TRIP13 is a predictor for poor prognosis and regulates cell proliferation, migration and invasion in prostate cancer

Accepted Manuscript TRIP13 is a predictor for poor prognosis and regulates cell proliferation, migration and invasion in prostate cancer

Liming Dong, Honglin Ding, Yanpei Li, Dongwei Xue, Zhi Li, Yili Liu, Teng Zhang, Jian Zhou, Ping Wang PII: DOI: Reference:

S0141-8130(18)34661-0 doi:10.1016/j.ijbiomac.2018.09.168 BIOMAC 10603

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

2 September 2018 22 September 2018 25 September 2018

Please cite this article as: Liming Dong, Honglin Ding, Yanpei Li, Dongwei Xue, Zhi Li, Yili Liu, Teng Zhang, Jian Zhou, Ping Wang , TRIP13 is a predictor for poor prognosis and regulates cell proliferation, migration and invasion in prostate cancer. Biomac (2018), doi:10.1016/j.ijbiomac.2018.09.168

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ACCEPTED MANUSCRIPT Title: TRIP13 is a predictor for poor prognosis and regulates cell proliferation, migration and invasion in prostate cancer

Authors Liming Dong1, Honglin Ding2, Yanpei Li1, Dongwei Xue1, Zhi Li3, Yili Liu1, Teng

Institutions

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Zhang4, Jian Zhou4, Ping Wang1*

1. Department of Urology, The Fourth Affiliated Hospital of China Medical

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University, Shenyang 110000, Liaoning, China.

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2. Department of Urology, The Affiliated Hospital of Chifeng Medical College, Chifeng 024000, Inner Mongolia, China.

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3. Department of Ethnpharmacology, School of Pharmaeutical Scineces, China Medical University, Shenyang 110000, Liaoning, China.

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4. Department of Urology, Tachengqu People’s Hospital, Tacheng 834700,

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Xinjiang, China.

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*Corresponding Author:

Ping Wang, Department of Urology, The Fourth Affiliated Hospital of China

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Medical University, No.4 Chongshan Road East, Huanggu District, Shenyang 110000, Liaoning, China. E-mail: [email protected]

Running Title: the role of TRIP13 in prostate cancer

Key words: TRIP13; prostate cancer; biomarker; oncogene; EMT

ACCEPTED MANUSCRIPT Abstract Thyroid hormone receptor interactor 13 (TRIP13) has been reported to be overexpressed in serval types of human cancers, and regulate tumor cell proliferation, migration and invasion. However, the role of TRIP13 in prostate cancer was still unclear. In our study, the correlation between TRIP13 expression

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and clinical parameters including prognosis was evaluated in 160 prostate cancer

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patients. Moreover, the MTT assay, cell migration and invasion assays were

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performed to assess the effect of TRIP13 on prostate cancer cell biological behaviour. In our results, the expression status of TRIP13 was observed to be

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elevated in prostate cancer tissue samples through analyzing microarray

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(GSE55945). Furthermore, mRNA and protein TRIP13 expression were confirmed to be overexpressed in prostate cancer tissue samples and cell lines. High-

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expression of TRIP13 was correlated with present lymph node involvement, distant metastasis, high Gleason score, levels of serum PSA and poor prgnosis in prostate

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cancer patients. The gain-of-function and loss-of-function studies suggested that TRIP13 functioned as oncogene to regulate prostate cancer cell proliferation,

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migration, invasion through controlling YWHAZ and epithelial-mesenchymal

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transition (EMT)-associated genes. In conclusion, TRIP13 is correlated with

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clinical progression and poor prognosis, and serves as oncogene in prostate cancer.

Introduction

ACCEPTED MANUSCRIPT Prostate cancer is the most common cancer in males and ranks the third most common cause of cancer-related death among men in western developed countries [1]. Despite of a sharp reduction in prostate cancer incidence of about 10% annually from 2010 to 2014 in the United States, there is still an estimated 164,690 new prostate cancer cases and 29,430 prostate cancer deaths in 2018 [2]. Although

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prostate cancer is the seventh most common cancer in China, a significantly

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increasing incidence and mortality trend from 2000 to 2011 was observed in

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prostate cancer patients [3]. In China, a huge population base and aging of population lead to a large and rising number of new prostate cancer cases [4]. The

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plasma prostate-specific antigen (PSA) determination greatly improved the rate of

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early diagnosis of prostate cancer [5, 6]. However, it has little value in predicting clinical outcome in prostate cancer patients [7]. Thus, it is needed to identify novel

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biomarkers to evaluate disease progression and prognosis and guide prostate cancer management.

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Thyroid hormone receptor interactor 13 (TRIP13) was originally suggested to involve in meiotic recombination, spindle assembly checkpoint and chromosome

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synapsis [8, 9]. Recently, TRIP13 has been reported to be overexpressed in serval

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types of human cancers, and regulate tumor cell proliferation, survival and invasion [10, 11]. Firstly, we analyzed a microarray (GSE55945) including human

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prostate cancer tissues and normal prostate tissues, and observed TRIP13 was overexpressed in prostate cancer tissues compared with normal prostate tissues. Then, we confirmed that TRIP13 was steadily up-regulated in prostate cancer tissue samples through qRT-PCR. Based on published reports and the microarray result, we supposed that TRIP13 expression is associated with disease progression and prognosis, and functions as an oncogene in prostate cancer. In order to investigate the clinical significance of TRIP13, we detected TRIP13 expression in one hundred and sixty prostate cancer patients through immunohistochemistry, and

ACCEPTED MANUSCRIPT estimated the associations between TRIP13 expression and clinical parameters. In addition, the MTT assay, cell migration and invasion assays were performed to assess the effect of TRIP13 on prostate cancer cell biological behaviour.

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Materials and Methods Microarray database analysis

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The data set of human exon array (GSE55945) was downloaded from the public Gene Expression Omnibus. The microarray GSE55945 contained eight human

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normal prostatic tissue samples and thirteen human prostate cancer tissue samples.

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The differentially expressed genes in human normal prostatic tissues and prostate

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cancer tissues were filtered by transcriptome microarray analysis.

Ethics statement

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The study methodologies were approved by the ethics committee of Tachengqu People’s Hospital and Fourth Affiliated Hospital of China Medical University and

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Affiliated Hospital of Chifeng Medical College. The written informed consents

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were obtained from each patient, and the procedures of this study were complied

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with the Declaration of Helsinki.

Clinical sample collection Paraffin-embedded clinical prostate cancer tissue specimens and paired normal prostatic tissue specimens from 160 patients were collected at Fourth Affiliated Hospital of China Medical University and Affiliated Hospital of Chifeng Medical College. Thirty fresh clinical tissue samples were obtained from Fourth Affiliated Hospital of China Medical University, and immediately stored in liquid nitrogen for qRT-PCR. Non-tumor tissue samples were gained from at least 3 cm distant of

ACCEPTED MANUSCRIPT the matched tumor tissues. All clinical tissues were confirmed by pathologists. None of the patients had received anti-tumor therapy before diagnose.

Quantitative RT-PCR (qRT-PCR) RNA isolation and TRIP13 expression determination were carried out according to

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previous description [12]. All the primers were listed as follows: TRIP13 forward, 5’-CTGTCTCTGGCAGTGGACAAG-3’;

GAPDH

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and

TTGGTTTGCAGAAGGGATTC-3’

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reverse,

forward,

5’5’-

GCTCTCTGCTCCTCCTGTTC-3’; reverse, 5’-ACGACCAAATCCGTTGACTC-

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3’. The GAPDH gene was used as gene internal control.

Immunohistochemistry staining and evaluation of staining

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Immumohistochemistry was performed as described [12] previously with a rabbit anti-TRIP13 antibody at concentration of 1:100 (Abcam, USA). Five random fields

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in each slide were selected and evaluated independently by two pathologists. Any disagreements were arbitrated by the third pathologists. A semi-quantitative

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scoring system was used to evaluate the staining results basing on the percentage

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and intensity of positively stained cells [13]. The intensity was scored as 0-3 (0 = negative; 1:weak; 2:moderate; 3:strong). The staining extent was scored as 1-4 (1:0%

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to 25%; 2:26% to 50%; 3：51% to 75%; 4：76% to 100%). The final score was calculated by multiplying intensity and extent score. All cases were classified into low-expression of TRIP13 group (0 to 7 scores) and high expression of TRIP13 group (8 to 12 scores).

Western Blot Western blotting was carried out according as described [12]. The membranes were blocked and then incubated with the primary antibodies: TRIP13 (Abcam,

ACCEPTED MANUSCRIPT USA), YWHAZ (Abcam, USA), E-Cadherin (Cell Signaling Technology, USA), Vimentin (Cell Signaling Technology, USA), β-catenin (Cell Signaling Technology, USA) or β-actin (CWBIO, China). The homologous HRP-conjugated antibodies (CWBIO, China) were used as the secondary antibody. Signals were detected using ECL chemiluminescence kit (CWBIO, China), and analyzed by

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Quantity One Software (Bio-Rad, USA). Each experiment was performed in

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triplicate.

Cell culture

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The human prostate cancer cell lines (LNCaP, PC3, DU145 and 22Rv1) and a

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normal human prostate epithelial cell line (P69) were purchased from the Chinese Academy of Sciences. P69, LNCaP, PC3, DU145 and 22Rv1 cells were

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maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS, GIBCO, USA). RWPE-1 cell was cultured in Keratinocyte Serum Free Medium

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(K-SFM, Invitrogen, USA) supplemented with bovine pituitary extract and human

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recombinant epidermal growth factor. All cell lines were cultured at 37℃ in a

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humidified atmosphere of 5% CO2.

Cell transfection

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The small interference RNA for depressing TRIP13 expression (siRNA-TRIP13) and negative control (siRNA-NC) were obtained from GenePharma (Shanghai, China). The siRNA-TRIP13 sequence was 5’-GCAAAUCACUGGGUUCUAC-3’ and the siRNA-NC sequence was 5’-GACUACGAUACGAGUUCUC-3’. Fulllength TRIP13 cDNA was amplified and cloned into pcDNA3.1 express vector (pcDNA- TRIP13). Non-targeting pcDNA3.1 express vector was used as negative control (pcDNA-NC). Cell transfection was conducted by using lipofectamine

ACCEPTED MANUSCRIPT 3000 reagent (Invitrogen, USA) in Opti-MEM (Gibco, USA) according to a previous description [12].

Cell proliferation assay The 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetra-zolium bromide (MTT, Sigma,

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USA) assay was used for detecting cell proliferation according to previous

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description [12]. After transfection, cells were grown in a 96-well plate for 24h,

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48h, 72h and 96h. Each experiment was performed in triplicate.

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Cell migration and invasion assays

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In vitro cell migration and invasion assays were conducted according to a previous description [12]. Cell migration and invasion experiments were performed using 12

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plates with 8.0μm pore diameter polycarbonate membrane insert in a transwell apparatus (Corning, USA). Prior to migration and invasion experiments, cells were

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starved of FBS for 24 hours. 2×104 LNCaP cells or 1.5×104 DU145 cells were suspended in serum-free medium and seeded in the upper wells. Lower chambers

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contained media with FBS to assess the migratory behavior of cultured cells, or

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without FBS as a negative control accordingly. For the invasion assay, the 24-well transwell units were coated with Matrigel (BD Biosciences, NJ, USA). The steps

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were the same as the cell migration assay described above. Each experiment was performed in triplicate.

Statistical analysis The SPSS 17.0 software and GraphPad prism 5.0 software were used for statistical analysis and make diagrams. The association between prostate cancer clnical parameters and TRIP13 expression was examined by Chi-square test. Log-rank test and univariate cox’s regression model were used for survival analysis. The

ACCEPTED MANUSCRIPT prognostic significance of various variables was analyzed by univariate and multivariate Cox regression analysis. The Student’s T-test was used for comparisons of two independent groups. A value of P < 0.05 was considered statistically significant.

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Results

TRIP13 expression is increased in prostate cancer tissues and cells

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In order to investigate the expression of TRIP13 in prostate cancer tissues, we assessed TRIP13 expression in microarray (GSE55945) contained eight human

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normal prostatic tissue samples and thirteen human prostate cancer tissue samples.

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We observed TRIP13 expression was elevated in prostate cancer tissue samples compared with normal prostatic tissue samples (Figure 1A). Furthermore, we

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confirmed TRIP13 mRNA and protein expression in tissues through qRT-PCR and immumohistochemistry, respectively. Compared with normal prostatic tissues,

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TRIP13 mRNA expression was significantly elevated in prostate cancer tissues (P<0.001, Figure 1B). Meanwhile, the result of immumohistochemistry showed

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levels of TRIP13 protein expression in prostate cancer tissues (55.0%, 88/160) was

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increased in comparison to normal prostatic tissues (22.5%, 9/40, P<0.001, Table

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We further measured TRIP13 mRNA and protein expression in prostate cancer cell lines (22Rv1, DU145, PC3, and LNCaP) and normal human prostate epithelial cell line (P69) via qRT-PCR and western blot, respectively. Compared with normal human prostate epithelial cell line, levels of TRIP13 mRNA expression were markedly risen in prostate cancer cell lines (P<0.001, Figure 1C). In addition, we found that prostate cancer cell lines exhibited high expression of TRIP13 protein in comparison to normal human prostate epithelial cell line (Figure 1D).

ACCEPTED MANUSCRIPT TRIP13 protein expression is correlated with clinical progression in prostate cancer patients In order to investigate the clinical value of TRIP13 in prostate cancer patients, we performed immumohistochemistry to measured TRIP13 expression in 160 prostate cancer clinical tissue samples (Figure 2A-H). As summarized in Table 2, TRIP13

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expression was not associated with age (≤65 years vs. ＞65 years, P=0.096), smoking (No vs. Yes, P=0.214) and advanced clinical T stage (T1-T2 vs. T3-T4;

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P=0.493). However, TRIP13 protein high-expression was obviously correlated with present lymph node involvement (No vs. Yes; P=0.001), distant metastasis

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(No vs. Yes; P=0.022), high Gleason score (≤8 vs. ＞8, P=0.016) and levels of

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serum PSA (≤20 ng/ml vs. ＞20 ng/ml, P=0.005).

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TRIP13 protein expression is correlated with poor overall survival in prostate

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cancer patients

We further investigated the prognostic value of TRIP13 protein expression in

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prostate cancer patients, and estimated the correlation between TRIP13 protein expression and overall survival in 160 prostate cancer cases through Kaplan-Meier

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analysis and log-rank test. We found high TRIP13 protein expression was correlated with short overall survival in prostate cancer patients (P=0.001, Figure

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2I). In addition, univariate analyses suggested clinical T stage (P<0.493), lymph node involvement (P<0.001), distant metastasis (P<0.001), high Gleason score (P=0.001) and levels of serum PSA (P=0.027) and TRIP13 expression (P=0.001) were unfavorable prognostic factors for prostate cancer patients (Table 3). Besides, we conducted multivariate Cox regression models adjusted to clinical T stage, lymph node involvement, distant metastasis, high Gleason score and levels of serum PSA, and found high-expression of TRIP13 was an independent poor

ACCEPTED MANUSCRIPT prognostic factor for overall survival in prostate cancer (HR=1.820, 95%CI: 1.1802.808, P=0.007, Table 3).

TRIP13 regulates prostate cancer cell proliferation, migration and invasion In order to explore the effect of TRIP13 in prostate cancer cell, loss-of-function

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and gain-of-function studies of TRIP13 were performed. Based on the expression

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of TRIP13 in prostate cancer cell lines (22Rv1, DU145, PC3, and LNCaP), we

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found that TRIP13 expression was the highest in LNCaP and the lowest in DU145 (Figure 1C). Thus, LNCaP and DU145 were used for loss-of-function study and

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gain-of-function study, respectively. These efficiencies of siRNA-TRIP13 and

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pcDNA-TRIP13 were confirmed by qRT-PCR and western blot (Figure 3A-B). The MTT assay showed that knocking down TRIP13 expression significantly

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suppressed cell proliferation, and TRIP13 overexpression dramatically enhanced cell proliferation (P<0.05, Figure 3C). The results of migration assay indicated

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knocking down TRIP13 expression markedly depressed cell migration, and TRIP13 overexpression obviously accelerated cell migration (P<0.001, Figure 3D).

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Similar to results of cell migration assay, invasion assay suggested that knocking

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down TRIP13 expression obviously inhibited cell invasion, and TRIP13

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overexpression significantly induced cell invasion (P<0.001, Figure 3E).

The molecular mechanism of TRIP13 in regulating prostate cancer cell proliferation, migration and invasion The published report has showed TRIP13 promote tumor cell proliferation, migration, invasion and epithelial mesenchymal transition (EMT) process through interacting with YWHAZ [13]. Thus, the effects of TRIP13 on YWHAZ, βcatenin, E-Cadherin and Vimentin protein expression were assessed by western blot in prostate cancer cells. We observed that knocking down TRIP13 expression

ACCEPTED MANUSCRIPT obviously inhibited the protein expression of YWHAZ, β-catenin and Vimentin, and elevated E-Cadherin protein expression (Figure 4). Contrarily, TRIP13 overexpression notably enhanced the protein expression of YWHAZ, β-catenin and

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Vimentin, and decreased E-Cadherin protein expression (Figure 4).

Discussion

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TRIP13 was originally showed to acts as key role in dsDNA break repair and mitotic checkpoint complex inactivation [14]. Recent decade, TRIP13 has been

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suggested to be overexpressed in various human cancers such as lung cancer [15],

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breast cancer [16], colorectal cancer [17], head and neck cancer [18], chronic lymphocytic leukemia [19, 20], lymphoma [21] and Wilms tumor [22]. Moreover,

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Wang Dazhi et al analyzed GEO database, and found TRIP13 expression was overexpressed in twelve kinds of malignancies including breast cancer, gastric

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cancer, colon cancer, renal cancer, esophageal carcinoma, lung cancer, liver cancer, ovarian cancer, pancreatic cancer, tongue cancer, prostate cancer and

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vulvar cancer [23]. In our study, we assessed TRIP13 expression in microarray

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(GSE55945) contained eight human normal prostatic tissue samples and thirteen human prostate cancer tissue samples, and observed TRIP13 expression was

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elevated in prostate cancer tissue samples compared with normal prostatic tissue samples. Furthermore, we confirmed TRIP13 mRNA and protein were overexpressed in prostate cancer tissues and cell lines. The clinical significance of TRIP13 expression was further estimated in prostate cancer. We assessed the association between TRIP13 protein expression and clinical parameters in 160 prostate cancer cases, and observed high-expression of TRIP13 protein was correlated with present lymph node involvement, distant metastasis, high Gleason score and levels of serum PSA in prostate cancer patients.

ACCEPTED MANUSCRIPT Similar, Sheng Nengquan et al reported TRIP13 was associated with CEA, CA19-9 and pTNM (pathologic primary tumor, lymph nodes, distant metastasis) classification in colorectal cancer patients [13]. Besides, TRIP13 expression was correlated with estrogen receptor (ER) status, progesterone receptor (PR) status, p53 mutation, Elston histological grade, tumor size and lymph nodes metastasis in

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breast cancer patients [23]. In lung cancer, TRIP13 overexpression was correlated

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with sex, advanced T stage, present lymph node metastasis and distant metastasis

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in lung cancer patients [23]. Similar results in hepatocellular carcinoma patients also were reported that high levels of TRIP13 were associated with large tumor

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size, advanced American Joint Committee on Cancer (AJCC) stage and Barcelona

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Clinic Liver Cancer Group (BCLC) stage [23]. These results consistently suggested that TRIP13 overexpression was associated with clinical progression in

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human cancer.

In past ten years, TRIP13 overexpression has been suggested to predict

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unfavorable prognosis in lung cancer [15, 23], breast cancer [16, 24], colorectal cancer [13, 25], breast cancer [23], liver cancer [23] and gastric cancer [23]. The

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prognostic significance of TRIP13 in prostate cancer was seldom reported. In our

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study, we found TRIP13 expression was negatively related with overall survival, and served as an independent unfavorable prognostic factor in prostate cancer

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patients. In breast cancer, Nieto-Jiménez Cristina et al and Maurizio Elisa et al congruously indicated that patients with high-expression of TRIP13 had short relapse-free survival, distant metastasis-free survival and overall survival [16, 24]. In colorectal cancer patients, Abdul Aziz NA et al TRIP13 was able to statistically predict the survival compared to the conventional Dukes’ classification [25]. Moreover, Sheng Nengquan et al demonstrated that colorectal cancer patients with TRIP13 high-expression exhibited shorter survival than those with low-expression of TRIP13, and high-expression of TRIP13 served as an independent prognostic

ACCEPTED MANUSCRIPT marker for colorectal cancer patients [13]. Li Wei et al reported that high levels of TRIP13 expression was correlated with lower overall survival in lung adenocarcinoma cases [15]. Besides, Wang Dazhi revealed that TRIP13 overexpression indicated unfavorable prognosis in breast cancer, liver cancer, gastric cancer and lung cancer patients [23].

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TRIP13 has been found to function as oncogene in lung cancer [15], colorectal

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cancer [13, 17], head and neck cancer [18], chronic lymphocytic leukemia [19, 20].

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In lung adenocarcinoma, Li Wei et al indicated that TRIP13 enhanced cell proliferation, clonogenicity, and migration while suppressing apoptosis and G2/M

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phase shift in vitro [15]. Moreover, TRIP13 has been suggested to promote

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colorectal cancer cell proliferation, invasion and migration in vitro and subcutaneous tumor formation in vivo [13, 17]. Zhou Keshu et al showed inhibition

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of TRIP13 repressed chronic lymphocytic leukemia cell proliferation, and induced cell apoptosis [19, 20]. In addition, Banerjee Rajat et al found that TRIP13

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overexpression in head and neck cancer cell leads to aggressive status, treatmentresistant tumors and enhanced repair of DNA damage [18]. However, the

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biological function of TRIP13 in prostate cancer was still unknown. Our study

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firstly suggested that knocking down TRIP13 expression depressed cell proliferation, migration and invasion, and TRIP13 overexpression enhanced cell

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proliferation, migration and invasion. Meanwhile, we further investigated the potential molecular mechanism of TRIP13 in regulating prostate cancer cell proliferation, migration and invasion. Sheng Nengquan et al found TRIP13 promote tumor cell proliferation, migration, invasion and EMT process through interacting with YWHAZ [13]. Recent researches proved that YWHAZ acted as a key role in tumor progression and served as a prognostic marker for human cancers [26-28]. In prostate cancer, YWHAZ has been suggested to be overexpressed in tumor tissues, and associated with malignancy and lymph node metastasis [29].

ACCEPTED MANUSCRIPT Thus, we detected the effects of TRIP13 on YWHAZ, β-catenin, E-Cadherin and Vimentin protein expression through western blot, and found TRIP13 positively regulated YWHAZ, β-catenin and Vimentin expression, and negatively modulated E-Cadherin expression. Thus, TRIP13 functioned as oncogene to regulate prostate cancer cell proliferation, migration, invasion and EMT process through modulating

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YWHAZ and EMT-associated genes. Interestingly, YWHAZ is a novel androgen-

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responsive gene that activates androgen receptor transcriptional activity [29]. In

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our study, TRIP13 positively regulated YWHAZ expression, which suggested that TRIP13 may be associated with androgen receptor and resistance for anti-androgen

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and androgen receptor in prostate cancer.

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therapy. In our future research, we will focus on the relationship between TRIP13

In conclusion, high-expression TRIP13 is associated with clinical progression,

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and served as an independent prognostic marker for prostate cancer patients. TRIP13 promotes prostate cancer cell proliferation, migration and invasion

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Conflict of interest

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through modulating YWHAZ and EMT-associated genes.

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The authors declare no conflict of interest.

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Acknowledgements

This work was supported by funding from Shenyang Natural Science Foundation (No.20158960239).

ACCEPTED MANUSCRIPT References

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1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A: Global cancer statistics, 2012. CA: a cancer journal for clinicians 2015, 65(2):87-108. 2. Siegel RL, Miller KD, Jemal A: Cancer statistics, 2018. CA: a cancer journal for clinicians 2018, 68(1):7-30. 3. Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ, He J: Cancer statistics in China, 2015. CA: a cancer journal for clinicians 2016, 66(2):115-132. 4. Chen W, Zheng R, Zeng H, Zhang S: The updated incidences and mortalities of major cancers in China, 2011. Chinese journal of cancer 2015, 34(11):502-507. 5. Etzioni R, Tsodikov A, Mariotto A, Szabo A, Falcon S, Wegelin J, DiTommaso D, Karnofski K, Gulati R, Penson DF et al: Quantifying the role of PSA screening in the US prostate cancer mortality decline. Cancer causes & control : CCC 2008, 19(2):175-181. 6. Fleshner K, Carlsson SV, Roobol MJ: The effect of the USPSTF PSA screening recommendation on prostate cancer incidence patterns in the USA. Nature reviews Urology 2017, 14(1):26-37. 7. Fedewa SA, Ward EM, Brawley O, Jemal A: Recent Patterns of Prostate-Specific Antigen Testing for Prostate Cancer Screening in the United States. JAMA internal medicine 2017, 177(7):1040-1042. 8. Roig I, Dowdle JA, Toth A, de Rooij DG, Jasin M, Keeney S: Mouse TRIP13/PCH2 is required for recombination and normal higher-order chromosome structure during meiosis. PLoS genetics 2010, 6(8). 9. Li XC, Schimenti JC: Mouse pachytene checkpoint 2 (trip13) is required for completing meiotic recombination but not synapsis. PLoS genetics 2007, 3(8):e130. 10. Vader G: Pch2(TRIP13): controlling cell division through regulation of HORMA domains. Chromosoma 2015, 124(3):333-339. 11. Ye Q, Rosenberg SC, Moeller A, Speir JA, Su TY, Corbett KD: TRIP13 is a proteinremodeling AAA+ ATPase that catalyzes MAD2 conformation switching. eLife 2015, 4. 12. Dong L, Li Y, Xue D, Liu Y: PCMT1 is an unfavorable predictor and functions as an oncogene in bladder cancer. IUBMB life 2018, 70(4):291-299. 13. Sheng N, Yan L, Wu K, You W, Gong J, Hu L, Tan G, Chen H, Wang Z: TRIP13 promotes tumor growth and is associated with poor prognosis in colorectal cancer. Cell death & disease 2018, 9(3):402. 14. Zanders S, Sonntag Brown M, Chen C, Alani E: Pch2 modulates chromatid partner choice during meiotic double-strand break repair in Saccharomyces cerevisiae. Genetics 2011, 188(3):511-521. 15. Li W, Zhang G, Li X, Wang X, Li Q, Hong L, Shen Y, Zhao C, Gong X, Chen Y et al: Thyroid hormone receptor interactor 13 (TRIP13) overexpression associated with tumor progression and poor prognosis in lung adenocarcinoma. Biochemical and biophysical research communications 2018, 499(3):416-424. 16. Nieto-Jimenez C, Alcaraz-Sanabria A, Paez R, Perez-Pena J, Corrales-Sanchez V, Pandiella A, Ocana A: DNA-damage related genes and clinical outcome in hormone receptor positive breast cancer. Oncotarget 2017, 8(38):62834-62841.

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17. Kurita K, Maeda M, Mansour MA, Kokuryo T, Uehara K, Yokoyama Y, Nagino M, Hamaguchi M, Senga T: TRIP13 is expressed in colorectal cancer and promotes cancer cell invasion. Oncology letters 2016, 12(6):5240-5246. 18. Banerjee R, Russo N, Liu M, Basrur V, Bellile E, Palanisamy N, Scanlon CS, van Tubergen E, Inglehart RC, Metwally T et al: TRIP13 promotes error-prone nonhomologous end joining and induces chemoresistance in head and neck cancer. Nature communications 2014, 5:4527. 19. Zhou K, Zhang W, Zhang Q, Gui R, Zhao H, Chai X, Li Y, Wei X, Song Y: Loss of thyroid hormone receptor interactor 13 inhibits cell proliferation and survival in human chronic lymphocytic leukemia. Oncotarget 2017, 8(15):25469-25481. 20. Zhou KS, Zhang Q, Zhang WT, Liu YY, Wu SS, Zhou J, Wei XD, Song YP: [Study on the expression of TRIP13 mRNA in chronic lymphocytic leukemia B lymphocyte and the molecular mechanism of TRIP13 mediated JVM-2 cell proliferation and apoptosis]. Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 2017, 38(7):618-622. 21. van Kester MS, Borg MK, Zoutman WH, Out-Luiting JJ, Jansen PM, Dreef EJ, Vermeer MH, van Doorn R, Willemze R, Tensen CP: A meta-analysis of gene expression data identifies a molecular signature characteristic for tumor-stage mycosis fungoides. The Journal of investigative dermatology 2012, 132(8):2050-2059. 22. Yost S, de Wolf B, Hanks S, Zachariou A, Marcozzi C, Clarke M, de Voer R, Etemad B, Uijttewaal E, Ramsay E et al: Biallelic TRIP13 mutations predispose to Wilms tumor and chromosome missegregation. Nature genetics 2017, 49(7):1148-1151. 23. Dazhi W, Mengxi Z, Fufeng C, Meixing Y: Elevated expression of thyroid hormone receptor-interacting protein 13 drives tumorigenesis and affects clinical outcome. Biomarkers in medicine 2017, 11(1):19-31. 24. Maurizio E, Wisniewski JR, Ciani Y, Amato A, Arnoldo L, Penzo C, Pegoraro S, Giancotti V, Zambelli A, Piazza S et al: Translating Proteomic Into Functional Data: An High Mobility Group A1 (HMGA1) Proteomic Signature Has Prognostic Value in Breast Cancer. Molecular & cellular proteomics : MCP 2016, 15(1):109-123. 25. Abdul Aziz NA, Mokhtar NM, Harun R, Mollah MM, Mohamed Rose I, Sagap I, Mohd Tamil A, Wan Ngah WZ, Jamal R: A 19-Gene expression signature as a predictor of survival in colorectal cancer. BMC medical genomics 2016, 9(1):58. 26. Chen CH, Chuang SM, Yang MF, Liao JW, Yu SL, Chen JJ: A novel function of YWHAZ/beta-catenin axis in promoting epithelial-mesenchymal transition and lung cancer metastasis. Molecular cancer research : MCR 2012, 10(10):1319-1331. 27. Liu S, Jiang H, Wen H, Ding Q, Feng C: Knockdown of tyrosine 3monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) enhances tumorigenesis both in vivo and in vitro in bladder cancer. Oncology reports 2018, 39(5):2127-2135. 28. Chang CC, Zhang C, Zhang Q, Sahin O, Wang H, Xu J, Xiao Y, Zhang J, Rehman SK, Li P et al: Upregulation of lactate dehydrogenase a by 14-3-3zeta leads to increased glycolysis critical for breast cancer initiation and progression. Oncotarget 2016, 7(23):35270-35283. 29. Murata T, Takayama K, Urano T, Fujimura T, Ashikari D, Obinata D, Horie-Inoue K, Takahashi S, Ouchi Y, Homma Y et al: 14-3-3zeta, a novel androgen-responsive gene, is upregulated in prostate cancer and promotes prostate cancer cell proliferation and

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survival. Clinical cancer research : an official journal of the American Association for Cancer Research 2012, 18(20):5617-5627.

ACCEPTED MANUSCRIPT Figure legends Figure 1. TRIP13 expression is increased in prostate cancer tissues and cells (A) TRIP13 expression was analyzed in the microarray GSE55945 contained eight human normal prostatic tissue samples and thirteen human prostate cancer tissue samples. (B) The expression of TRIP13 mRNA in thirty pairs of prostate cancer

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tissue and normal prostatic tissue samples were detected by qRT-PCR. (C) The

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expression of TRIP13 mRNA was measured in four human prostate cancer cell

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lines (LNCaP, PC3, DU145 and 22Rv1) and a normal human prostate epithelial

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cell lines (P69) through qRT-PCR.

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Figure 2. Immunohistochemical staining of TRIP13 in prostate cancer and normal prostatic tissue samples expression

was

detected

in

normal

prostatic

tissues

by

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TRIP13

immunohistochemistry: (A) negative expression, (B) weak expression, (C)

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moderate expression and (D) strong expression. TRIP13 expression was detected in prostate cancer tissues by immunohistochemistry: (E) negative expression, (F)

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weak expression, (G) moderate expression or (H) strong expression. (I) Kaplan-

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Meier survival analysis of overall survival duration in 160 prostate cancer patients according to TRIP13 protein expression, and suggested TRIP13 protein expression

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were negatively correlated with the overall survival of prostate cancer patients.

Figure 3. TRIP13 regulates prostate cancer cell proliferation, migration and invasion (A-B) These efficiencies of siRNA-TRIP13 in LNCaP cell and pcDNA-TRIP13 in DU145 cell were confirmed by qRT-PCR and western blot. (C) The proliferation curve of TRIP13 siRNA infected LNCaP cell and pcDNA-TRIP13 infected DU145 cell compared with control group, were determined by MTT assay at 24 h, 48 h, 72

ACCEPTED MANUSCRIPT h and 96h. (D-E) Cell transwell migration and invasion experiments were used to estimate the effect of TRIP13 on cell migration and invasion in LNCaP and DU145 cells. The graph shows the average number of migrated or invasive cells per field. (*, P<0.05; **, P<0.001 )

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Figure 4. The molecular mechanism of TRIP13 in regulating prostate cancer cell proliferation, migration and invasion

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The effect of TRIP13 on YWHAZ, β-catenin, E-Cadherin and Vimentin protein expression were assessed by Western blot in prostate cancer cells transfected with

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siRNA-TRIP13 or pcDNA-TRIP13.

ACCEPTED MANUSCRIPT Table 1 Expression of TRIP13 protein between prostate cancer tissues and normal prostate tissues TRIP13 cases

40

expression(%)

88(55.0)

72(45.0)

9(22.5)

31(77.5)

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Normal prostate tissues

expression(%)

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160

Low

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Prostate cancer tissues

P

High

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Group

<0.001

ACCEPTED MANUSCRIPT Table 2 Associations between TRIP13 protein expression and clinicopathological parameters in prostate cancer Characteristics

n

High expression(%)

Low expression(%)

≤65

62

29(46.8)

33(53.2)

＞65

98

59(60.2)

39(39.8)

No

54

26(48.1)

P

106

62(58.5)

73

38(52.1)

87

Lymph node involvement 130

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No Yes

≤8

44(41.5)

35(47.9) 0.493 37(42.5)

67(51.5) 0.001

25(83.3)

5(16.7)

66(48.9)

69(51.1)

25

19(76.0)

6(24.0)

70

31(44.3)

39(55.7)

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Gleason score

0.214

30

135

Yes

63(48.5)

28(51.9)

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Distant metastasis No

50(57.5)

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T3-T4

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T1-T2

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Clinical T stage

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Smoking

Yes

0.096

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Age(y)

0.022

0.016 ＞8

90

57(63.3)

33(36.7)

63

26(41.3)

37(58.7)

Serum PSA (ng/ml) ≤20

0.005

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62(63.9)

35(36.1)

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ACCEPTED MANUSCRIPT Table 3 Univariate and multivariate Cox regression analyses of overall survival in prostate cancer Univariate analysis

Multivariate analysis

Parameter HR

95%CI

P

HR

1.279

0.841-1.945

0.250

0.966

0.632-1.477

0.874

3.109

1.996-4.843

3.224

1.974-5.264

Smoking

<0.001

1.163

0.325-4.154

0.817

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2.172-5.818

<0.001

1.175

0.322-4.293

0.807

1.962

1.295-2.971

0.001

1.902

1.171-3.090

0.009

1.605

1.054-2.444

0.027

0.998

0.627-1.588

0.992

1.998

1.310-3.045

0.001

1.820

1.180-2.808

0.007

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Serum PSA (ng/ml)

<0.001

3.555 （No vs. Yes）

Gleason score

1.761-4.835

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Distant metastasis

（≤8 vs. ＞8）

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Lymph node involvement （No vs. Yes）

2.918

<0.001

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（T1-T2 vs. T3-T4）

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(No vs. Yes)

Clinical T stage

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(≤65 vs. ＞65)

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Age

95%CI

（≤20 vs. ＞20）

TRIP13 expression （Low vs. High ） HR, hazard ratio; 95% CI, 95% confidence interval; PSA, prostate-specific antigen

Figure 1

Figure 2

Figure 3

Figure 4