Am J Cancer Res 2018;8(10):2076-2087 www.ajcr.us /ISSN:2156-6976/ajcr0084894 Original Article Abnormal expression of YEATS4 associates with poor prognosis and promotes cell proliferation of hepatic carcinoma cell by regulation the TCEA1/DDX3 axis Song You2,3*, Fuqiang Wang1,2*, Qing Hu4, Pengtao Li1,2, Changmao Zhang2,3, Yaqi Yu2, Yi Zhang2, Qiu Li2, Qing Bao2, Pingguo Liu1,2, Jie Li1,2* 1Department of Hepatobiliary Surgery, Zhongshan Hospital of Xiamen University, Xiamen, Fujian, China; 2Fujian Provincial Key Laboratory of Chronic Liver Disease and Hepatocellular Carcinoma (Xiamen University Affiliated Zhongshan Hospital), Xiamen, Fujian, China; 3Graduate College of Fujian Medical University, Fuzhou, Fujian, China; 4Medicine Clinical Laboratory of Xiamen Xianyue Hospital, Xiamen, Fujian, China. *Equal contributors. Received August 31, 2018; Accepted September 14, 2018; Epub October 1, 2018; Published October 15, 2018 Abstract: YEATS domain containing 4 (YEATS4) is usually amplified and functions as an oncogene in several ma- lignancies, such as colorectum, ovarian, breast and lung. However, the biological role of YEATS4 in hepatocellular carcinoma (HCC) has not yet been discussed. Herein, we found that YEATS4 was significantly upregulated in HCC compared to para-cancerous tissues, and was associated with poor prognosis, large tumor size, poor differentia- tion and distant metastasis. In addition, YEATS4 promoted HCC cell proliferation and colony formation by binding to and increasing the transcriptional activity of the TCEA1 promoter. Concurrently, upregulation of TCEA1 increased the stability of the DDX3 protein, a member of the DEAD box RNA helicase family, and augmented the proliferative and colony forming ability of HCC cells. Furthermore, YEATS4 accelerated tumor growth in vivo in a xenograft HCC model. Taken together, our study provides evidence for the first time on the potential role of the YEATS4/TCEA1/ DDX3 axis in regulating HCC progression, and presents YEATS4 as a promising therapeutic target and prognosis maker for HCC. Keywords: YEATS4, TCEA1, DDX3, hepatic carcinoma, prognosis, proliferation Introduction the Wnt/β-catenin/TCF signaling pathway [5, 6]. YEATS4 is also upregulated in NSCLC and Hepatocellular carcinoma (HCC) is a malignant contributes to cancer cell proliferation and tumor of the liver, and has a high mortality rate tumor growth by inhibiting p53 expression [7]. with an annually increasing incidence world- Furthermore, it has been linked with increas- wide [1]. The poor prognosis of HCC patients is ed drug resistance in ovarian cancer [8]. Dow- largely attributed to the difficulty in early diag- nregulation of YEATS4 by miR-218 sensitized nosis, as well as the high metastasis, invasion colorectal cancer cells to L-OHP-induced cell and recurrence of this cancer [2]. Therefore, it apoptosis by weakening cytoprotective auto- is vital to explore the pathways involved in HCC phagy [9]. However, the precise function of initiation, and thereby identify early specific bio- YEATS4 in HCC, and underlying mechanisms markers and potential therapeutic targets for and patient prognosis is unclear. HCC. SOX is an important transcriptional elongation YEATS4 was first identified in the nucleolix [3]. factor that can directly bind to RNA polymerase High sequence homology to the human MLLT1 II, and allow it to read through various transcrip- and MLLT3 proteins indicated a transcription tion arrest sites [10, 11]. SOX has 3 distinct iso- factor function [4]. Recently, YEATS4 has been forms: the ubiquitously expressed TCEA1 that shown to be involved in the genesis and pro- was identified in the network-based protein- gression of various cancers. In the pancreatic protein interactions (PPI) for meningioma rela- and gastric cancers, it acts as an oncogene via tive to healthy controls, the testis-specific SOX2 YEATS4 promotes cell proliferation [12], and SOX3 which controls murine embry- Technology). The blots were then incubated onic stem cell (mESC) fate and maintains its with horseradish peroxidase-conjugated sec- pluripotency by regulating the Lefty1-Nodal- ondary antibodies (Pierce) and visualized by ch- Smad2 pathway [13]. TCEA1 is also up-regulat- emiluminescence. Experiments were repeated ed in HCC [14], although its potential role in triplicate independently. TCEA1 in liver cancer is not completely clear. Human tissue specimens DDX3 (DEAD box protein 3) belongs to the DEAD box RNA helicase family that are charac- Tumor samples were obtained with informed terized by the presence of a helicase domain consent from HCC patients at Zhongshan Hos- and involved in RNA post-transcriptional pro- pital Xiamen University, between 2007 and cession. They are also members of the DEAD/ 2011. The study was approved by the Xiamen H-box family [15], which are aberrantly expr- University Medical Ethics Committee. Overall essed in various solid and hematological ma- survival was calculated from the date of sur- lignancies [16-18]. Studies have shown that gery to the date of death or final follow-up. high-levels of DDX3 could promote proliferation and neoplastic transformation of immortalized Hematoxylin-eosin and immunohistochemistry human breast cancer epithelial cells [19]. In staining addition, studies have hinted to DDX3 growth- regulatory functions of DDX3 in hepato-carcino- Tissues were fixed with 10% neutral formalin, genesis and progression [20, 21]. embedded in paraffin, and 3-μm-thick sections were prepared by the pathological technologist. In this study, we showed that YEATS4 transcrip- For Hematoxylin-Eosin (HE) staining, sections tionally activated TCEA1 expression TCEA1 by were deparaffinized and hydrated with a gra- binding to its promoter, TCEA1 and the latter dient alcohol. After soaking in PBS, sections upregulated DDX3 levels by increasing protein were stained with HE. For IHC, sections were stability. These interactions significantly accel- stained using the streptavidin-peroxidase (S-P) erated HCC cells proliferation and growth in method. In brief, sections were deparaffiniz- vitro and in vivo, thereby indicating that up-reg- ed, hydrated and soaked in 3% H2O2 for 10 ulation of YEATS4 is associated with poor prog- minutes at room temperature, and then incu- nosis in HCC patients. bated with Pygo2 polyclonal antibody (1:3000, ab109001, Abcam) at 4°C overnight. Biotiny- Materials and methods lated secondary antibody and diaminobenzi- dine (DAB) were purchased from Maixin Bio- Cell culture technology (Fuzhou, China). Human liver cancer cell lines were purchased Plasmid construction and lentivirus prepara- from the cell bank of Shanghai Institute of Cell tion Biology and cultured in medium (HyClone) sup- plemented with 10% fetal calf serum (Gibco), For target gene knockdown, control and shRNA 100 IU/ml penicillin and 100 μg/ml streptomy- sequences (shown in Table 1) against YEATS4 cin (Millipore). were cloned into the pLKO.1-Puro vector. For over-expression, genomic sequence of the Immunoblotting YEATS4 and TCEA1 coding region was respec- tively cloned into the backbone of the plv-Puro Cells were harvested and protein was extracted vector vector downstream of the CMV promot- using RIPA buffer (Sigma) with 1% v/v protease er. Then 293T cells were transfected with the inhibitor cocktail (Sigma) and 1% v/v phospha- above mentioned plasmids and packaging vec- tase inhibitor cocktail (Sigma). Equal amounts tors by using the Turbofect Transfection Re- of protein lysates were separated by SDS-PAGE agent (Thermo). Infected cells were cultured for and were transferred onto PVDF membranes. selection with puromycin (InvivoGen). The filters were probed with the following spe- cific primary antibodies: anti-YEATS4 and anti- RNA extraction and real-time PCR DDX3 (Abcam), anti-TCEA1 and anti-PCNA (Pro- teintech Group), anti-ha, anti-flag and anti-β- RNA was extracted from tissues or cell samples actin (Cell Signaling Technology) (Cell Signaling using the Trizol reagent (Invitrogen) according 2077 Am J Cancer Res 2018;8(10):2076-2087 YEATS4 promotes cell proliferation Table 1. YEATS4 abnormal expression correlates with clinic-patho- unting Kit-8 (CCK-8) assays logical factors of HCC patients were performed using the Kit Clinical-pathology Factors A abnormal expression N X2 P (#YB-K001, Yiyuan Biotechno- logies, Guangzhou, China) ac- Age (yr) cording to the manufacturer’s < 55 20 (80.0%) 25 0.125 0.724 instructions for the purpose of ≥ 55 40 (83.3%) 48 detecting cell proliferation ab- Gender ility. Optical density (OD) val- Male 35 (83.3%) 42 0.088 0.768 ues were measured at a wa- Female 25 (80.6%) 31 velength of 450 nm by a micro- Tumor size plate reader. All experiments < 5 cm 21 (70.0%) 30 5.172 0.023* were performed three times, ≥ 5 cm 39 (90.7%) 43 and the average results were Metastasis calculated. Yes 38 (92.7%) 41 7.033 0.008** EdU assay No 22 (68.8%) 32 Differentiation The EdU incorporation assay Poor 28 (93.3%) 30 5.074 0.034* was performed using the EdU Moderate 12 (85.7%) 14 assay kit (RiboBio, Guangzhou, Well 20 (71.4%) 28 China), according to the manu- facturer’s instructions. Briefly, Liver cirrhosis status cells were cultured in triplica- No 19 (73.1%) 26 2.261 0.200 te in 24-well plates. The cells Yes 41 (87.2%) 47 were then incubated with 50 Serum HBV level (cps/ml) nM EdU for an additional 2 h at < 1000 26 (83.9%) 31 0.166 0.684 37°C. The cells were fixed with ≥ 1000 28 (80.0%) 35 4% formaldehyde for 15 min at Serum AFP level (ug/L) room temperature and treat- < 400 27 (79.4%) 34 0.279 0.597 ed with 0.5% Triton X-100 for ≥ 400 32 (84.2%) 38 20 min at room temperature to *p < 0.05; **P < 0.01. permeabilize them. After three washes with phosphate-buff- ered saline (PBS), the cells we- to the manufacturer’s instructions. Primers re incubated with the Apollo reaction cocktail were designed and synthesized by BGI-Tech. (100 l/well) for 30 min. After being washed th- The sequences of the primer pairs are shown in ree times with phosphate buffered saline (PBS), Table S1.