ZNF692 promotes proliferation and cell mobility in lung adenocarcinoma

Accepted Manuscript ZNF692 promotes proliferation and cell mobility in lung adenocarcinoma Quanli Zhang, Xiufen Zheng, Qi Sun, Run Shi, Jie Wang, Biqing Zhu, Lin Xu, Guangqin Zhang, Binhui Ren PII:

S0006-291X(17)31311-6

DOI:

10.1016/j.bbrc.2017.06.180

Reference:

YBBRC 38086

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 22 June 2017 Accepted Date: 28 June 2017

Please cite this article as: Q. Zhang, X. Zheng, Q. Sun, R. Shi, J. Wang, B. Zhu, L. Xu, G. Zhang, B. Ren, ZNF692 promotes proliferation and cell mobility in lung adenocarcinoma, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.06.180. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

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ZNF692

promotes

proliferation

and

cell

mobility

in

lung

adenocarcinoma Quanli Zhanga,b,c 1, Xiufen Zhenga,b,c 1, Qi Sunb, 1, Run Shi b,c, Jie Wang b,c,

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Biqing Zhu b,c, Lin Xu a,b,c *，Guangqin Zhanga**, Binhui Ren b,c * a

Department of Clinical Pharmacy, China Pharmaceutical University,

Nanjing, China

Jiangsu Key Laboratory of Molecular and Translational Cancer Research,

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b

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Nanjing, China c

Department of Thoracic Surgery, Jiangsu Cancer Hospital, Institue

Affiliated to Nanjing Medical University, Cancer Institute of Jiangsu Province, Baiziting 42, Xuanwu District, Nanjing, 210009, PR China

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210009, PR China * Corresponding author.

** Corresponding authors.

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E-mail addresses: [email protected] (Q.Zhang), [email protected]

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(X.Zheng), [email protected] (Q. Sun), [email protected] (R. Shi), [email protected] (J. Wang), [email protected] （ B.Zhu ） , [email protected]

(L.Xu),

[email protected]

[email protected] (B. Ren), 1

Equal contributors.

Abstract

1

(G.

Zhang),

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By analyzing The Cancer Genome Atlas (TCGA) datasets, we discovered that the zinc finger protein 692 (ZNF692) were over-expressed in Lung adenocarcinoma (LUAD) tissues compared to adjacent non-tumor tissues

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(P<0.0001). In this study, we investigated the function of ZNF692 in the progression of LUAD. We found that ZNF692 knockdown inhibited LUAD cells proliferation, migration, and invasion both in vitro and in And

LUAD

cell apoptosis

was

induced

following

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vivo.

the

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down-regulation of ZNF692. Our results show that ZNF692 is over-expressed in LUAD tissues compared to adjacent normal tissues, and hyper-expression of ZNF692 in LUAD is an independent risk factor for worse overall survival in LUAD patients (HR: 8.800, 95%CI:

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1.082-71.560, P=0.042) by Tissue Microarray stain assay (TMA). GO analysis indicated that most genes were enriched in metabolic process which were associated highly with ZNF692 levels. Collectively, our

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results suggested that ZNF692 may serve as a potential oncogene and

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biomarker in LUAD by influencing cell metabolism. Keywords: ZNF692, TCGA, lung adenocarcinoma (LUAD), cell metabolism

Introduction

Lung cancer is one of the most commonly diagnosed cancers and the leading cause of cancer-related deaths worldwide, with a five-year survival rate of less than 15%, though patients accept the standard 2

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therapies[1]. Lung adenocarcinomas (LUAD) has become the most common subtype of lung cancer in recent years [2]. Lung carcinogenesis and progression caused by the dysregulation of many cancer-related

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genes are intricate biological processes [3]. Therefore, we are supposed to do a further exploration to identify new biomarkers for LUAD and have better understanding of the molecular mechanisms underlying lung cancer

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progress, which may benefit the individualized treatment and improve the

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prognosis thereafter [4].

Through an analysis of the TCGA database, we identified the zinc finger protein 692 (ZNF692) as lung cancer-related gene which was differentially expressed between LUAD and normal tissues (P<0.0001) [5,

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6]. Our previous microarray analysis yielded similar result (details shown in Table S1) (P<0.0001) [7]. ZNF692 is located on chromosome 1q44, and was first identified as a transcription factor bound to the promoter

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elements of Phosphoenolpyruvate carboxykinase (PEPCK) [8]. Recent

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research showed that ZNF692 has different RNA splicing events within various types of hepatocellular carcinoma [9]. Additionally, ZNF692 has been reported to be related with the relapse of Wilms tumor [10]. However，the expression profile and molecular function of ZNF692 in LUAD remain unknown. In this research, we demonstrate that ZNF692 is a potential oncogene which promotes the proliferation and migration of LUAD cell in vitro and in vivo. Furthermore, over-expression of ZNF692 3

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may be a biomarker indicating the progression of LUAD. Materials and methods Cell lines, culture conditions, siRNA transfection

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A549, H1299, SPCA-1, H1975 cells and human bronchial epithelial cells (HBE) were obtained from American Type Culture Collection (ATCC, USA). All cells were cultured in RPMI 1640 medium (KeyGEN, Nanjing,

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China) while SPCA-1 and HBE cells were cultured in DMEM

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(KeyGEN, Nanjing, China) medium, which were supplemented with 10% fetal bovine serum (GIBCO-BRL , Invitrogen, Carlsbad ,CA ,USA) in humidified air at 37

with 5%CO2. A549 and H1299 cells were

transfected with small-interfering (SI) or negative control (NC) sequences

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using Lipofectamine RNAi MAX (Invitrogen, USA) according to the manufacture’s protocol. Transfection efficiency was evaluated by quantitative real-time RT-PCR and western blot. Two siRNAs were

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designed, whose sequences were as follows: SI1 for ZNF692: sense antisense

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5’-UCUGGUGCUCUUGUCUCAUTT-3’,

5’-AUGAGACAAGAGCACCAGATT-3’; SI2 for ZNF692 : sense 5’-CCUUGUCAGCAUCCUCAUUTT-3’,

antisense

5’-UGAGGAUGCUGACAAGGTT-3’8; And the following Nonsense siRNA

was

used

as

negative

control(NC):

sense

5′-

UUCUCCGAACGUGUCACGUTT-3′,

antisense

5

′

-ACGUGACACGUUCGGAGAATT -3 ′

. The human ZNF692

4

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targeting small hairpin RNA sequence was designed in accordance with SI2. Total RNA extraction and qRT-PCR analysis

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Total RNA was extracted from cultured cells, using TRIzol (Invitrogen, Carlsbad, CA, USA). For qRT-PCR, RNA was reverse transcribed to c DNA by using a Reverse Transcription Kit (Takara, cat: RRO36A),

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according to the manufacturers’ instructions. PCR analyses were

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performed with SYBR Select Master Mix (Applied Biosystems, Cat: 4472908). The PCR primers sequences are listed in Table1. The qRT-PCR data collection was performed using a QuantStudioTM 6 Flex Real-Time PCR System. The qRT-PCR reaction included an initial denaturation step

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at 95°C for 10 min, followed by 40 cycles of 92°C for 15 sec and 60°C for 1 min. Each sample was run in triplicate, and the relative expression was calculated and normalized using the 2-∆∆Ct method relative to β-actin.

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Cell proliferation assay

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Cell proliferation (cell index) was monitored using the real time xCELLigence analysis system (RTCA), following the research protocol afforded by the manufacturer (Roche Applied Science and ACEA Biosciences) [11]. The xCELLigence system consists of four main components: the RTCA analyzer, the RTCA DP station, the RTCA computer with integrated software and disposable E-plate 16. Each of the 16 wells on the E-Plate 16 contains an integral sensor electrode array. 5

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The transfected cells were placed in E-Plate 16 plate (10000 cells per well) and maintained in 150 ul medium containing 10% FBS. The E-Plate 16 plate was put on the RTCA device, and the signal of cell proliferation was

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recorded. EDU assay

A549 and H1299 cells were placed in 96-well plates (8000/well) after

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transfection with SI-ZNF692 or SI-NC. 6 hours later, EDU incorporation

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assay was performed according to the manufacture’s protocol (EdU, Ribobio, Guangzhou, China)[12]. Colony formation assay

For the colony formation assay, the transfected cells were placed in

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6-well plates (100 cells per well) and maintained in media which contains 10% FBS, replacing medium every five days. After 15 days, cells were stained with 0.1% crystal violet after fixed with 4% methanol for twenty

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minutes. Visible stained colonies were counted and each experiment

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was repeated three times.

Basic cell migration assay Transfected cells (40,000 cells) were placed in the upper chamber of trans-well insert (8 mm pores, Millipore, Billerica, MA) containing 200ul of serum free RPMI1640 medium. The bottom chambers were filled with 500ul RPMI1640 containing 10% FBS. After 24 hours of incubation, cells fixed with methanol on the filter membrane were stained with crystal 6

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violet, and then photographed. Migration was assessed by counting the number of stained cell nuclei from 5 random fields per filter in each group.

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Cell invasion assay For invasion assays, transfected cells (40,000 cells) were plated in the top chamber with a matrigel-coated membrane (BD bioscience) in 200ul

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serum-free RPMI 1640. The bottom chambers were filled with 800ul

incubation. Wound healing assay

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RPMI1640 containing 10% FBS. Invasion was determined after 48h

For wound healing assay, transfected cells were placed in 6-plated when

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cells grew to approximated full confluence, and the sterile wound gaps were created with a 200-µl pipette tip. The cells were maintained in serum-free RPMI 1640 medium. The spacing of gap was photographed at

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0 h and 24 h by microscope.

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Apoptosis assay

For apoptosis assay, transfected cells were washed and resuspended at a concentration of 1 × 106 cells/ml, and analyzed using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences). After incubation at room temperature in the dark for 20 min, the cells were analyzed by a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ) following manufacturer’s protocol. 7

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Xenograft study Twenty-two male nude mice (4-5 weeks old) were conducted in SPF Laboratory Animal Center at Nanjing Medical University and

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manipulated in accordance with NIH animals use guidelines and protocols approved by Nanjing Medical University Animal Care Committee. A549 cells were transfected with sh-ZNF692 or sh-NC using

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Lipofectamine 3000 (Invitrogen). After 36 hours, 1.0ⅹ106 cells were

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injected subcutaneously in the mice’s axilla. Tumor volume was estimated using calipers every week ((length*width^2)/2). In the fifth week after injection, animals were sacrificed. Tumor nodes were obtained and measured volume and weight to evaluate the proliferation of cells, and

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then immunohistochemically stained with Ki67 and ZNF692. Tissue sample and Immunohistochemistry Paired LUAD cancer tissues and adjacent non-tumor tissues were

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obtained from patients who underwent surgery of LUAD between July

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2004 and June 2008 at the Cancer Institute of Jiangsu Province. All paired tumor and non-tumor tissues were confirmed by two experienced pathologists. This study was approved by the Research Ethics Committee of Nanjing Medial University (Nanjing, Jiangsu, China). For IHC assay based on a tissues microarray, 182 formalin-fixed paraffin-embedded (FFPE) paired tissues samples from 91 patients were used (after excluding missing date, 83 tumor and pairs were included in 8

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further analysis). Tissue sections were deparaffnized and rehydrated using graded alcohol, endogenous peroxidase activity was blocked by incubation in 3% H2O2. A ZNF692 rabbit polyclonal antibody (NOVUS,

pathologist

scored

immune-staining

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NBP2-37973) was administrated for the positive cells, and two independently.

As

describe

before[13], the staining intensity was evaluated as the basis of 4 grades:

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0 (negative staining), 1 (weak staining), 2 (moderate staining), or 3

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(strong staining). The product (percentage of positive cells and respective intensity scores) was used as the final staining score (a minimum value of 0 and a maximum value of 300).

Protein preparation and western blot

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Cells were collected and treated with lysis buffer on ice (KeyGEN, Nanjing, China), and the concentration of total protein lysate was quantified by BCA protein assay kit (KeyGEN, Nanjing, China). Equal

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amounts of protein lysates were separated by 10% SDS-PAGE gels and

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transferred to a PVDF membrane. The membrane was incubated with the antibodies: ZNF692 (NOVUS, NBP2-37973 1:1000), β-actin (Cell Signaling, 8H10D10 1:1000). After washing in TBST, the membrane was incubated with goat anti-rabbit HRP-conjugated secondary antibody (1:10000; Abcam) or goat anti-mouse HRP-conjugated secondary antibody (1:10000; Abcam) for 2h at room temperature. The blots were visualized by ECL detection (Thermo Scientific). 9

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Data source and bioinformatics analysis A TCGA dataset named TCGA_LUAD_exp_HiSeqV2-2015-02-24 was downloaded

from

UCSC

cancer

browser

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(https://genome-cancer.ucsc.edu/). The dataset contains a list of 511 LUAD samples and 58 adjacent normal tissue samples. Expression of ZNF692 were obtained from the “genomicMatrix” file, and all values

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were normalized. Student’s t-test was used to evaluate ZNF692

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expression in tumor and para-tumor tissues. Chi-square test was used to analyze the association between clinical characteristics and ZNF692 expression. Kaplan-Meier analysis, log-rank test and Cox regression analysis were used to evaluate the prognostic value of ZNF692 in LUAD

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patients. A list of 1498 genes (details shown in Table S1) which have highest co-expression correlation (Pearson r value >0.3) with ZNF692 in the

TCGA

LUAD

dataset

was

submitted

to

WebGestalt

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(http://bioinfo.vanderbilt.edu/webgestalt/login.php) for Gene Ontology

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(GO) pathway enrichment analysis[14]. Statistical Analysis Data are presented as means ± S.D. Student’s t test, one-way ANOVA, Kaplan-Meier survival analysis, chip-square test and cox regression analysis were used to analyze the date (SPSS Statistics, version 20.0, Chicago, Ill). P<0.05 were considered statistically significant. The data graphs were made with GraphPad Prism 6.0 software. 10

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Results ZNF692 is overexpressed in lung adenocarcinoma tissues and cells. By analyzing the TCGA_LUAD_exp_HiSeqV2-2015-02-24 dataset, the

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mean expression values of ZNF692 are 8.941 ± 0.04295(N=511) in LUAD tissues and 7.349 ± 0.09618(N=58) in adjacent normal tissues (Figure

1A).

Immunohistochemistry

showed

that

ZNF692

was

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significantly elevated in lung adenocarcinoma tissues compared with

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para-carcinoma tissues (p<0.0001；Figure 1B and 1C). The expression profile of ZNF692 was detected in different LUAD cell lines. ZNF692 was hyper-expressed in A549 and H1299 cell lines as compared with normal human bronchial epithelial (HEB) cells (Figure 1D and 1E). To explore

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the biological function of ZNF692 in vitro, two different siRNAs of ZNF692 (SI1, SI2) were utilized to knockdown ZNF692. Both siRNA constructs were able to effectively decrease ZNF692 mRNA and protein

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levels (Figure 1F and 1G).

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Aberrant expression of ZNF692 correlates with LUDA cell proliferation, migration, invasion in vitro As shown in Figure 2A, the xCELLigence data revealed that knockdown of ZNF692 inhibited cell proliferation of A549 and H1299 cells compared with the control cells. Moreover, the EDU assay and the colony formation assay showed the similar results (Figure 2B and 2C). The results of wound healing assay and trans-well assay indicated that 11

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ZNF692 siRNA (SI-ZNF692) transfected cells had impaired migration abilities than those transfected with control siRNA-NC (SI-NC) (Figure 2D, Figure 3A). Knockdown of ZNF692 inhibited cell invasion

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capacities in A549 and H1299 cell lines, based on the matrigel invasion assay (Figure 3B). SI-ZNF692 treatment increased cell apoptosis compared with control group (Figure 3C). These date demonstrated that

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ZNF692 promotes LUAD cells proliferation, migration, invasion, and

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inhibits LUAD cell apoptosis.

Knockdown of ZNF692 suppresses tumor growth in vivo We used a nude mouse xenograft assay with A549 cells. As shown in Figure 3D, average volume of tumors were smaller in the sh-ZNF692

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treated group compared with the control group. IHC analysis showed that tumors derived from sh-ZNF692 transfected cells had weaker staining of Ki-67 than those in the sh-NC group (Figure 4A). These data suggested

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that ZNF692 knockdown could reduce tumor growth in vivo.

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Overexpression of ZNF692 predicts poor prognoses and could be regarded as an independent predictor for overall survival of lung adenocarcinoma cancer patients ZNF692 protein was increased in tumor tissues compared with their

relative normal tissues (in 63/66 tissues examined). High ZNF692 expression (as determined by a cut-off score of 114) was detected in 49 (72.1%) of the 68 lung cancer tissues, compared with only 19 (26.8%) of 12

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71 adjacent normal tissue samples[13]. A chi-square test indicated that the expression level of ZNF692 in LUAD was correlated with patients’ TNM stage (P=0.0002), lymph node metastasis (P= 0.0198) and T stage

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(P= 0.0415). There were no correlations between ZNF692 expression and other factors, such as gender (male, female), age (<60, ≥60) (Table 2). The ZNF692 staining score was also increased along with advanced TNM

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stage in the LUAD tissues (P<0.0001; Figure 4B, Figure 4C). .

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Multivariate analyses indicated that elevated ZNF692 was associated with poorer overall survival rate (HR: 8.800, 95%CI: 1.082-71.560, P=0.042) (P<0.0001; Figure 4D).

Bioinformatics analysis indicates ZNF692 is related with metabolic

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process

We picked out a list of 1499 genes which have highest correlation values with ZNF692 from TCGA LUAD dataset (details shown in Table S2), then

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used KEGG pathway and GO analysis (WebGestalt) on them. The two

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analyses yielded similar results. Most of the genes were enriched in metabolic and biological regulation process. Given our results that ZNF692 have promoted cancer cell proliferation, migration, and invasion, we sought to hypothesize that ZNF692 might influence cancer progression via promoting metabolic process (Figure 4E). DISCUSSION The

zinc-finger

protein

692(ZNF692) 13

was

phosphorylated

by

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AMP-activated protein kinase (AMPK) [15], then transcribed PEPCK as a novel transcription factor [8]. Several studies revealed that PEPCK expression level was elevated three folds in lung cancer samples over

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normal lungs [15, 16]. In addition, AMPK is a central regulator of cell growth, reprogramming metabolism and coordinating autophagy [17, 18]. Previous researches have indicated that AMPK played a critical role in

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maintain energetic balance to survive metabolic stress or conditions of

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nutrient withdrawal by reprogramming metabolism in cancer cells [19-21]. Our studies was to research whether ZNF692 could influence the lung adenocarcinoma progression.

In this study, we provided the first evidence that over-expression of

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ZNF692 occurred in lung adenocarcinoma and positively correlated with more aggressive clinic-pathological features at both mRNA and protein levels.

The

results

of

cell

phenotypes

assays

suggested

that

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knocking-down ZNF692 inhibited the proliferation, migration and

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invasion capacities of LUAD cells. Moreover, ZNF692 knockdown reduced tumor growth in vivo. The prognostic value of ZNF692 had been also evaluated. Kaplan-Meier analysis revealed that LUAD patients with elevated ZNF692 had a worse prognosis, and multivariate Cox regression analysis suggested that hyper-expression of ZNF692 was an independent risk factor for shorter overall survival in LUAD patients. GO enrichment analysis showed that ZNF692 was correlated with metabolic process, 14

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which maybe a reason for the promotion of cancer progression. In conclusion, our study suggested that ZNF692 was overexpressed in LUAD tissues and relevant to clinic-pathological characteristics with a

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poorer prognosis. ZNF692 could facilitate LUAD cells proliferation, migration and invasion, as well as decelerate the apoptosis in vitro. These results revealed that ZNF692 plays an oncogenic role in LUAD, and it

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may serve as a potential oncogene and biomarker in LUAD by

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influencing cell metabolism. Ethics approval and consent to participate

The Ethics Boards of the Cancer Institute of Jiangsu Province approved this study. Informed written consent was obtained from all patients

Funding

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included in this study.

This research was supported by the Natural Science Foundation of China

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(81672869, 81372321), Jiangsu Provincial Special Program of Medical

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Science Funding (BL2012030), Jiangsu Provincial Science Foundation (BK20161596) , Jiangsu Provincial Medical Outstanding Talent (Lin Xu) and Jiangsu Provincial Medical Youth Talent (QNRC2016657, Binhui Ren).

Authors' contributions Lin Xu, Guangqing Zhang and Binhui Ren designed the study. Quanli zhang, Xiufen Zheng and Qi Sun performed all experiments. Binhui Ren 15

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and Guangqing Zhang provided the funds. Run Shi, Jie Wang and Biqing Zhu performed the statistical analysis, and Quanli zhang wrote the manuscript. All authors read and approved the final manuscript.

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Acknowledgements We thank Wenjia Xia for technical guidance. And we thank Xing Huang (Department of Pathology, Jiangsu Cancer Hospital) for consultation IHC

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results. References

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Figure legend

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Figure 1, ZNF692 is hyper-expressed in LUAD tissues and cell lines. (A) ZNF692 is hyper-expressed in LUAD tissues compared with adjacent

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normal tissues in the TCGA database (P<0.0001). (B and C) Overexpression of the ZNF692 in LUAD tissues were detected by

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Immunohistochemistry. (D and E) ZNF692 mRNA and protein are upregulated in A549 and H1299 cell lines. (F and D) Both of the designed siRNAs showed significantly inhibitory efficiency, and SI-ZNF692-2 had a better efficiency than SI-ZNF692-1 in A549 and h1299 cell lines. Figure 2, Knockdown of ZNF692 inhibits LUAD cells proliferation and motility in vitro. (A) Depletion of ZNF692 undermined both A549 and H1299 cell lines proliferation. (B and C) Colony numbers of A549 and 17

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H1299 cells transfected with SI-ZNF692 were less than those transfected with SI-NC. (D) The wound heal assay showed that the migration abilities of H1299 and A549 cells were decreased by knockdown of

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ZNF692. Figure 3, Knockdown of ZNF692 alters LUAD cells migration, invasion and apoptosis in vitro. (A and B) Migratory and invasion rates of A549 or

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H1299 cell transfected with SI-ZNF692 are decreased with SI-NC. (C)

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SI-ZNF692 increases cell apoptosis as compared with control group. (D). Xenograft tumors obtained from sh-ZNF692 -transfected A549 cells treated mice had smaller tumor volumes and weights than those injected with sh-NC cells. .

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Figure 4, ZNF692 associated with a poor prognosis and metabolic processes in LUAD. (A) Sh-ZNF692 tumors from A549 cell lines has less dense Ki67 staining. (B, C and D)Immunohistochemistry analysis

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revealed that ZNF692 staining score was associated with TNM stage, and

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univariate survival analysis revealed that ZNF692 expression was associated with poorer prognosis (p<0.0001).(E) GO enrichment analysis uncovered that ZNF692 expression was highly correlated with genes enriched in the metabolic process and biological regulation.

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ACCEPTED MANUSCRIPT Table 1 Sequences of primers. Gene Sense Anti-sense ZNF692 TTCCGCACTAGCAGCAACC AAACCCGCATATCTCACACTG ACTB GAAATCGTGCGTGACATTAA AAGGAAGGCTGGAAGAGTG

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Table 2 Correlation between ZNF692 protein expression and clinicopathologic characteristic Groups ZNF692 High expression Low expression Gender Male 10 5 Female 38 13 Age <60 20 8 ≥60 28 10 Differentiation I-II 27 11 III-IV 21 7 T stage T1-T2 34 17 T3-T4 14 1 Lymph node metastasis N0 26 15 N1-N3 24 3 TNM stage I-II 24 18 III-IV 24 0 *Signifcant correlation

Pearson χ2

P-value

0.36

0.5488

0.04

0.8389

0.13

0.7219

4.16

0.0415*

5.43

0.0198*

14.14

0.0002*

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Table 3 Analysis of independent correlation factors of lung cancer prognosis with Cox multivariate analysis SE Wald DF P-value HR 95%CI Lower Upper Age 0.408 0.633 1.000 0.426 0.723 0.325 1.608 Gender 0.438 0.290 1.000 0.590 0.790 0.335 1.864 Differentiation (III0.432 0.725 1.000 0.395 0.692 0.297 1.615 IV vs I-II) T stage (T1-T2 vs T3-T4) 0.401 2.584 1.000 0.108 1.905 0.868 4.181 N stage (N0 vs N1-N3) 0.480 1.224 1.000 0.269 1.700 0.664 4.355 TNM stage (III-IV vs 0.464 8.312 1.000 0.004* 3.816 1.535 9.484 I-II) ZNF692 expression 1.069 4.137 1.000 0.042* 8.800 1.082 71.560 (high vs low) SE: standard error; DF: degree of freedom; HR: hazard ratio; CI: confdence interval; Lower: lower limit; Upper: upper limit. *Signifcant correlation

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