cancer RNA-seq (Uterus

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corpus endometrial carcinoma); gene symbol: TMEFF2; survival: overall survival (OS)/progression-free

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survival (PFS); follow-up threshold: 120 months. At the same time, the relationship between TMEFF2 and

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the prognosis of EC was further analyzed using UALCAN.

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2.4. LinkedOmics analysis

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The LinkedOmics database (http://www.linkedomics.org/login.php) [24] is a multi-group database

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and clinical database based on a web platform. The LinkFinder module of LinkedOmics was used to study

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differentially expressed genes associated with TMEFF2 in the TCGA UCEC dataset (n = 176), and the

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results were statistically analyzed using the Pearson correlation coefficient. LinkFinder statistically

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analyzed each differentially expressed gene associated with TMEFF2, and the results were presented in the

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form of a volcano plot, heat map, or a scatter plot. First, the Link-Interpreter module of LinkedOmics

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standardizes and sorts all differentially expressed genes in LinkFinder and then enriches and analyzes the

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signaling pathway and network regulation of the above genes. This process uses gene set enrichment

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analysis (GSEA) for kinase–target network enrichment, microRNA (miRNA)–target network enrichment,

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and transcription factor–target network enrichment. The latter two network analyses are based on the

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Molecular Signatures Database. The grade standard is the false discovery rate (FDR) <0.05, and 500

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simulations are carried out. The filter conditions were as follows: cancer cohort: TCGA_UCEC; search

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dataset: TCGA_UCEC (RNAseq); search dataset attribute: TMEFF2; search target dataset: TCGA_UCEC

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(RNAseq); search statistical method: Pearson correlation test.

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2.5. GeneMANIA analysis

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GeneMANIA (http://www.genemania.org) [25] is a flexible, user-friendly web interface that can

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predict gene functions and analyze gene lists. GeneMANIA is also used to construct protein–protein

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interaction (PPI) networks, and identify protein–DNA interactions, signaling pathways, physiological and

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biochemical reactions, gene and protein expression, and protein domains; it allows phenotypic screening, 4

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and the data are updated regularly. We used GeneMANIA to visualize the functions and regulatory

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networks of molecules of TMEFF2 with significant differences after LinkedOmics analysis.

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2.6. Metascape analysis

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Metascape (http://metascape.org) [26] is a free, user-friendly gene list analysis tool for gene

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annotation and analysis, which can not only complete pathway enrichment and biological process

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annotation but also analyze gene-related protein networks. In this study, Metascape was used as a tool to

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analyze the enrichment of TMEFF2 and its related differentially expressed genes by process and pathway.

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The conditions included: P < 0.01, a minimum count of 3, and the enrichment factor >1.5 to obtain

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significant statistical differences. The PPI enrichment analysis in Metascape uses the following databases:

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BioGrid, InWeb_IM, and OmniPath. In addition, the Molecular Complex Detection (MCODE) algorithm

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is used to mine molecules with deeper network regulation relationships.

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2.7. Paraffin-embedded tissue samples

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A total of 135 paraffin-embedded tissue samples were collected from the Department of Obstetrics

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and Gynecology, Shengjing Hospital affiliated to China Medical University, from 2007 to 2013. All tissue

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sections were pathologically diagnosed by pathologists at the Shengjing Hospital affiliated to China

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Medical University. There were 75 cases of EC, 24 cases of atypical hyperplasia (mild hyperplasia, n = 8;

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moderate hyperplasia, n = 9; and severe hyperplasia, n = 7), and 36 cases of normal endometrium

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(secretory endometrium, n = 15; and proliferative endometrium, n = 21). The normal endometrial samples

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were collected from patients with no fertility requirements, who underwent a total hysterectomy of the

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uterus or the uterus and bilateral adnexa due to cervical lesions. The atypical hyperplasia and normal

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endometrium groups did not have any uterine myomas, ovarian chocolate cysts, or other

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estrogen-dependent diseases. The patients in the EC group were 36–79 years of age (average: 58.79 years);

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those in the atypical hyperplasia group were 30–66 years of age (average: 45.08 years); and the subjects in

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the normal endometrium group were 23–58 years of age (average: 43.76 years), with no statistically

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significant differences among the groups (P > 0.05). The pathological types of EC were as follows: 37

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cases of endometrioid carcinoma, 23 cases of serous carcinoma, and eight cases of clear-cell carcinoma.

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There were seven cases of other pathological types (three cases of mucinous carcinoma, two cases of

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squamous cell carcinoma, two cases of undifferentiated carcinoma, and one case of small-cell carcinoma). 5

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According to the pathological grade, there were 19, 24, and 32 cases of well, moderately, and poorly

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differentiated cancer, respectively. According to the 2009 International Federation of Gynecology and

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Obstetrics (FIGO) staging system, there were 56 cases of stages I–II and 19 cases of stages III–IV. Lymph

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node metastasis was judged as follows: no metastasis (48 cases), metastasis (14 cases), and no

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lymphadenectomy (13 cases). There were 48 cases of muscular invasion <1/2 and 27 cases of muscular

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invasion ≥1/2. All cases were primary EC patients, with complete clinical and pathological data and no

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preoperative radiotherapy, chemotherapy, and hormone therapy. The study was approved by the Research

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Ethic Board of China Medical University.

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2.8. Immunohistochemistry

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Consecutive sections (5 µm thick) were cut from the endometrial tissue specimens. The expression of

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TMEFF2 was detected using an immunohistochemical streptavidin–peroxidase method. Human prostatic

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hyperplasia tissues served as positive control for TMEFF2, and phosphate-buffered saline (PBS) served as

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a negative control for the TMEFF2 rabbit polyclonal primary antibody (Abcam, USA, Cat #ab133562).

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The working dilution of the primary antibody was 1:75, and the staining procedure was carried out

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according to the manufacturer's instructions. The presence of stained buffy granules in the cell membrane

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and cytoplasm was considered positive TMEFF2 expression. Based on the staining intensity, not

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pigmentation, light yellow, brown yellow, and dark brown colors were scored 0, 1, 2, and 3, respectively.

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The percentage of stained cells observed in the whole section, <5%, 5–25%, 26–50%, 51–75%, and >75%,

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was scored as 0, 1, 2, 3, and 4, respectively. The above two scores were multiplied to obtain the final score

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as follows: 0 to 2, (−); 3 to 4, (+); 5 to 8, (++); and 9 to 12, (+++). To control errors, the stained images

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were independently observed by two senior pathologists who had no knowledge of the patient's data.

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2.9. Cell culture and cell transfection

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EC cell lines (HEC-1A, HEC-1B, and Ishikawa) were purchased from the Shanghai Cell Culture

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Collection and were cultured in McCoy’s 5A medium with 10 % fetal bovine serum (FBS) at 37 °C in a

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humidified atmosphere with 5 % CO2. Before transfection, Ishikawa cells at the exponential growth phase

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were digested with 0.25 % trypsin. When the cells grew to 60-70 % confluency, they were transfected with

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a TMEFF2 small interfering RNA (siRNA) using a Lipofectamine 3000 transfection kit (Thermo Fisher

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Scientific, USA). After 48 h of transfection, the interference effect was detected, and the cells were 6

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collected and used for further analysis. The TMEFF2 siRNA (Genema, Shanghai, China) sequences were

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as follows: 5′-CUCUGCAUCACAAGGAAAUTT-3′ and 5′-AUUUCCUUGUGAUGCAGAGTT-3′.

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2.10.

Western blotting

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Cells were lysed at 4 °C for 30 min with ice-cold RIPA lysis buffer supplemented with

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phenylmethylsulfonyl fluoride. The protein lysates were centrifuged at 4 °C, 12,000 rpm for 30 min, and

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the total protein concentration in the supernatant was determined using a bicinchoninic acid assay (BCA.

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Thermo Fisher Scientific, USA). Proteins were separated by 10 % sodium dodecyl sulfate polyacrylamide

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gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride membrane (PVDF)

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(EMD Millipore, Billerica, MA, USA). After being blocked with 5 % skim milk or bovine serum albumin

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for 2 h, membranes were incubated overnight at 4 °C with the following primary antibodies: anti-TMEFF2

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(1:1,000; Abcam, Cat# Cat #ab133562); anti-E-cadherin (1:2,000; Proteintech, Wuhan, China, Cat#

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20874-1-AP); anti-matrix metalloproteinase (MMP) 2 (1:1,000; Proteintech, Cat# 10373-2-AP);

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anti-MMP9 (1:1,000; Proteintech, Cat# 10375-2-AP); anti-vimentin (1:4,000; Proteintech, Cat#

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10366-1-AP); anti-mitogen-activated protein kinase (MAPK) kinase (MEK1/2; 1:1,000; Cell Signaling

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Technology (CST), Danvers, MA, USA, Cat# 4694); anti-phospho-MEK1/2 (1:1,000; 9154S);

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anti-ERK1/2 (1:1,000; CST, Cat# 9102S); anti-phospho-ERK1/2 (1:1,000; CST, Cat# 9101S);

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anti-phosphoinositide 3-kinase (PI3K) p85 (1:1,000; CST, Cat# 4292S), anti-phospho-PI3K (1:500; CST,

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Cat# 4228S), anti-protein kinase B (also known as AKT; 1:1,000; CST, Cat# 4691S), anti-phospho-AKT

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(1:1,000; CST, Cat# 4060S); and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2,000;

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ZSGB-BIO, Beijing, China, Cat# TA-08). The membranes were washed three times with Tris-buffered

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saline with Tween 20 and then incubated with goat anti-rabbit/mouse secondary antibody (1:5,000;

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ZSGB-BIO, Cat# ZB-2301/ZB-2305) for 2 h at room temperature. Protein bands were detected using the

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Immobilon® western chemiluminescent horseradish peroxidase substrate (Millipore, Billerica, MA, USA).

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The experiments were repeated three times.

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2.11.

Cell proliferation assay

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Cells in the logarithmic growth phase were seeded in 96-well plates at a density of 3,000 cells/well.

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After 6 h of incubation at 37 °C, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

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solution (20 µL, 5 µg/mL; Solarbio, Beijing, China) was added to each well, and the plate was incubated at 7

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37 °C for 4 h. The culture medium was removed, and 150 µL of dimethyl sulfoxide was added. After 10

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min of shaking, absorbance was measured at 0, 24, 48, 72, and 96 h time points. For each treatment, five

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parallel wells were used, and the experiment was repeated three times.

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2.12.

Wound healing assay

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Cells in the logarithmic growth phase were seeded in a six-well plate. When cell confluence reached

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90 %, a wound was gently scratched using a 100-µL micropipette tip. The cells were washed twice with

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PBS and incubated with a serum-free medium for 24 h. The distance of migration was measured under a

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microscope. The experiment was repeated three times.

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2.13.

Transwell assay

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Cell invasion was assessed using a Transwell assay. The upper Transwell chamber (Corning, Inc.,

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Corning, NY, USA) was covered with a Matrigel solution (70 µL; BD) and dried overnight at 37 °C. Cells

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(2 × 105 per 200 µL) were seeded in the upper chamber with a serum-free medium, and 500 µL of medium

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containing 10 % serum was added to the lower chamber. After incubation for 48 h at 37 °C, the Transwell

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chambers were washed three times with PBS, and cells were fixed with 4 % paraformaldehyde for 30 min

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at room temperature. After being stained with crystal violet for 30 min, the Matrigel and cells from the

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upper surface of the chamber were gently removed with a cotton swab. The number of tumor cells that

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infiltrated the Transwell chamber membrane was counted under a microscope. The experiment was

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repeated three times.

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2.14.

Statistical analysis

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All data were statistically analyzed using the SPSS 21.0 software (IBM Corporation, Armonk, NY,

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USA) and expressed as the mean ± standard deviation. Student’s t-test and a chi-squared test were used to

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evaluate differences between two groups, whereas more than two groups were compared using one-way

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analysis of variance. A survival curve was generated using the Kaplan–Meier method and a log-rank test.

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The Cox model was employed to analyze the patient prognosis. Differences were considered statistically

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significant at bilateral P values < 0.05.

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3. Results 8

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3.1. TMEFF2 expression analyzed using the Oncomine database and UALCAN

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The Oncomine database includes data from studies of TMEFF2 in 272 different types of tumors.

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There were 26 studies that showed statistically significant differences in TMEFF2 mRNA levels between

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tumor and normal tissues, of which 8 studies showed significantly increased and 18 studies showed

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significantly decreased levels of TMEFF2 mRNA expression. The TMEFF2 mRNA expression was

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significantly higher in all kidney cancers, lymphomas, myelomas, and prostate cancers and lower in all

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brain and CNS cancers, breast cancers, and head and neck cancers than in normal tissues (Fig. 1A). To

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investigate the expression of TMEFF2 in different endometrial cancers, TCGA Endometrium (Reporter ID:

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02-192645440) was selected for analysis, which included a total of 354 endometrial cancer samples, 25

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normal endometrial samples, and 338 blood samples. The results showed that the DNA copy numbers of

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TMEFF2 in endometrial serous adenocarcinoma and mixed endometrial adenocarcinoma were

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significantly higher than those in the control group (both P < 0.05). Although the fold change was < 2, the

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gene rank was still within the top 10 % based on the DNA copy numbers. The TMEFF2 DNA copy number

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was also higher in the endometrial endometrioid adenocarcinoma than in the control group (P < 0.001), but

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the gene rank was not in the top 10 % (Fig. 1B–E).

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A subgroup analysis of 546 cases of UCEC in the TCGA database was performed using UALCAN.

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The results showed that the expression levels of TMEFF2 mRNA in primary endometrial cancer tissues

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were lower than those in the healthy control group (P = 0.03; Fig. 2A). However, when different

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histological subtypes were considered, the expression levels of TMEFF2 mRNA in endometrial serous

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adenocarcinoma and mixed endometrial adenocarcinoma were significantly higher than those in healthy

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controls (both P < 0.05). The TMEFF2 mRNA expression was lower in endometrial endometrioid

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adenocarcinoma than in the healthy control group, but the difference was not statistically significant (Fig.

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2B). We further analyzed the levels of TMEFF2 gene methylation and found that irrespective of the sample

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type, age, race, weight, cancer stage, and tumor grade, TMEFF2 exhibited low levels of methylation in

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UCEC, however, which were higher than those in the healthy control group (Fig. 2C–H).

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3.2. Relationship between TMEFF2 mRNA expression and EC prognosis

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The KM Plotter online analysis tool was used to set truncated data for overall survival (OS), and a

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total of 535 endometrial cancer cases in the database met these conditions. The KM Plotter showed that OS 9

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was significantly lower in endometrial cancer patients with high TMEFF2 expression than in patients with

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low TMEFF2 expression (HR = 2.55 (1.69–3.85), log-rank P = 3.8e−06) (Fig. 2I). When truncated data

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were set to progression-free survival (PFS), a total of 417 endometrial cancer cases in the database met

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these conditions. The KM Plotter showed that PFS was significantly lower in endometrial cancer patients

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with high TMEFF2 expression than in those with low TMEFF2 expression (HR = 1.89 (1.12–3.18),

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log-rank P = 0.015) (Fig. 2J). Compared with endometrial cancer patients with low/medium TMEFF2

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expression (407 cases), those with a high TMEFF2 expression (136 cases) also had a significantly shorter

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survival time based on UALCAN (P = 0.00024; Fig. 2K). The results of these online survival analyses

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suggest that endometrial cancer patients with high TMEFF2 expression have a poor prognosis.

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3.3. TMEFF2 protein expression in different endometrial tissues

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TMEFF2 was primarily stained in the cell membrane and cytoplasm; some staining was also seen in

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the nuclei. The positive expression rates of TMEFF2 in the endometrial cancer group and atypical

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hyperplasia endometrial group were 92 % (69/75) and 70.83 % (14/24), respectively, which were both

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significantly higher than the rates of positive expression in the normal endometrium group (50 %, [18/36];

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both P < 0.05). The positive expression rate in the endometrial cancer group was also higher than that in

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the atypical hyperplasia endometrial group (P < 0.05). Further, the high positive expression rate of

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TMEFF2 in the endometrial cancer group was 73.33 % (55/75), which was significantly higher those that

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in atypical hyperplasia endometrial group (41.67 %, 10/24) and the normal endometrium group (33.33 %,

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12/36) (both P < 0.05). The positive expression rates of TMEFF2 in the moderate and severe atypical

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hyperplasia endometrial groups were 77.78 % (7/9) and 71.43 % (5/7), respectively, both of which were

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higher than that in the mild hyperplasia group (62.50 %, 5/8), but the difference was not statistically

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significant (both P > 0.05). The positive expression rate in the secretory endometrium group was 47.62 %

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(11/21), which was higher than that in the proliferative endometrium group (40 %, 6/15), but the difference

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was not statistically significant (P > 0.05; Fig. 3A–E and Table 1).

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3.4. Correlation between TMEFF2 expression and the clinical pathological parameters

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of endometrial cancer

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75 cases of endometrial cancer were divided into low (−/+) and high (++/+++) TMEFF2 expression

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groups based on the TMEFF2 expression in endometrial cancer tissues. The results showed that the high 10

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positive expression rate of TMEFF2 in stages III-IV endometrial carcinoma patients was 95% (18/19),

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which was higher than that in stages I-II (66%, 37/56) (P < 0.05). The high expression rate of TMEFF2

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gradually increased as the degree of differentiation decreased; specifically, the high expression rate in the

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poor differentiation group was 87.50% (29/32), which was higher than those in the well differentiation

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(47.37%, 9/19) and well-moderate differentiation (62.79%, 27/43) groups (both P < 0.05). The high

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expression rate of TMEFF2 was 75% (18/24) in the moderate differentiation group, which was also higher

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than that in the well differentiation group, but the difference was not statistically significant (P > 0.05). In

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the lymph node metastasis group, the high expression rate of TMEFF2 was 100% (14/14), which was

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higher than that in the non-metastasis group (68.75%, 33/48) (P < 0.05). No statistically significant

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differences in the TMEFF2 expression with respect to the age at diagnosis, pathological type, progesterone

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receptor (PR), estrogen receptor (ER), or the depth of myometrial invasion (all P > 0.05; Table 2) was

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observed.

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3.5. TMEFF2 expression predicts poor prognosis in endometrial carcinoma

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The 75 cases of endometrial cancer patients were followed until January 30, 2018. The longest and

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shortest survival time were 60 and 3 months, respectively. Among the 75 patients with endometrial

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carcinoma, 20 died and 4 were lost to follow-up. Univariate Kaplan–Meier analysis showed that the

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average survival time was 49.4 months in the TMEFF2 high positive expression group and 59.4 months in

295

the TMEFF2 low positive expression group. High positive expression of TMEFF2 were significantly

296

associated with a shortened overall survival (OS) (P = 0.011). These results further confirmed those of the

297

online survival analysis. In addition, FIGO stage (I–II vs. III–IV), depth of myometrial invasion (<1/2 vs.

298

≥1/2), age at presentation (<59 years vs. ≥59 years), lymph node metastasis (negative vs. positive), ER

299

status (negative vs. positive), and PR status (negative vs. positive) were all correlated with a poor prognosis

300

(all P < 0.05) (Fig. 3F-L and Table 3). The Cox regression model was used to analyze risk factors for the

301

prognosis of endometrial cancer patients. The age, FIGO stage, lymph node metastasis, depth of

302

myometrial invasion, ER status, PR status, and TMEFF2 expression affected the prognosis of endometrial

303

cancer patients (all P < 0.05). The multivariate Cox regression analysis showed that FIGO stage, lymph

304

node metastasis, and the depth of myometrial invasion were independent prognostic risk factors in

305

endometrial cancer patients (Table 4). 11

306

3.6. Functional enrichment of TMEFF2 in patients with EC

307

The mRNA sequencing data of 176 UCEC patients, available in the TCGA database, were analyzed

308

using the function module in LinkedOmics. As shown in the volcano plot, there were 1,769 genes with a

309

significant positive correlation with TMEFF2 (dark red dots) and 538 genes with a significant negative

310

correlation with TMEFF2 (dark green dots) (FDR < 0.01). The heat-maps show the top 50 gene sets were

311

significantly positively and negatively correlated with TMEFF2 (Fig. 4A–C). These results indicate that

312

TMEFF2 has a wide range of functions in regulating metabolism, protein activity, and intercellular

313

communications. Statistical scatter plots of individual genes showed a significant positive correlation

314

between TMEFF2 and LHX8 (positive rank #1, Pearson correlation = 0.68, P = 8.189e−16), PAX3

315

(Pearson correlation = 0.53, P = 2.354e−14), and ASXL3 (Pearson correlation = 0.51, P = 4.405e−13)

316

expression (Fig. 4D−F), reflecting important roles of these genes in embryonic development, maintenance

317

of normal CNS function, and regulation of protein activity.

318

Metascape was used for GO and KEGG enrichment analyses of the functions of TMEFF2 and its

319

associated differentially expressed genes. The GO results showed that TMEFF2 and its associated

320

differentially expressed genes were primarily located in axons and dendrites and in both pre- and

321

postsynaptic sites and were also structural components of the extracellular matrix, involved in the

322

formation of ion channel complexes, etc. (Fig. 5A-B and Supplementary Table S1). The biological

323

processes and molecular functions of TMEFF2 and its associated genes included CNS development and

324

regulation, intercellular adhesion, cellular signal transduction, synthesis of proteoglycan complexes,

325

transmembrane signal transduction, ion channel regulation, and binding to chromatin and RNA polymerase

326

II regulatory region sequence-specific DNA, as well as to tubulin, actin, the ephrin receptor, and amyloid-β

327

(Fig. 5C-F and Supplementary Table S2-S3). The KEGG enrichment analysis showed that the signaling

328

pathways in which TMEFF2 and its associated differentially expressed genes participate included axon

329

guidance, the Hippo signaling pathway, the cyclic GMP (cGMP)/protein kinase G (PKG) signaling

330

pathway, MAPK signaling pathways, and Hedgehog signaling pathway (Fig. 5G-H and Supplementary

331

Table S4). In addition, we used Metascape for PPI enrichment analysis to better understand the

332

relationship between TMEFF2 and UCEC. A list of important genetic components in the PPI network and

333

MCODE is shown in Fig. 5I-J. The four most important MCODE components were extracted from the PPI

334

network, and pathway and enrichment process analyses were independently applied to each MCODE 12

335

component. The results showed that the biological functions of the MCODE components primarily

336

included axon guidance, cholinergic synapse, endocytosis, neuroactive ligand–receptor interactions, the

337

cGMP/PKG signaling pathway, and pathways in cancer.

338

3.7. TMEFF2 networks of kinase, miRNA, and transcription factor targets in UCEC

339

To further investigate the functional targets of TMEFF2 in endometrial cancer, the kinase, miRNA,

340

and transcription factor target networks that significantly and positively correlated with TMEFF2 were

341

generated using GSEA. The top five most important genes associated with the kinase target networks

342

included polo-like kinase 2 (PLK2), protein kinase, X-linked (PRKX), protein kinase cAMP-activated

343

catalytic subunits gamma (PRKACG) and beta (PRKACB), and cyclin-dependent kinase 5 (CDK5). The

344

molecules associated with the miRNA target networks included CAGTAT_miR-200b/miR-200c/miR-429,

345

ACATATC_miR-190, ATATGCA_miR-448, CAGTGTT_miR-141, miR-200a, and TAGCTTT_miR-9.

346

The transcription factor target networks primarily included FOXD3_0, NRSF_01, CDX2_Q5, PAX4_04,

347

and CDP_02 (Table 5 and Supplementary Tables S5–S7). Furthermore, the protein interaction network

348

constructed using GeneMANIA revealed mutual regulation of genes associated with PLK2,

349

miRNA-200b/200c/429, and FOXD3_0. The PLK2-related gene set is primarily involved in the

350

protein-regulating topology, functions of unfolded proteins, calcium ion binding, regulation of

351

neurotransmitter levels, and dopamine metabolism. The gene set of the transcription factor FOXD3_0 is

352

primarily involved in the regulation of protein binding and EMT and is closely associated with the brain,

353

vascular, and cell development processes. The gene set of miRNA-200b/200c/429 is primarily involved in

354

axonal fasciculation, protein kinase A activation, and establishment and maintenance of cell polarity

355

(Supplementary Figure S1).

356

3.8. Downregulated TMEFF2 expression inhibits proliferation, migration, and invasion

357

in endometrial cancer cells

358

To further investigate the effects of TMEFF2 expression on the proliferation, invasion, and migration

359

of endometrial carcinoma cells, we first determined the expression of TMEFF2 in 3 endometrial cancer

360

cell lines (Ishikawa, HEC-1A, and HEC-1B). The results showed that the expression of TMEFF2 in

361

Ishikawa cell line was higher than that in the other two cell lines (Fig. 6A). Further, the expression of

362

TMEFF2 in Ishikawa cells was inhibited by RNA interference (Fig. 6B). Then, changes in the proliferation, 13

363

migration, invasion of Ishikawa cell were measured by MTT, scratch assay, and Transwell assay,

364

respectively. The proliferation capacity decreased after the inhibition of the TMEFF2 expression in

365

Ishikawa cells (P < 0.05; Fig. 6C). The scratch and Transwell assays demonstrated that the migration and

366

invasion abilities of Ishikawa cells were significantly inhibited after TMEFF2 downregulation compared

367

with that in the control group (both P < 0.05; Fig. 6D-E).

368

3.9. Downregulation of TMEFF2 suppresses EMT process and inhibits MAPK and

369

PI3K-AKT signaling pathways

370

To explore the mechanism underlying the effect of TMEFF2 on malignant behaviors of endometrial

371

carcinoma cell, the expression of EMT-related molecules (E-Cadherin, Vimentin, MMP2, and MMP9) was

372

detected by western blotting and immunocytochemistry. Our study indicated that the expression of

373

E-Cadherin increased and that of Vimentin, MMP2, and MMP9 decreased after the inhibition of the

374

TMEFF2 expression (P < 0.05) (Fig. 7A-B). We further measured the key node molecules of MAPK and

375

PI3K/AKT signaling pathways in Ishikawa cells; significant downregulation of p-PI3K, p-AKT, p-MEK,

376

and p-ERK was observed after TMEFF2 was inhibited (P < 0.05), whereas the expression of PI3K, AKT,

377

MEK, and ERK was not significantly changed (Fig. 7C). These results demonstrate that downregulated

378

TMEFF2 expression could suppress the EMT process and inhibit MAPK and PI3K-AKT signaling

379

pathways in endometrial cancer cells.

380

Discussion

381

EC is a group of epithelial malignant cancers that occur in the endometrium, the development and

382

progression of which are multifactorial biological processes, involving multiple genes and comprising

383

many steps and stages. Type I endometrial cancer develops from endometrial hyperplasia and has a good

384

prognosis, owing to hormone levels and the endocrine environment. Type II endometrial cancer can result

385

from de novo cancerous transformation of a normal endometrium and results in a poor prognosis [27,28].

386

However, specific pathogenesis of endometrial cancer is still unclear, and there is a lack of specific

387

molecular markers for its early diagnosis. Therefore, it is especially important to investigate the molecular

388

mechanisms and therapeutic targets that affect the development and progression of endometrial cancer.

389

TMEFF2 is a single-pass transmembrane protein whose functions and mechanisms are still unclear. 14

390

Owing to its unique structural features, TMEFF2 can play important roles in many physiological and

391

pathological processes in the organism. The follistatin structural domains in the extracellular domain of

392

TMEFF2 bind and regulate a variety of growth factors, including the TGFβ family, PDGFs, and VEGF

393

[4,6,7]. In pancreatic cancer, TMEFF2 inhibits the cancer cell proliferation by inhibiting the expression of

394

phosphorylated STAT3, MCL1, and VEGF through binding to SHP-1. VEGF plays an essential role in the

395

processes of angiogenesis, proliferation, invasion, and metastasis of malignant cancers. In recent years,

396

targeted therapy with anti-angiogenic drugs targeting VEGF has been rapidly developed, and a variety of

397

drugs have been approved for clinical treatment of malignant cancer, including monoclonal VEGF

398

antibodies (bevacizumab and ramucirumab), soluble VEGF receptor drugs (aflibercept and conbercept),

399

and others [29]. The EGF-like structural domain of the extracellular domain of TMEFF2 promotes

400

phosphorylation of a tyrosine residue in ERB-4 [4], suggesting that the domain may function similarly to

401

other members of the EGF superfamily. Similarly, many anti-EGF/EGF receptor targeted drugs have been

402

approved for clinical treatment, such as gefitinib, afatinib as well as AZD9291 [30], indicating that

403

TMEFF2 has a potential clinical value for targeted therapy in malignant cancers. Thus, in the present study,

404

we investigated the expression of the TMEFF2 mRNA and protein in endometrial cancer and the specific

405

mechanisms involved.

406

The TMEFF2 DNA copy numbers in endometrial cancer tissues were significantly higher than those

407

in the control group in the TCGA endometrium dataset. We further found that the expression of TMEFF2

408

mRNA in endometrial serous adenocarcinoma (115 cases) and mixed endometrial adenocarcinoma (22

409

cases) was significantly higher than that in the control group. The levels of TMEFF2 mRNA in

410

endometrioid adenocarcinoma (409 cases) were lower than those in the healthy control group, but the

411

difference was not statistically significant. Studies have shown that endometrioid adenocarcinoma is

412

hormone-dependent, and TMEFF2 expression is regulated by androgens [18]. Based on the above

413

paradoxical results of a decreased TMEFF2 mRNA expression in endometrioid adenocarcinoma, it seems

414

reasonable to speculate that TMEFF2 expression may also be regulated by estrogens or other related

415

molecules in endometrial adenocarcinoma. We performed immunohistochemistry to further confirm that

416

TMEFF2 was highly expressed in endometrial cancer tissues, and its high expression was associated with

417

the FIGO stage, degree of differentiation, and lymph node metastasis. The results also showed that the

418

positive expression rate of TMEFF2 in the atypical endometrial hyperplasia group was significantly higher

419

than that in the normal endometrium group, which may be used to investigate the relationship between 15

420

TMEFF2 expression and the malignant progression of endometrial cancer, and to monitor the treatment

421

outcomes, prognosis, and malignant transformation in patients with atypical endometrial hyperplasia.

422

These studies indicate that TMEFF2 expression is associated with the development and progression of

423

endometrial cancer.

424

To further analyze the relationship between TMEFF2 mRNA expression and the prognosis of

425

endometrial cancer patients, we demonstrated that OS and PFS of patients with high TMEFF2 expression

426

were significantly shorter than those of patients with low expression. We confirmed that patients with a

427

high TMEFF2 expression, advanced FIGO stage, myometrial invasion, lymph node metastasis, and

428

positive for PR and ER had a significantly shorter OS and a poor prognosis in EC samples. These results

429

are strongly consistent with the TMEFF2 expression data in various bioinformatics databases and further

430

indicate that TMEFF2 can potentially be used as a molecular marker for clinical diagnosis and assessment

431

of prognosis for endometrial cancer patients and may also play an important role in the invasion,

432

metastasis, and other malignant biological behaviors in endometrial cancer.

433

DNA methylation is an important modification process in genomes of eukaryotic cells. It regulates the

434

gene expression by interacting with transcription factors, altering the chromatin structure, and regulating

435

the biological and genetic information at the epigenetic level and plays a very important role in the

436

development and progression of cancer. The methylation of the TMEFF2 gene promoter region has been

437

confirmed in a variety of tumors, including clear cell renal cell carcinoma, lung cancer, breast cancer, and

438

urothelial carcinoma [31-35], which results in a decreased TMEFF2 expression in these cancers and

439

suggests that it is closely associated with the epigenetic regulation of cancer. We further analyzed the levels

440

of TMEFF2 methylation in endometrial cancer and found that based on the sample type, age, race, weight,

441

cancer stage, and tumor grade, TMEFF2 methylation was low in UCEC but still higher than that in the

442

healthy control group. These data indicate that the methylation of the TMEFF2 gene promoter region is

443

closely associated with the development and progression of endometrial cancer; however, the relationship

444

between DNA methylation and mRNA and protein expression of TMEFF2 in endometrial cancer needs

445

further investigation.

446

In recent years, many studies have shown that TMEFF2 can affect the development, progression,

447

malignancy, and other behaviors of cancer cells by regulating various molecular mechanisms and

448

associated signaling pathways. The full length TMEFF2 and a recombinant form of TMEFF2 exert

449

different effects on the ERK and AKT signaling pathways in prostate cells and promote the proliferation of 16

450

prostate cancer cells in an ADAM17-dependent manner [19,36]. In HEK293 cells, the extracellular domain

451

of TMEFF2 can induce ERK1/2 phosphorylation through EGF receptor-dependent pathways, which

452

promotes the cell proliferation [36]. In addition, TMEFF2 could regulate cell cycle and was associated

453

with recurrence risk in prostate cancer [37]. TMEFF2 can also inhibit the proliferation by activating the

454

JAK/STAT signaling pathway in colon cancer [38]. The c-Myc protein can bind to the TMEFF2 promoter

455

region and inhibit transcription of the gene, making TMEFF2 a target for colon cancer therapeutic drugs

456

[39]. Decreased TMEFF2 expression is associated with advanced stage and a poor prognosis in patients

457

with gastric cancer. In addition, TMEFF2 inhibits the STAT3 activation and DNA binding by binding to

458

SHP-1, which affects the development and progression of gastric cancer [40]. In this study, we performed

459

GO and KEGG pathway enrichment analyses of TMEFF2 and its associated genes and found that these

460

genes were primarily involved in biological processes such as the regulation and development of CNS,

461

intercellular adhesion, cellular signal transduction, and the synthesis of proteoglycan complexes. As a

462

transmembrane protein, TMEFF2 performs its biological functions by binding to a variety of proteins,

463

including tubulin, actin, the ephrin receptor, and amyloid-β. In addition, TMEFF2 can participate in axon

464

guidance, the Hippo signaling pathway, neuroactive ligand–receptor interaction, cGMP/PKG signaling

465

pathway, MAPK signaling pathways, phospholipase, and other signaling pathways in cancer. We speculate

466

that TMEFF2 can affect the development and progression of endometrial cancer by regulating these

467

pathways.

468

EMT refers to a biological process in which epithelial-like cells are transformed into mesenchymal

469

cells through specific mechanisms, resulting in the migration, invasion, and widespread dissemination of

470

cancer cells. In this process, cancer cells lose tight junctions and cell–cell adhesion and acquire the ability

471

to invade and migrate. In addition, the secretion of extracellular MMPs increases, which creates conditions

472

for distant cell metastasis [41]. EMT plays an extremely important role in the invasion and metastasis of

473

malignant cancers; it was confirmed that TMEFF2 plays a role in the malignant biological behavior of

474

endometrial cancer cells. In addition, the EMT process was suppressed after downregulation of TMEFF2

475

expression in endometrial cancer cells. Therefore, we can speculate that TMEFF2 affects the malignant

476

biological behavior of endometrial cancer by regulating EMT process. Our study also showed that the

477

activation of the MAPK and PI3K/AKT signaling pathways were inhibited after downregulation of

478

TMEFF2 expression in endometrial cancer cells. Many studies have shown that the activation of the

479

MAPK signaling pathway affects the proliferation, apoptosis, differentiation, transformation, migration, 17

480

and invasion of cancer cells by regulating nuclear transcription factors, enzymes, and cytoskeletal proteins.

481

Abnormal activation of the PI3K/AKT signaling pathway can also promote the growth, invasion, and

482

migration of cancer cells; both pathways can participate in EMT of endometrial cancer [42]. Thus, we

483

hypothesized that TMEFF2 might affect the development and progression of endometrial cancer and the

484

EMT process by regulating the MAPK and PI3K/AKT signaling pathways. However, the specific

485

mechanism of this process requires further investigation.

486

4. Conclusions

487

In summary, we demonstrated, for the first time, using multiple databases such as Oncomine and

488

Metascape, as well as in vitro experiments, that TMEFF2 is highly expressed in EC and associated with a

489

shortened survival time and poor prognosis in endometrial carcinoma patients. We also found that

490

TMEFF2 could affect malignant biological behaviors such as proliferation, invasion, and migration, as

491

well as EMT in EC cells. Moreover, TMEFF2 can regulate the activities of MAPK and PI3K-AKT

492

signaling pathways in EC; suggesting that TMEFF2 plays an important role in the development,

493

progression, and malignant behavior and can potentially be used as a tumor marker in EC. Our findings are

494

of great significance for investigation of the pathogenesis, as well as for early diagnosis and prognosis of

495

endometrial cancer, and may help identify therapeutic targets for EC.

496

Availability of data and material

497

The datasets used and analysed during the current study are available from the corresponding author on

498

reasonable request.

499

Declaration of competing interest

500

The authors declare that there are no conflicts of interest.

501

Acknowledgment

502

This work was supported by grants from the National Natural Science Foundation of China (No. 81672590

503

and No. 81472437) and Shengjing Freedom researchers’ plan (No. 201804).

504 505 506 507 508 18

509 510

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tumor stage and survival outcome of clear cell renal cell carcinoma, Cancer Biomarkers. 19 (2017)

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with non-small cell lung cancer, Mol. Cells. 34 (2012) 171–176. https://doi:10.1007/

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promoter hypermethylation biomarkers in breast cancer - A short report, Cell. Oncol. 37 (2014)

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297–303. https://doi:10.1007/s13402-014-0189-1.

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carcinoma, upper tract urothelial carcinoma, bladder carcinoma, and urothelial carcinoma with gross

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hematuria using selected urine-DNA methylation biomarkers: A prospective, single-center study, Urol.

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Oncol. Semin. Orig. Investig. 36 (2018) 342.e15-342. e23. https://doi:10.1016/j.urolonc.2018.04.001.

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[36] X. Chen, R. Overcash, T. Green, D. Hoffman, A.S. Asch, M.J. Ruiz-Echevarría, The tumor suppressor

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activity of the transmembrane protein with epidermal growth factor and two follistatin motifs 2

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(TMEFF2) correlates with its ability to modulate sarcosine levels, J. Biol. Chem. 286 (2011)

619

16091–16100. https://doi:10.1074/jbc.M110.193805.

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cancer, BMC Cancer. 19 (2019) 423. https://doi:10.1186/s12885-019-5592-6.

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Cell Biol. 34 (2015) 541–549. https://doi:10.1089/dna.2014.2730.

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630

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and ERK pathways, Cancer Biol. Ther. 20 (2019) 295–306. https://doi:10.1080/15384047.2018.152

637

9096.

638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 23

658 659

Figure legends

660

Fig. 1. TMEFF2 expression in patients with endometrial carcinoma.

661

(A) Levels of TMEFF2 mRNA in different types of tumors in the Oncomine database. (B-E) Levels of

662

TMEFF2 DNA copy number in chips of endometrial carcinoma in TCGA endometrium. TCGA: The

663

Cancer Genome Atlas; TMEFF2: transmembrane protein with EGF-like and two follistatin-like domains.

664 665

Fig. 2. Expression and methylation of TMEFF2 in patients with endometrial carcinoma and correlation

666

analysis with survival prognosis.

667

(A) Relative expression of TMEFF2 mRNA in normal and UCEC samples. (B) Relative expression of

668

TMEFF2 mRNA in normal and different pathological types of UCEC patients. (C) Levels of TMEFF2

669

methylation in normal individuals and UCEC samples. (D) Levels of TMEFF2 methylation in normal

670

individuals of any age and in UCEC patients aged 21–40, 41–60, 61–80, or 81–100 years. (E) Levels of

671

TMEFF2 methylation in normal individuals of any ethnicity or in UCEC patients of Caucasian,

672

African-American or Asian ethnicity. (F) Levels of TMEFF2 methylation in normal individuals of any

673

weight or in UCEC patients with normal weight, extreme weight, obese, extreme obese. (G) Levels of

674

TMEFF2 methylation in normal individuals or in UCEC patients in stages 1, 2, 3 or 4. (H) Levels of

675

TMEFF2 methylation in normal individuals or UCEC patients with grade 1, 2, 3 or 4. (I) correlation of

676

TMEFF2 expression with overall survival (OS) in Kaplan-Meier Plotter. (J) correlation of TMEFF2

677

expression with progression free survival (PFS) in Kaplan-Meier Plotter. (K) correlation of TMEFF2

678

expression with OS in UALCAN. The Beta value indicates level of DNA methylation ranging from 0

679

(un-methylated) to 1 (fully methylated); different beta value cut-offs were considered to indicate

680

hypo-methylation (Beta-value: 0.25-0.3) or hyper-methylation (Beta value: 0.5 - 0.7). UCEC: Uterus

681

Corpus Endometrial Carcinoma; TMEFF2: transmembrane protein with EGF-like and two follistatin-like

682

domains. *P < 0.05, ** P < 0.01, and *** P < 0.001.

683 684

Fig. 3. Expression and prognosis of TMEFF2 in different endometrial tissues

685

(A-E) TMEFF2 expression in different endometrial tissues: (A) Endometrial carcinoma tissue. (B)

686

Atypical hyperplasia endometrium. (C) Proliferative endometrium. (D) Secretory endometrium. (E)

687

TMEFF2 expression in different endometrial tissues. (F-L) Relationship between high expression of

688

TMEFF2 (F); FIGO stage (G); myometrial invasion (H); lymph node metastasis (I); PR (J); ER (K) and

689

age (L) and prognosis of patients with endometrial cancer. TMEFF2: transmembrane protein with

690

EGF-like and two follistatin-like domains; FIGO: International Federation of Gynecology and Obstetrics;

691

PR: Progesterone receptor; ER: estrogen receptor. *P < 0.05, ** P < 0.01, and *** P < 0.001.

692 693 24

694 695 696

Fig. 4. Differentially expressed genes in correlation with TMEFF2 in endometrial carcinoma.

697

(A) Correlations between TMEFF2 and genes differentially expressed in UCEC. (B-C) Genes positively

698

and negatively correlated with TMEFF2 in UCEC as heat maps showing (TOP 50). Red indicates positive

699

correlation genes and green indicates negative correlation genes. (D-F) Correlation of TMEFF2 expression

700

with expression of LHX8 (D), PAX3 (E) and ASXL3 (F) with Pearson test in the scatter plot. TMEFF2:

701

transmembrane protein with EGF-like and two follistatin-like domains; UCEC: Uterus Corpus Endometrial

702

Carcinoma; LHX8: LIM homeobox 8; PAX3: paired box 3; ASXL3: ASXL transcriptional regulator 3.

703 704

Fig. 5. Significantly enriched GO annotations and KEGG pathways of TMEFF2 related genes in UCEC.

705

(A-B) Top 20 of cellular component enrichment related to TMEFF2 related genes with bar graph and

706

network. (C-D) Top 20 of biological process enrichment related to TMEFF2 related genes with bar graph

707

and network. (E-F) Top 20 of molecule function enrichment related to TMEFF2 related genes with bar

708

graph and network. (G-H) KEGG enriched terms with bar graph and network. (I) Protein–protein

709

interaction (PPI) network and four most significant MCODE components form the PPI network. (J)

710

Independent functional enrichment analysis of three MCODE components. TMEFF2: transmembrane

711

protein with EGF-like and two follistatin-like domains; UCEC: Uterus Corpus Endometrial Carcinoma;

712

GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; MCODE: Molecular Complex

713

Detection. Above results were colored by p-value, where terms containing more genes tend to have a more

714

significant P-value.

715 716

Fig. 6. Downregulation of TMEFF2 inhibited proliferation, invasion, and metastasis capacities of

717

endometrial carcinoma cell Ishikawa in vitro.

718

(A) Detection of TMEFF2 expression in different endometrial carcinoma cell lines by western blotting. (B)

719

The inhibition degree of TMEFF2 expression in Ishikawa by western blotting. (C) Influence of TMEFF2

720

downregulation on the proliferation ability was measured by MTT in Ishikawa. (D) Influence of TMEFF2

721

downregulation on the migration ability was measured by Scratch assay in Ishikawa. (E) Influence of

722

TMEFF2 downregulation on the invasion ability was measured by Transwell assay in Ishikawa. TMEFF2:

723

transmembrane

724

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H- tetrazolium bromide; Untreated: untreated group; Mock

725

transfected: empty-plasmid transfected group; TMEFF2-siRNA: TMEFF2-small interfering RNA. *P <

726

0.05, **P < 0.01, and ***P < 0.001

protein

with

EGF-like

and

two

follistatin-like

domains;

MTT:

727 728 729 730 25

731

Fig. 7. Downregulation of TMEFF2 inhibited the expression of EMT-related molecules and MAPK and

732

PI3K-AKT signaling pathways in Ishikawa cells.

733

(A-B) The effect of inhibition of TMEFF2 expression on EMT related molecules was detected by western

734

blotting and immunocytochemistry. (C) The effect of inhibition of TMEFF2 expression on MAPK and

735

PI3K-AKT signaling pathways were detected by western blotting. TMEFF2: transmembrane protein with

736

EGF-like and two follistatin-like domains; EMT: epithelial–mesenchymal transition. *P < 0.05, **P < 0.01,

737

and *** P < 0.001.

738 739

Supplementary figure 1 Protein-protein interaction network (PPI) of PLK2、miR-200B/200C/429 and

740

FOXD3_0 target networks with GeneMANIA.

741

(A) PPI network and functional analysis of PLK2 kinases related gene set. (B) PPI network and functional

742

analysis miR-200B/200C/429 related gene set. (C) PPI network and functional analysis transcription factor

743

FOXD3_0 related gene set. Different colors of the network edge indicate the bioinformatics methods

744

applied: physical interactions, pathway, Co-expression, Predicted, Physical Interactions, Co-localization,

745

Genetic Interactions. PPI: Protein-protein interaction; PLK2: polo like kinase 2; FOXD3: forkhead box D3.

746

26

Table 1 Expression of TMEFF2 in different endometrial tissues Low Groups

Cases

Malignant Atypical Severe Moderate Mild Normal Proliferative Secretory

75 24 7 9 8 36 15 21

（-） 6 7 2 2 3 18 9 11

High （+）

（++）

（+++）

14 7 2 2 3 6 4 2

34 8 2 4 2 9 2 7

21 2 1 1 0 3 0 1

Positive rate (%)

High expression rate (%)

92.00 a,b 70.83e 71.43 77.78 62.50 50.00 40.00 47.62

73.33c,d 41.67 42.86 55.56 25.00 33.33 13.33 38.10

a, b

positive TMEFF2 cases in malignant group, compared with atypical and normal group, both P < 0.05 (Pa = 0.020, Pb < 0.001)

c, d

high positive TMEFF2 cases in malignant group, compared with atypical and normal group, both P < 0.05 (Pc = 0.004, Pd < 0.001)

e

positive TMEFF2 cases in atypical hyperplasia group compared with normal endometrium group, P < 0.05 (Pe = 0.044)

Table 2 Relationships between expression of TMEFF2 and the clinical pathological parameters of 75 endometrial cancer patients Low Groups

Cases

High

(-)

(+)

(++)

(+++)

Positive rate (%)

P

High expression rate (%)

P

Age at diagnosis ＜59

37

4

5

17

11

89.19

≥59

38

2

8

17

10

92.11

I-II III-IV Pathologic type

56 19

6 0

13 1

23 11

14 7

89.29 100.00

P > 0.05

66.07 94.74

P I-II/III-IV = 0.015

Endometrioid Serous

37 23

2 2

9 3

14 11

12 7

94.59 91.30

P > 0.05

70.27 78.26

P > 0.05

P > 0.05

75.68

P > 0.05

71.05

FIGO stage

Clear cell

8

1

1

5

1

87.50

75.00

7

1

1

4

1

85.71

71.43

19 24 32

4 1 1

6 5 3

4 9 21

5 9 7

78.95 95.83 96.88

P >0.05

47.37 75.00 87.50

+ Unknown PR c

39 27 9

3 2 1

5 8 1

17 12 5

14 5 2

92.31 92.59 88.89

P > 0.05

79.49 62.96 77.78

P > 0.05

+ Unknown

38 28 9

3 2 1

6 7 1

16 13 5

13 6 2

92.11 92.86 88.89

P > 0.05

76.32 67.86 77.78

P > 0.05

＜1/2

48

6

9

20

13

87.50

≥1/2 LN metastasisd

27

0

5

14

8

100.00

81.48

-

48

6

9

20

13

87.50

68.75

+

14

0

0

10

4

100.00

Unknown

13

0

5

4

4

100.00

Others

a

Differentiation Well Moderate Poor

P P

well vs Poor=0.002

well-mod vs Poor=0.017

ER b

Muscular invasion P > 0.05

P > 0.05

68.75

100.00

P > 0.05

P = 0.041

61.54

a

“Others” indicated 3 mucous carcinomas, 2 squamous carcinomas, 2 undifferentiated carcinomas, 1 small cell carcinoma

b

9 patients without ER detection

c

9 patients without PR detection

d

13 patients without lymphadenectomy

Table 3 Kaplan-Meier analysis of the prognosis of endometrial cancer Variable

Characteristics

(Log-rank) P-value

Age at diagnosis

＜59 y vs. ≥59y

0.04

FIGO stage

I-II vs. III-IV

＜0.001

LN metastasis

Negative vs. Positive

＜0.001

Muscular invasion

＜1/2 vs. ≥1/2

＜0.001

Differentiation grade

well-mod vs. Poor

0.354

PR

Negative vs. Positive

0.01

ER

Negative vs. Positive

0.021

TMEFF2

Low vs. High

0.011

Table 4 Univariate and multivariate Cox regression analysis of patients with endometrial cancer

Univariate analysis

Multivariate analysis

Variables P -value

Hazard ratio (95% CI）

P -value

Age at diagnosis(＜59 y vs. ≥59)

0.05

2.603(0.999-6.785)

0.754

0.987 (0.908-1.702）

FIGO stage (I-II vs. III-IV)

<0.001

8.214 (3.235-20.857)

0.001

121.3 (6.724-2188.130)

Differentiation grade (well-mod vs. Poor)

0.362

1.504(0.626-3.614)

0.207

2.354 (0.624-8.885)

Muscular invasion (<1/2 vs. ≥1/2)

0.002

4.423(1.761-11.112)

0.007

47.539 (2.917-774.797)

LN metastasis (Negative vs. Positive)

0.001

5.411(1.941-15.086)

0.035

0.066 (0.005-0.827)

PR (Negative vs. Positive)

0.02

0.2230(0.066-0.795)

0.996

0.994 (0.090-10.970)

ER (Negative vs. Positive)

0.033

0.260(0.075-0.898)

0.034

0.137 (0.022-0.859)

TMEFF2 (Low vs. High)

0.037

8.509(1.138-63.631)

0.962

63724.663 (0-3.058E+204)

Hazard ratio (95% CI）

Table 5 Kinase, miRNA and transcription factor-target networks of TMEFF2 in endometrial carcinoma in LinkedOmics.

Enriched Category

GeneSet

LeadingEdgeNum

FDR

Kinase Target

Kinase_PLK2 Kinase_PRKX Kinase_PRKACG Kinase_PRKACB Kinase_CDK5 CAGTATT,MIR-200B,MIR-200C,MIR-429 ACATATC,MIR-190 ATATGCA,MIR-448 CAGTGTT,MIR-141,MIR-200A TAGCTTT,MIR-9 V$FOXD3_01 V$NRSF_01 V$CDX2_Q5 V$PAX4_04 V$CDP_02

3 24 20 23 25 195 17 97 120 94 69 41 70 71 33

0.090 0.095 0.103 0.121 0.136 0.000 0.000 0.000 0.000 0.000 0.000 0.001 0.002 0.002 0.002

miRNA Target

Transcription Factor Target

LeadingEdgeNum: the number of leading edge genes; FDR: false discovery rate from Benjamini and Hochberg from gene set enrichment analysis (GSEA); V$: the annotation found in Molecular Signatures Database (MSigDB) for transcription factors (TF).