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Differentially expressed gene profiles in the serum before and after the ultrasound-guided ethanol sclerotherapy in patients with ovarian endometriomas
Differentially expressed gene profiles in the serum before and after the ultrasound-guided ethanol sclerotherapy in patients with ovarian endometriomas Lu-Lu Wang, Huai-Qiu Cai, Xiao-Qiu Dong, Li-Wei Zhang, Shan-Shan Jiang, Ning Zhao, Xiao-Hui Shao, Si-Ming Wang, Li Zhu, Tong Zhang PII: DOI: Reference:
S0009-9120(15)00216-7 doi: 10.1016/j.clinbiochem.2015.06.003 CLB 9045
To appear in:
Clinical Biochemistry
Received date: Revised date: Accepted date:
4 April 2015 31 May 2015 1 June 2015
Please cite this article as: Wang Lu-Lu, Cai Huai-Qiu, Dong Xiao-Qiu, Zhang Li-Wei, Jiang Shan-Shan, Zhao Ning, Shao Xiao-Hui, Wang Si-Ming, Zhu Li, Zhang Tong, Differentially expressed gene profiles in the serum before and after the ultrasoundguided ethanol sclerotherapy in patients with ovarian endometriomas, Clinical Biochemistry (2015), doi: 10.1016/j.clinbiochem.2015.06.003
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ACCEPTED MANUSCRIPT Differentially expressed gene profiles in the serum before and after the ultrasound-guided ethanol sclerotherapy in patients
Lu-Lu Wang
a 1
, Huai-Qiu Cai
a 1
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with ovarian endometriomas
, Xiao-Qiu Dong a*, Li-Wei Zhang a,
Shan-Shan Jiang a, Ning Zhao a, Xiao-Hui Shao a, Si-Ming Wang a, Li Zhu , Tong Zhang c
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Department of Ultrasonography, the Fourth Hospital of Harbin Medical
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a
Both authors are contributed equally to this paper.
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1
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b
b
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University, Harbin, P. R. China
Department of Obstetrics and Gynecology, the Fourth Hospital of Harbin
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Medical University, Harbin, P. R. China c
KangChen Bio-technology Corp., Shanghai, P. R..China
*
Corresponding author. Yiyuan Str 37, Harbin, Heilongjiang Province 150001,
P. R. China. Tel.: +86 451 82576706; fax: +86 451 82576705. E-mail address: [email protected]
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ACCEPTED MANUSCRIPT ABSTRACT Objectives: To detect the differentially expressed genes and subsequently
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identify disease-related signatures and potential biomarkers for patients with
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ovarian endometriomas in the serum before and after the ultrasound-guided ethanol sclerotherapy in patients with ovarian endometriomas. Design and methods: Venous blood samples were collected from nine
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patients with ovarian endometriomas before and after ultrasound-guided
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ethanol sclerotherapy, and the serum were isolated after centrifugation. NimbleGen human gene expression microarrays analysis was conducted to
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analyse gene ontology categories (GO terms) and signalling pathways of
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differentially expressed genes. The accuracy of some typical genes from
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microarray analysis was verified by quantitative PCR (qPCR). Results: Approximately 45,033 genes were analysed by NimbleGen human
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gene expression microarrays, which identified 447 genes that showed differential expressions before and after therapy. Of these, 225 genes were up-regulated and 222 genes were down-regulated. The GO terms of the down-regulated genes were strongly associated with the pathogenesis of ovarian endometriomas; 15 down-regulated genes showed overlaps in both signalling pathways and GO terms. Among these, six genes showed statistical significance including IL6, CD36, JUNB, B4GALT1, HES1, and NR4A1, which were also validated by qPCR analysis. Conclusions: There were differentially expressed genes in the serum before 2
ACCEPTED MANUSCRIPT and after ultrasound-guided ethanol sclerotherapy in patients with ovarian endometriomas. Notably, the expressions of IL6, CD36, JUNB, B4GALT1,
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HES1, and NR4A1, which are strongly associated with the pathogenesis of
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ovarian endometriomas, were significantly down-regulated after ethanol sclerotherapy. This may not only help us understand EMs pathogenesis, but also provide potential biomarkers for verifying the effects of ethanol
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sclerotherapy.
Keywords: ovarian endometriosis; ultrasound-guided; ethanol sclerotherapy;
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differentially expressed genes; DNA microarray
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ACCEPTED MANUSCRIPT Introduction Ovarian endometriosis, which is as the most common type of endometriosis
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(EMs), is a refractory gynaecological disease [1]. Although the pathogenesis
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mechanism of EMs is not clear, studies have shown that the generation and development of EMs involves processes of adhesion, aggression and angiogenesis which is called the "3 A Process" [2]. And these processes are
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associated with many genes which are up-regulated in the patients with EMs
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[3-5], including interleukin-6 (IL-6), IL-8, TNF-α, monocyte chemoattractant protein-1 (MCP-1), macrophage migration inhibitory factor (MIF) and
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vascular endothelial growth factor(VEGF) in the peritoneal fluid [6-9], MIF,
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IL-6 and IL-8 in serum [10,11] and IL-6 in stromal cells derived from
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endometriotic tissues [12]. Recently studies showed that the serum IL-6 level was reduced in patient with EMs after drug therapy or laparoscopic surgery
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[13,14]. However, EMs-related gene expression and its signatures in serum are still unclear before and after the ultrasound-guided ethanol sclerotherapy in patients with ovarian endometriomas. Ultrasound-guided
ethanol
sclerotherapy,
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minimally
invasive
technology, has been increasingly recognized by clinicians as a reliable surgical alternative for treating ovarian endometriomas with many advantages, including minimal trauma, fewer risks and complication, lower costs and decreased recurrence rates in recent years [15,16]. Under ultrasound guidance, there are three key procedures of the therapy which consist of the cystic fluid 4
ACCEPTED MANUSCRIPT aspiration, saline washings and 95% ethanol retention. Initially, the steps of cystic fluid aspiration and saline washings are to eliminate the cystic fluid
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which could stimulate ectopic cell proliferation. Secondly, ethanol (95%), as a
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sclerosing agent, is retained in the cystic cavity to inactivate the cyst wall cells and to sclerose capillaries [17]. With the wide clinical applications of the technology, reports on its methodology and efficacy have gradually increased
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over recent years [18]. Althrough most of criteria for efficacy were based on
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using ultrasound to detect lesion characteristics or the changes in ovarian function and hormone levels [19,20]. However, the role of EMs-related gene
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in serum remains undefined. Therefore, this study aimed to identify
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EMs-related gene expression profile in the serum and its association with the
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pathogenesis of ovarian endometriomas before and after ultrasound-guided
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ethanol sclerotherapy for patients with ovarian endometriomas.
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Materials and methods
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Subjects
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Among the patients with ovarian endometriomas who underwent ultrasound-guided ethanol sclerotherapy at our hospital from December 2011 to December 2013, nine subjects were selected based on the following criteria:
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1) Unilateral solitary ovarian endometriosis without any treatments. 2) Size of
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ovarian endometriosis: 4.0 cm ≤ diameter ≤ 6.0 cm. 3) No adenomyosis and other gynecological diseases. 4) Normal serum CA125 (0–35 mIU/mL). 5)
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Lesions disappeared after three months. 6) No alcoholic allergies, normal
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results in routine blood and urine tests, and normal lung, liver and kidney
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functions. The age of patients ranged from 27 to 49 years (mean: 36.5 ± 1.5 years), and all patients were married. Five subjects had ovarian
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endometriomas in their left ovaries and four subjects had them on their right ovaries. All patients were verbally informed of the treatment process and the risks involved, and signed written informed consents. The Ethics Committee of our hospital (The Fourth Hospital of Harbin Medical University, Harbin, P. R. China) approved the study design of the present investigation.
Equipment and materials Equipment and materials used in the ethanol sclerotherapy The following equipment and materials were used: a Mylab 90 Color 6
ACCEPTED MANUSCRIPT Doppler ultrasonography unit (Esaote, Italy), transvaginal probe (Model: EC123 9-5, frequency 7-12 MHz), percutaneous transhepatic cholangiography
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(PTC) needle (16/18 G × 20 cm, Origin: Nagano, Japan), local anaesthetic:
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2% lidocaine (5 mL: 0.1 g, Origin: Shanghai, China), washing solution: 0.9% saline (250 mL: 2.25 g, Origin: Tonghua, China), and hardener: 95% medical
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alcohol (Origin: Dezhou, China).
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Ultrasound-guided ethanol sclerotherapy
Transvaginal ultrasonography was performed before treatment. The location,
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size, and sonographic features of the ovarian endometriosis were recorded,
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and the images were captured. The malignancy was excluded based on the
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clinical diagnosis and laboratory tests. After a routine gynecological disinfection, the cyst was punctured under ultrasound guidance using a PTC
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needle as described previously [15,18,21]. The cystic fluid was aspirated and the cyst was repeatedly washed with saline. When the cystic fluid was clear, 95% ethanol was used for washing and 20-50 mL of the ethanol was retained. After the patient returned to a normal state, the needle was removed. Ultrasound examination was performed three months after the treatment.
Equipment and materials for microarray analysis and PCR verification The Human 12135K Gene Expression Array was manufactured by Roche NimbleGen (Madison, WI, USA). Double stranded (ds)-cDNA was cleaned 7
ACCEPTED MANUSCRIPT and labelled in accordance to the NimbleGen Gene Expression Analysis protocol (NimbleGen Systems, Inc., Madison, WI, USA). PCR primers were
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synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). Reverse
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transcription of purified RNA was performed using SuperScript III Reverse Transcriptase (2,000U, Invitrogen, USA). Real-time qPCR was carried out on
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a Bio-Rad Real-time PCR Detector (Bio-Rad Chromo4TM, USA).
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Samples for microarray analysis and PCR verification Collection and processing of blood samples: Two tubes of 5 mL venous
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blood from the patients were collected before treatment and three months after
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treatment. After blood collection, the blood samples were immediately
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centrifuged (temperature: room temperature, speed: 1000 r/min) and the serum was divided into two tubes as Tube 1 and Tube 2, which were immediately
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stored at -80°C until an analysis. The 2 mL serum in Tube 1 was transferred to an RNAse-free cryovial for NimbleGen human gene expression microarrays analysis. The 2 mL serum in Tube 2 was transferred to an RNAse-free Eppendorf (EP) tube for PCR verification.
NimbleGen human gene expression microarrays analysis RNA quantity and quality were measured by using a NanoDrop ND-1000. RNA integrity was assessed by standard denaturing agarose gel electrophoresis. The microarray experiments were performed by KangChen Bio-technology 8
ACCEPTED MANUSCRIPT Corp. (Shanghai, China) according to the standard protocol. The Human 12 135K Gene Expression Array was manufactured by Roche NimbleGen. Briefly,
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total RNA of each sample was used for labelling and array hybridization using
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the following steps: 1) Reverse transcription using Invitrogen SuperScript ds-cDNA synthesis kit. 2) ds-cDNA labelling with NimbleGen one-color DNA labelling kit. 3) Array hybridization using the NimbleGen Hybridization System,
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followed by washing with the NimbleGen wash buffer kit, and 4) Array
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scanning using the Axon GenePix 4000B microarray scanner (Molecular Devices Corporation). Scanned images (TIFF format) were then imported into
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the NimbleScan software (Version 2.5) for grid alignment and expression data
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analysis. Expression data were normalized using quantile normalization and the
software.
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Robust Multichip Average (RMA) algorithm included in the NimbleScan
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Scanned images (TIFF format) were then imported into NimbleScan software (Version 2.5) for grid alignment and expression data analysis. Expression data were normalized through quantile normalization and the Robust Multichip Average (RMA) algorithm included in the NimbleScan software. All gene level files were imported into Agilent GeneSpring GX software (Version 11.5.1) for further analysis. Differentially expressed genes with statistical significance between the two groups were identified through Volcano Plot filtering. Differentially expressed genes were identified through Fold Change filtering. Pathway Analysis and GO analysis were applied to determine the roles of these 9
ACCEPTED MANUSCRIPT differentially expressed genes played in these biological pathways or GO terms. Finally, Hierarchical Clustering was performed to show distinguishable gene
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expression profiling among samples (Microarray data are available at
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www.ncbi.nlm.nih.gov/geo/, accession numbers GSE69310).
Quantitative real-time PCR (qPCR)
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Total RNA from human serum was extracted using a RNA extraction kit.
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Reverse transcription of purified RNA was performed using SuperScript III reverse transcriptase following the manufacturer’s instructions (Invitrogen). 2
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× SuperArray PCR master mix, to verify the accuracy of the microarray data,
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six differentially expressed genes were chosen for mRNA quantification of
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expression levels by real-time PCR. Real-time qPCR was carried out in triplicate on a Bio-Rad Chromo4TM Real-time PCR Detector (Bio-Rad). The
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PCR parameters are described in Table 1. GAPDH was used as an internal control. Data analysis was performed using the MJ Opticon Monitor Analysis Software (Bio-Rad), and fold change of mRNA expression was calculated using the △△Ct method. The reaction without first-strand cDNA was used as negative control.
Statistical analysis Data are presented as mean ± standard deviation. The Student’s t-test was used each time with the SPSS 19.0 software package, and the P-value was 10
ACCEPTED MANUSCRIPT calculated. A difference was determined to be statistically significant when the P-value was < 0.05. The gene concentration of each sample is generated
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directly by the Rotor-Gene Real-Time Analysis Software 6.0.
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NimbleGen human gene expression microarrays analysis
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Results
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Among the 45,033 genes identified from the gene expression profile microarray analysis, 447 differentially expressed genes were screened out (P < 0.05). Among these, 225 genes were up-regulated, and 222 genes were
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down-regulated. A "volcano plot" was constructed by plotting the
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differentially expressed genes between the P values and fold changes (Fig. 1).
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expressed genes
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The main GO terms and signalling pathways of up-regulated differentially
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There were 225 up-regulated genes cover 169 GO terms. Among these, six GO terms showed high enrichment scores (ES) and high frequencies of
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occurrence. The GO terms with the highest ES was the respiratory electron transport chain, which had the highest level of statistical significance (P = 0.00005, Fig. 2). Up-regulated genes exerted their effects through eight signalling pathways. The higher ES the pathway had, the more statistically significant the pathway was. The pathway of folate biosynthesis showed the highest ES, with the highest level of statistical significance (P = 0.0008, Fig. 3).
The main GO terms and signalling pathways of down-regulated differentially 12
ACCEPTED MANUSCRIPT expressed genes There were 222 down-regulated genes cover 480 GO terms, of which 11
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were strongly associated with ovarian endometriomas, including vasculature
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development, inflammatory response, chemotaxis, cell adhesion and epithelium development. Among these 11 GO terms, vasculature development showed the smallest P value, the smallest false discovery rate (FDR), and the
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highest ES, indicating the highest level of statistical significance (P = 0.0002,
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Table 2 and Fig. 4). Down-regulated genes exerted their effects through 13 signalling pathways that were statistically significant (P < 0.05). The
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mitogen-activated protein kinase (MAPK) pathway showed the smallest
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absolute FDR (It is similar to its P value, thus reflecting an error rate), the
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smallest P value, and the highest ES, suggesting the highest level of statistical
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significance (P = 0.0004) (Table 3 and Fig. 5).
Selection of typical genes with significantly down-regulated differentially expressed genes and their GO terms Among the down-regulated differentially expressed genes, 15 ovarian endometriosis- associated genes showed overlaps in both signalling pathways and GO terms (Fold change ≥ 2). Among these, IL6, CD36, JUNB, B4GALT1, HES1, and NR4A1 were closely related to ovarian endometriomas. The most statistically significant gene was IL-6 (P = 0.0006). Each gene exerted their GO terms through one or more signalling pathways (Table 4). 13
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Identification of typical differentially expressed genes by qPCR
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PCR analysis was performed on six genes. The product lengths were
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between 95 and 282 bp, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as control. The forward and reverse primer sequences of the six genes are listed in Table 1. The result showed fold-change relationships when
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the six genes were compared with the control genes, and their post-treatment
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expressions were down-regulated. Their fold changes in the microarray analysis ranged from -2.17 to -3.46. The fold changes of qPCR were between
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-1.05 and -4.03, consistent with the negative values from the microarray
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analysis. This verified the consistency of the down-regulated differentially
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expressed genes. In addition, the fold changes of PCR were all ≥ 1, quantitatively confirming that the six down-regulated genes were statistically
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significant (Fig.6).
Discussion
In the present study, a potential EMs-related gene expression profile in the serum from the patients with ovarian endometriomas was identified before and after ultrasound-guided ethanol sclerotherapy. More importantly, the expressions of IL6, CD36, JUNB, B4GALT1, HES1, and NR4A1, which are strongly associated with the pathogenesis of ovarian endometriomas, were significantly down-regulated after ethanol sclerotherapy. 14
ACCEPTED MANUSCRIPT Ultrasound-guided ethanol sclerotherapy is more effective way to treat ovarian endometriomas than drug therapy or laparoscopic surgery [15]. Through
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ultrasound-guided ethanol sclerotherapy, the cystic fluid containing a large
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number of macrophages and transforming growth factor is almost completely removed. The cells of cyst wall are inactivated and losed secretory function and the vascular are sclerosed and occlused by 95% ethanol. Then cyst wall become
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solidification, hardening, adhesion and closure. In the procress, various genes
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expression have been changed.
From the gene expression profile microarray analysis, 447 differentially
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expressed genes were detected. Among these, 225 differentially expressed genes
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were up-regulated and 222 genes were down-regulated. These differentially
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expressed genes cover various GO terms through multiple signalling pathways. Among 169 GO terms exerted by the 225 up-regulated genes, there were six
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with higher ES (Higher ES indicates that the corresponding GO term and signalling pathway are more statistically significant.) and higher frequencies of occurrence. The GO term “respiratory electron transport chain” had the highest ES (ES = 4.30). There were eight signalling pathways that showed statistical significance, in which up-regulated genes were involved. The pathway of folate biosynthesis was the most statistically significant (ES = 3.12). However, the GO terms exerted by these up-regulated genes such as the electron transport chain and cell respiration were not strongly associated with the occurrence and development of ovarian endometriomas. 15
ACCEPTED MANUSCRIPT Among the 480 GO terms exerted by 222 down-regulated genes, 11 functions were determined to be strongly associated with ovarian
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endometriomas, including vasculature development, immune responses to
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inflammation, chemotaxis, cell adhesion, and epithelium development. Among these, vasculature development had the smallest FDR (FDR = 0.0142, FDR is the absolute fold change of error rate, which has the same meaning as
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the P value. A smaller FDR means a higher statistical significance.). On the
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other hand, the highest ES (ES = 3.73) was found in vasculature development, indicating the GO term of vasculature development had the highest level of
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statistical significance. There were 13 signalling pathways with statistical
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significance, in which down-regulated genes were involved. Among these, the
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MAPK pathway was the most statistically significant (FDR = 0.0920, ES = 3.44). Among the down-regulated genes involved in 11 GO terms, 15
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down-regulated genes not only showed GO terms that were strongly associated with the pathogenesis of ovarian endometriomas, but also exerted their functions through 13 signalling pathways. These overlapped both in terms of GO terms and signalling pathways. Among these, six genes were excluded because they had higher frequencies of occurrence in GO terms and signalling pathways, and their sites were close to the front of the signalling pathways. These six genes were IL6, CD36, JUNB, B4GALT1, HES1, and NR4A1. Each gene was involved in one or more GO terms, and played a role through one or more signalling pathways. 16
ACCEPTED MANUSCRIPT IL-6 was the gene with the highest level of statistical significance among the six genes. It has been widely studied in the research of the pathogenic ovarian
endometriomas
[22].
As
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of
endometrium
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mechanism
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immunoregulatory gene, IL-6 promotes the metastasis, localization, and invasive growth of ectopic endometrium. Some studies have shown that the IL-6 level secreted by the ectopic endometrial stromal cells of the patients
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with EMs was six-fold higher than that secreted by the normal endometrium
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[23,24]. In this study, the ovarian endometriosis-associated GO terms, in which IL-6 was involved, included inflammatory response, chemotaxis,
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epithelium development, and vasculature development. The five other genes
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identified in this study were rarely reported genes in the field of ovarian
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endometriosis research. CD36 is commonly reported in the studies of atherosclerosis and tumour diseases, with the biological effects of
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inflammation, angiogenesis, tumour, and lipid metabolism [25-27]. JUNB is often reported in liver cancer [28] and has been extensively studied in cancer research; it also plays an important role in tumour neo-vascularisation, angiogenesis, tumour formation, metastasis, and invasion [29,30]. B4GALT1 is among the glycosyltransferases that have been extensively studied [31]. It imparts an adhesion molecule-like effect. HES1, which is involved in cell differentiation [32], is reported in the researches on promoting neural stem cell proliferation [33]. NR4A1 is an immediate early gene product found in pheochromocytoma cells and fibroblasts [34], and functions in proliferation 17
ACCEPTED MANUSCRIPT and differentiation [35]. However, in this study, CD36 and JUNB were involved in cell adhesion and vasculature development, respectively. They
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have also been associated with angiogenesis in ovarian endometriomas and
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tumour-like growth. Both B4GALT1 and HES1 are involved in epithelium growth, cell adhesion, and vasculature development. NR4A1 plays a role in chemotaxis and angiogenesis. These results suggest that these gene
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expressions in the serum from the patients with ovarian endometriomas may
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be related to adhesion, differentiation, chemotaxis, and vascularization in the growth process of ovarian endometriomas. After alcohol sclerotherapy, the
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expressions of these genes were down-regulated, and their corresponding GO
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terms were also inhibited, supporting an identification of potential
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EMs-related gene network. Although we do not provide direct evidence for these gene functions in disease development and progression of EMs, the
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expression levels of these genes in the serum from the patients may be useful as a biomarker for predicting the prognosis in disease recurrence, progression and therapeutic efficacy.
Conclusions In the present study, we identified differentially expressed gene profiles before and after the ethanol sclerotherapy in the serum from the patients with ovarian endometriomas. Notably, the expressions of IL6, CD36, JUNB, B4GALT1, HES1, and NR4A1, which are strongly associated with the 18
ACCEPTED MANUSCRIPT pathogenesis of ovarian endometriomas, were significantly down-regulated after ethanol sclerotherapy. This identification may help us in understanding
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EMs pathogenesis and provide potential biomarkers for clinical application.
Acknowledgements
The sponsors of this study were the National Natural Science Foundation of
Innovation
Research
Program
Fund
of
China
(grant
no.
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Bureau
NU
China (grant no. 81271646 to X. Q. Dong), Harbin Science and Technology
2012RFXXS057) and Heilongjiang Key Technologies R&D Program funds of
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[27] Collot-Teixeira S, Martin J, McDermott-Roe C, Poston R, McGregor JL. CD36 and macrophages in atherosclerosis. Cardiovasc Res 2007; 75: 468-77. [28] Nausch N, Florin L, Hartenstein B, Angel P, Schorpp-Kistner M, Cerwenka A. Cutting edge: the AP-1 subunit JunB determines NK cell-mediated target cell killing by regulation of the NKG2D-ligand RAE-1epsilon. J Immunol 2006; 176:7-11. 23
ACCEPTED MANUSCRIPT [29] Schmidt D, Textor B, Pein OT, Licht AH, Andrecht S, Sator-Schmitt M, et al. Critical role for NF-kappaB-induced JunB in VEGF regulation and
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tumor angiogenesis. EMBO J 2007; 26:710-9.
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[30] Wang L, Zhang R, Meng YL, Yu GR, Wang T, Zhao J, et al. Expression and functional analysis of transcriptional factor JunB in hepatic cancer cells. Chin J CellMol Immunol 2010; 26: 5-8.
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[31] Hathaway HJ. Cell surface beta1,4-galactosyltransferase function in
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mammary gland morphogenesis: insights from transgenic and knockout mouse models. J Mammary Gland Biol Neoplasia 2003; 8: 421-33.
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[32] Murata K, Hattori M, Hirai N, Shinozuka Y, Hirata H, Kageyama R, et al.
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Hes1 directly controls cell proliferation through the transcriptional
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repression of p27Kip1. Mol Cell Biol 2005; 25: 4262-71. [33] Yamamoto S, Nagao M, Sugimori M, Kosako H, Nakatomi H, Yamamoto
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N, et al. Transcription factor expression and Notch-dependent regulation of neural progenitors in the adult rat spinal cord. J Neurosci 2001; 21: 9814-23.
[34] Hazel TG, Nathans D, Lau LF. A gene inducible by serum growth factors encodes a member of the steroid and thyroid hormone receptor superfamily. Proc Natl Acad Sci 1988; 85: 8444-8. [35] Yu L, Su YS, Zhao J, Wang H, Li W. Repression of NR4A1 by a chromatin modifier promotes docetaxel resistance in PC-3 human prostate cancer cells. FEBS Lett 2013; 587: 2542-51. 24
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Figure Legends
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Fig.1. “Volcano plot” of differentially expressed genes
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Volcano Plots are useful tools for visualizing differential expression between two different conditions (fold-change values and P-values). Fold change stands for Multiple of postoperative vs preoperative. The upper-left
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region bounded by horizontal line y = a and vertical line x = -1 represents
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down-regulated genes; the up-right region bounded by horizontal line y = a
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and vertical line x = 1 represented up-regulated genes.
Terms
stands
for
gene
ontology
of
up-regulated
genes,
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GO
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Fig.2. Main GO terms exerted by up-regulated genes
Enrichment_Score stands for the Enrichment Score value of the GO terms of
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up-regulated genes. The vertical axis represents the full names of six GO terms; the horizontal axis represents the ES of the GO terms.
Fig.3. Signaling pathways in which the up-regulated genes were involved Signaling pathways stands for the name of the signalling pathways of up-regulated genes, Enrichment_Score stands for the Enrichment Score value of the signalling pathways of up-regulated genes. The vertical axis represents the full names of eight signalling pathways; the horizontal axis represents the ES of the signalling pathways. 25
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Fig.4. Ovarian endometriosis-associated GO terms exerted by down-regulated
stands
for gene
ontology of
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terms
down-regulated
genes,
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GO
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genes
Enrichment_Score stands for the Enrichment Score value of the GO terms of down-regulated genes, it equals "-log10 (P-value)". The vertical axis
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represents the full names of 11 GO terms; the horizontal axis represents the
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ES of the GO terms.
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Fig.5. Signalling pathways in which the down-regulated genes were involved
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Signalling pathways stands for the name of the signalling pathways of
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down-regulated genes, Enrichment_Score stands for the Enrichment Score value of the signalling pathways of down-regulated genes, it equals "-log10
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(P-value)". The vertical axis represents the full names of 13 signalling pathways; the horizontal axis represents the ES of the signalling pathways.
Fig.6. Comparison of fold changes in differentially expressed genes between microarray and qPCR The horizontal axis represents genes; the vertical axis represents fold changes; the dark column represents microarray analysis; the light-coloured column represents qPCR.
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Figure 6
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B4GALT1 HES1 NR4A1 GAPDH
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Product size (bp)
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JUNB
Annealing Tm (℃) 60
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CD36
F:5’TGACAAACAAATTCGGTACATCC3’ R:5’GTGCCTCTTTGCTGCTTTCA3’ F:5’ TGGTTCCGTACCCTGTTACTACC3’ R:5’ CGTCGGATTCAAATACAGCATAG3’ F:5’ GGAACAGCCCTTCTACCACGA3’ R:5’ GGCTCGGTTTCAGGAGTTTG3’ F:5’ATGTTTGCCTGGTCCGTGAG3’ R:5’GCCAGCACAGCATCTCCTTTA3’ F:5’GCCAGTTTGCTTTCCTCATTC3’ R:5’ TTCTCTCCCAGTATTCAAGTTCCT3’ F:5’GCAAGTGGGCGGAGAAGAT3’ R:5’CCTCGCCTGGCTTAGACCT3’ F:5’GGGAAACTGTGGCGTGAT3’ R:5’GAGTGGGTGTCGCTGTTGA3’
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IL6
Primer sequences
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Gene
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Table 1. Primer sequences and PCR parameters for different selected genes.
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282
60
101
60
235
60
253
60
126
60
299
"Gene" stands for the name of genes; “Primer sequences” stands for the base sequence of primers; "Annealing Tm" stands for annealing temperature, the unit is ℃; "Product size" stands for the length of the fragment after the primer for amplification; F: Forward primer, R: Reversed primer.
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Terms vasculature development
GO:0031349
0.0174
GO:0070741 GO:0060429
positive regulation of 0.0003 defense response response to interleukin-6 0.0091 epithelium development 0.0126
GO:1900046 GO:0006935
regulation of hemostasis chemotaxis
0.0160 0.0230
0.2084 0.2560
GO:0007155
cell adhesion
0.0246
GO:0006954
inflammatory response
0.0335
0.3338
GO:0050727
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FDR 0.0142
regulation of 0.0398 inflammatory response platelet activation 0.0399 epidermal growth factor 0.0493 receptor signaling pathway
0.3748
IL6, FOXA2 NKX2-5, B4GALT1, DVL1, ERRFI1, FOXA2, IL6, SPDEF, SOCS3, HES1, ETV5 THBD, FOXA2, CD36 IL6, RHOB, CACNB1, DVL1, NR4A1, HOXB9, CXCL2, CYR61, FOSL1, CMTM1 CD36, TNFRSF12A, BCAM, CYR61, TYRO3, KIFC3, FOXA2, RHOB, B4GALT1, HES1, NEDD9, PTPRS, LY6D, NPNT B4GALT1, IL6, OSM, TYRO3, IER3, FOS, CXCL2, IRAK2 IL6, OSM, TYRO3, IER3
0.3748 0.4416
THBD, TYRO3, RHOB, CD36, IL6 ERRFI1, NR4A1, TRIB3, RICTOR
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0.1545 0.1834
Genes VHL, SH2D2A, TNFRSF12A, NKX2-5, JUNB, CYR61, NR4A1, IL6, RHOB, HES1, B4GALT1, ERRFI1, SOCS3 CD36, DUSP4, ELK1, FOS, IRAK2, IL6, OSM, TYRO3
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GO.ID GO:0001944
GO:0030168 GO:0007173
0.2697
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P-value 0.0002
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Table 2. The GO terms associated with ovarian endometriomas of down-regulated genes.
"GO" stands for gene ontology, "GO.ID" stands for the ID of gene ontology term; "Terms" stands for the name of gene ontology term; "Pvalue" stands for the significance testing value of the GOID, P-value cut-off: 0.05; "FDR" stands for the false discovery rate of the GOID, using Benjamini & Hochberg (1995) method; "Genes" stands for the DE genes associated with the GOID. 34
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hsa04115 hsa05164 hsa04380
MAPK signaling pathway
0.0004
0.0920
Glycosphingolipid biosynthesis-lacto and neolacto series HTLV-I infection
0.0023
0.2718
CACNB1, DDIT3, DUSP2, DUSP4, DUSP5, ELK1, FOS, GADD45A, HSPA1A, NR4A1 B4GALT1, FUT1, FUT3
0.0053
0.2718
Adipocytokine signaling pathway Prion diseases Herpes simplex infection Legionellosis Dorso-ventral axis formation Maturity onset diabetes of the young Protein processing in endoplasmic reticulum p53 signaling pathway Influenza A Osteoclast differentiation
0.0054 0.0054 0.0107 0.0188 0.0250 0.0270 0.0277 0.0326 0.0330 0.0425
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Genes
0.2718 0.2718 0.4493 0.6727 0.6900 0.6900 0.6900
DVL1, ELK1, ETS2, FOS, FOSL1, HLA-C, IL6, MYBL1 CD36, PPARA, PRKAB1, SOCS3 ELK1, HSPA1A, IL6 FOS, GTF2IRD1, HLA-C, IL6, PER1, SOCS3 CXCL2, HSPA1A, IL6 CPEB1, ETS2 FOXA2, HES1 DDIT3, DNAJB1, ERN1, HSPA1A, PPP1R15A
0.6900 0.6900 0.8200
GADD45A, PMAIP1, TNFRSF10B DNAJB1, HSPA1A, IL6, SOCS3, TNFRSF10B FOS, FOSL1, JUNB, SOCS3
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hsa04920 hsa05020 hsa05168 hsa05134 hsa04320 hsa04950 hsa04141
FDR
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hsa05166
P-value
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hsa00601
Definition
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Pathway ID hsa04010
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Table 3. The 13 significant signalling pathways in which the down-regulated genes were involved.
"Pathway ID" stands for Pathway identifiers used in KEGG; "Definition" stands for the definition of the Pathway ID; P-value cut-off: 0.05; "FDR" stands for Absolute Fold Change, the absolute ratio (no log scale) of normalized intensities between two conditions; "Genes" stands for the DE genes associated with the Pathway ID.
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Gene_ name IL6
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Table 4. The down-regulated genes that had overlaps in both signalling pathways and GO terms.
Description
Signalling pathways
GO terms
0.0006
interleukin 6 (interferon, beta 2)
CD36
0.0352
JUNB B4GAL T1
0.0291 0.0164
HES1
0.0217
NR4A1
0.0024
CD36 molecule (thrombospondin receptor) jun B proto-oncogene UDP-Gal: betaGlcNAc beta 1,4galactosyltransferase, polypeptide 1 hairy and enhancer of split 1, (Drosophila) nuclear receptor subfamily 4, group A, member 1
HTLV-I infection, Prion diseases, vasculature development, positive regulation of defense Herpes simplex infection, response, response to interleukin-6, epithelium Legionellosis, Influenza A development,chemotaxis, inflammatory response, regulation of inflammatory response, platelet activation Adipocytokine signaling pathway positive regulation of defense response, regulation of hemostasis, cell adhesion, platelet activation Osteoclast differentiation vasculature development Glycosphingolipid vasculature development, epithelium development, cell biosynthesis-lacto and neolacto adhesion, inflammatory response series Maturity onset diabetes of the vasculature development, epithelium development, cell young adhesion MAPK signaling pathway vasculature development, chemotaxis, epidermal growth factor receptor signaling pathway
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P-value
P-value calculated from t-test, P-value cut-off: 0.05; "Description" stands for the full name of gene; "Signalling pathways" stands for the name of the pathways; "GO terms" stands for gene ontology.
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ACCEPTED MANUSCRIPT Highlights This article combines the clinical research and basic research, our aim is to
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serve the clinical treatment by combining basic research. Ovarian endometriosis,
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which is the most common type of endometriosis, is a refractory gynaecological disease. Ultrasound-guided interventional therapies have been used in the treatment of ovarian endometriosis in recent year. The paper was combine
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ultrasound-guided interventional therapy with NimbleGen human gene
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expression microarrays analysis to detect the differentially expressed genes in the sera before and after the therapy in patients with ovarian endometriomas to
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evalue the effect of the therapy from at the molecular level. And there is rare
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report on it recently. We believe that this study has a high clinical value.
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S0009-9120(15)00216-7 doi: 10.1016/j.clinbiochem.2015.06.003 CLB 9045
To appear in:
Clinical Biochemistry
Received date: Revised date: Accepted date:
4 April 2015 31 May 2015 1 June 2015
Please cite this article as: Wang Lu-Lu, Cai Huai-Qiu, Dong Xiao-Qiu, Zhang Li-Wei, Jiang Shan-Shan, Zhao Ning, Shao Xiao-Hui, Wang Si-Ming, Zhu Li, Zhang Tong, Differentially expressed gene profiles in the serum before and after the ultrasoundguided ethanol sclerotherapy in patients with ovarian endometriomas, Clinical Biochemistry (2015), doi: 10.1016/j.clinbiochem.2015.06.003
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.
ACCEPTED MANUSCRIPT Differentially expressed gene profiles in the serum before and after the ultrasound-guided ethanol sclerotherapy in patients
Lu-Lu Wang
a 1
, Huai-Qiu Cai
a 1
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with ovarian endometriomas
, Xiao-Qiu Dong a*, Li-Wei Zhang a,
Shan-Shan Jiang a, Ning Zhao a, Xiao-Hui Shao a, Si-Ming Wang a, Li Zhu , Tong Zhang c
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Department of Ultrasonography, the Fourth Hospital of Harbin Medical
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a
Both authors are contributed equally to this paper.
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1
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b
b
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University, Harbin, P. R. China
Department of Obstetrics and Gynecology, the Fourth Hospital of Harbin
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Medical University, Harbin, P. R. China c
KangChen Bio-technology Corp., Shanghai, P. R..China
*
Corresponding author. Yiyuan Str 37, Harbin, Heilongjiang Province 150001,
P. R. China. Tel.: +86 451 82576706; fax: +86 451 82576705. E-mail address: [email protected]
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ACCEPTED MANUSCRIPT ABSTRACT Objectives: To detect the differentially expressed genes and subsequently
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identify disease-related signatures and potential biomarkers for patients with
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ovarian endometriomas in the serum before and after the ultrasound-guided ethanol sclerotherapy in patients with ovarian endometriomas. Design and methods: Venous blood samples were collected from nine
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patients with ovarian endometriomas before and after ultrasound-guided
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ethanol sclerotherapy, and the serum were isolated after centrifugation. NimbleGen human gene expression microarrays analysis was conducted to
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analyse gene ontology categories (GO terms) and signalling pathways of
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differentially expressed genes. The accuracy of some typical genes from
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microarray analysis was verified by quantitative PCR (qPCR). Results: Approximately 45,033 genes were analysed by NimbleGen human
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gene expression microarrays, which identified 447 genes that showed differential expressions before and after therapy. Of these, 225 genes were up-regulated and 222 genes were down-regulated. The GO terms of the down-regulated genes were strongly associated with the pathogenesis of ovarian endometriomas; 15 down-regulated genes showed overlaps in both signalling pathways and GO terms. Among these, six genes showed statistical significance including IL6, CD36, JUNB, B4GALT1, HES1, and NR4A1, which were also validated by qPCR analysis. Conclusions: There were differentially expressed genes in the serum before 2
ACCEPTED MANUSCRIPT and after ultrasound-guided ethanol sclerotherapy in patients with ovarian endometriomas. Notably, the expressions of IL6, CD36, JUNB, B4GALT1,
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HES1, and NR4A1, which are strongly associated with the pathogenesis of
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ovarian endometriomas, were significantly down-regulated after ethanol sclerotherapy. This may not only help us understand EMs pathogenesis, but also provide potential biomarkers for verifying the effects of ethanol
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sclerotherapy.
Keywords: ovarian endometriosis; ultrasound-guided; ethanol sclerotherapy;
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differentially expressed genes; DNA microarray
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ACCEPTED MANUSCRIPT Introduction Ovarian endometriosis, which is as the most common type of endometriosis
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(EMs), is a refractory gynaecological disease [1]. Although the pathogenesis
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mechanism of EMs is not clear, studies have shown that the generation and development of EMs involves processes of adhesion, aggression and angiogenesis which is called the "3 A Process" [2]. And these processes are
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associated with many genes which are up-regulated in the patients with EMs
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[3-5], including interleukin-6 (IL-6), IL-8, TNF-α, monocyte chemoattractant protein-1 (MCP-1), macrophage migration inhibitory factor (MIF) and
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vascular endothelial growth factor(VEGF) in the peritoneal fluid [6-9], MIF,
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IL-6 and IL-8 in serum [10,11] and IL-6 in stromal cells derived from
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endometriotic tissues [12]. Recently studies showed that the serum IL-6 level was reduced in patient with EMs after drug therapy or laparoscopic surgery
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[13,14]. However, EMs-related gene expression and its signatures in serum are still unclear before and after the ultrasound-guided ethanol sclerotherapy in patients with ovarian endometriomas. Ultrasound-guided
ethanol
sclerotherapy,
an
minimally
invasive
technology, has been increasingly recognized by clinicians as a reliable surgical alternative for treating ovarian endometriomas with many advantages, including minimal trauma, fewer risks and complication, lower costs and decreased recurrence rates in recent years [15,16]. Under ultrasound guidance, there are three key procedures of the therapy which consist of the cystic fluid 4
ACCEPTED MANUSCRIPT aspiration, saline washings and 95% ethanol retention. Initially, the steps of cystic fluid aspiration and saline washings are to eliminate the cystic fluid
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which could stimulate ectopic cell proliferation. Secondly, ethanol (95%), as a
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sclerosing agent, is retained in the cystic cavity to inactivate the cyst wall cells and to sclerose capillaries [17]. With the wide clinical applications of the technology, reports on its methodology and efficacy have gradually increased
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over recent years [18]. Althrough most of criteria for efficacy were based on
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using ultrasound to detect lesion characteristics or the changes in ovarian function and hormone levels [19,20]. However, the role of EMs-related gene
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in serum remains undefined. Therefore, this study aimed to identify
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EMs-related gene expression profile in the serum and its association with the
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pathogenesis of ovarian endometriomas before and after ultrasound-guided
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ethanol sclerotherapy for patients with ovarian endometriomas.
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Materials and methods
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Subjects
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Among the patients with ovarian endometriomas who underwent ultrasound-guided ethanol sclerotherapy at our hospital from December 2011 to December 2013, nine subjects were selected based on the following criteria:
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1) Unilateral solitary ovarian endometriosis without any treatments. 2) Size of
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ovarian endometriosis: 4.0 cm ≤ diameter ≤ 6.0 cm. 3) No adenomyosis and other gynecological diseases. 4) Normal serum CA125 (0–35 mIU/mL). 5)
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Lesions disappeared after three months. 6) No alcoholic allergies, normal
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results in routine blood and urine tests, and normal lung, liver and kidney
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functions. The age of patients ranged from 27 to 49 years (mean: 36.5 ± 1.5 years), and all patients were married. Five subjects had ovarian
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endometriomas in their left ovaries and four subjects had them on their right ovaries. All patients were verbally informed of the treatment process and the risks involved, and signed written informed consents. The Ethics Committee of our hospital (The Fourth Hospital of Harbin Medical University, Harbin, P. R. China) approved the study design of the present investigation.
Equipment and materials Equipment and materials used in the ethanol sclerotherapy The following equipment and materials were used: a Mylab 90 Color 6
ACCEPTED MANUSCRIPT Doppler ultrasonography unit (Esaote, Italy), transvaginal probe (Model: EC123 9-5, frequency 7-12 MHz), percutaneous transhepatic cholangiography
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(PTC) needle (16/18 G × 20 cm, Origin: Nagano, Japan), local anaesthetic:
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2% lidocaine (5 mL: 0.1 g, Origin: Shanghai, China), washing solution: 0.9% saline (250 mL: 2.25 g, Origin: Tonghua, China), and hardener: 95% medical
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alcohol (Origin: Dezhou, China).
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Ultrasound-guided ethanol sclerotherapy
Transvaginal ultrasonography was performed before treatment. The location,
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size, and sonographic features of the ovarian endometriosis were recorded,
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and the images were captured. The malignancy was excluded based on the
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clinical diagnosis and laboratory tests. After a routine gynecological disinfection, the cyst was punctured under ultrasound guidance using a PTC
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needle as described previously [15,18,21]. The cystic fluid was aspirated and the cyst was repeatedly washed with saline. When the cystic fluid was clear, 95% ethanol was used for washing and 20-50 mL of the ethanol was retained. After the patient returned to a normal state, the needle was removed. Ultrasound examination was performed three months after the treatment.
Equipment and materials for microarray analysis and PCR verification The Human 12135K Gene Expression Array was manufactured by Roche NimbleGen (Madison, WI, USA). Double stranded (ds)-cDNA was cleaned 7
ACCEPTED MANUSCRIPT and labelled in accordance to the NimbleGen Gene Expression Analysis protocol (NimbleGen Systems, Inc., Madison, WI, USA). PCR primers were
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synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). Reverse
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transcription of purified RNA was performed using SuperScript III Reverse Transcriptase (2,000U, Invitrogen, USA). Real-time qPCR was carried out on
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a Bio-Rad Real-time PCR Detector (Bio-Rad Chromo4TM, USA).
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Samples for microarray analysis and PCR verification Collection and processing of blood samples: Two tubes of 5 mL venous
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blood from the patients were collected before treatment and three months after
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treatment. After blood collection, the blood samples were immediately
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centrifuged (temperature: room temperature, speed: 1000 r/min) and the serum was divided into two tubes as Tube 1 and Tube 2, which were immediately
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stored at -80°C until an analysis. The 2 mL serum in Tube 1 was transferred to an RNAse-free cryovial for NimbleGen human gene expression microarrays analysis. The 2 mL serum in Tube 2 was transferred to an RNAse-free Eppendorf (EP) tube for PCR verification.
NimbleGen human gene expression microarrays analysis RNA quantity and quality were measured by using a NanoDrop ND-1000. RNA integrity was assessed by standard denaturing agarose gel electrophoresis. The microarray experiments were performed by KangChen Bio-technology 8
ACCEPTED MANUSCRIPT Corp. (Shanghai, China) according to the standard protocol. The Human 12 135K Gene Expression Array was manufactured by Roche NimbleGen. Briefly,
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total RNA of each sample was used for labelling and array hybridization using
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the following steps: 1) Reverse transcription using Invitrogen SuperScript ds-cDNA synthesis kit. 2) ds-cDNA labelling with NimbleGen one-color DNA labelling kit. 3) Array hybridization using the NimbleGen Hybridization System,
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followed by washing with the NimbleGen wash buffer kit, and 4) Array
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scanning using the Axon GenePix 4000B microarray scanner (Molecular Devices Corporation). Scanned images (TIFF format) were then imported into
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the NimbleScan software (Version 2.5) for grid alignment and expression data
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analysis. Expression data were normalized using quantile normalization and the
software.
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Robust Multichip Average (RMA) algorithm included in the NimbleScan
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Scanned images (TIFF format) were then imported into NimbleScan software (Version 2.5) for grid alignment and expression data analysis. Expression data were normalized through quantile normalization and the Robust Multichip Average (RMA) algorithm included in the NimbleScan software. All gene level files were imported into Agilent GeneSpring GX software (Version 11.5.1) for further analysis. Differentially expressed genes with statistical significance between the two groups were identified through Volcano Plot filtering. Differentially expressed genes were identified through Fold Change filtering. Pathway Analysis and GO analysis were applied to determine the roles of these 9
ACCEPTED MANUSCRIPT differentially expressed genes played in these biological pathways or GO terms. Finally, Hierarchical Clustering was performed to show distinguishable gene
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expression profiling among samples (Microarray data are available at
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www.ncbi.nlm.nih.gov/geo/, accession numbers GSE69310).
Quantitative real-time PCR (qPCR)
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Total RNA from human serum was extracted using a RNA extraction kit.
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Reverse transcription of purified RNA was performed using SuperScript III reverse transcriptase following the manufacturer’s instructions (Invitrogen). 2
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× SuperArray PCR master mix, to verify the accuracy of the microarray data,
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six differentially expressed genes were chosen for mRNA quantification of
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expression levels by real-time PCR. Real-time qPCR was carried out in triplicate on a Bio-Rad Chromo4TM Real-time PCR Detector (Bio-Rad). The
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PCR parameters are described in Table 1. GAPDH was used as an internal control. Data analysis was performed using the MJ Opticon Monitor Analysis Software (Bio-Rad), and fold change of mRNA expression was calculated using the △△Ct method. The reaction without first-strand cDNA was used as negative control.
Statistical analysis Data are presented as mean ± standard deviation. The Student’s t-test was used each time with the SPSS 19.0 software package, and the P-value was 10
ACCEPTED MANUSCRIPT calculated. A difference was determined to be statistically significant when the P-value was < 0.05. The gene concentration of each sample is generated
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directly by the Rotor-Gene Real-Time Analysis Software 6.0.
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Results
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Among the 45,033 genes identified from the gene expression profile microarray analysis, 447 differentially expressed genes were screened out (P < 0.05). Among these, 225 genes were up-regulated, and 222 genes were
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down-regulated. A "volcano plot" was constructed by plotting the
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differentially expressed genes between the P values and fold changes (Fig. 1).
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expressed genes
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The main GO terms and signalling pathways of up-regulated differentially
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There were 225 up-regulated genes cover 169 GO terms. Among these, six GO terms showed high enrichment scores (ES) and high frequencies of
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occurrence. The GO terms with the highest ES was the respiratory electron transport chain, which had the highest level of statistical significance (P = 0.00005, Fig. 2). Up-regulated genes exerted their effects through eight signalling pathways. The higher ES the pathway had, the more statistically significant the pathway was. The pathway of folate biosynthesis showed the highest ES, with the highest level of statistical significance (P = 0.0008, Fig. 3).
The main GO terms and signalling pathways of down-regulated differentially 12
ACCEPTED MANUSCRIPT expressed genes There were 222 down-regulated genes cover 480 GO terms, of which 11
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were strongly associated with ovarian endometriomas, including vasculature
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development, inflammatory response, chemotaxis, cell adhesion and epithelium development. Among these 11 GO terms, vasculature development showed the smallest P value, the smallest false discovery rate (FDR), and the
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highest ES, indicating the highest level of statistical significance (P = 0.0002,
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Table 2 and Fig. 4). Down-regulated genes exerted their effects through 13 signalling pathways that were statistically significant (P < 0.05). The
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mitogen-activated protein kinase (MAPK) pathway showed the smallest
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absolute FDR (It is similar to its P value, thus reflecting an error rate), the
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smallest P value, and the highest ES, suggesting the highest level of statistical
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significance (P = 0.0004) (Table 3 and Fig. 5).
Selection of typical genes with significantly down-regulated differentially expressed genes and their GO terms Among the down-regulated differentially expressed genes, 15 ovarian endometriosis- associated genes showed overlaps in both signalling pathways and GO terms (Fold change ≥ 2). Among these, IL6, CD36, JUNB, B4GALT1, HES1, and NR4A1 were closely related to ovarian endometriomas. The most statistically significant gene was IL-6 (P = 0.0006). Each gene exerted their GO terms through one or more signalling pathways (Table 4). 13
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Identification of typical differentially expressed genes by qPCR
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PCR analysis was performed on six genes. The product lengths were
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between 95 and 282 bp, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as control. The forward and reverse primer sequences of the six genes are listed in Table 1. The result showed fold-change relationships when
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the six genes were compared with the control genes, and their post-treatment
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expressions were down-regulated. Their fold changes in the microarray analysis ranged from -2.17 to -3.46. The fold changes of qPCR were between
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-1.05 and -4.03, consistent with the negative values from the microarray
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analysis. This verified the consistency of the down-regulated differentially
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expressed genes. In addition, the fold changes of PCR were all ≥ 1, quantitatively confirming that the six down-regulated genes were statistically
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significant (Fig.6).
Discussion
In the present study, a potential EMs-related gene expression profile in the serum from the patients with ovarian endometriomas was identified before and after ultrasound-guided ethanol sclerotherapy. More importantly, the expressions of IL6, CD36, JUNB, B4GALT1, HES1, and NR4A1, which are strongly associated with the pathogenesis of ovarian endometriomas, were significantly down-regulated after ethanol sclerotherapy. 14
ACCEPTED MANUSCRIPT Ultrasound-guided ethanol sclerotherapy is more effective way to treat ovarian endometriomas than drug therapy or laparoscopic surgery [15]. Through
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ultrasound-guided ethanol sclerotherapy, the cystic fluid containing a large
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number of macrophages and transforming growth factor is almost completely removed. The cells of cyst wall are inactivated and losed secretory function and the vascular are sclerosed and occlused by 95% ethanol. Then cyst wall become
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solidification, hardening, adhesion and closure. In the procress, various genes
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expression have been changed.
From the gene expression profile microarray analysis, 447 differentially
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expressed genes were detected. Among these, 225 differentially expressed genes
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were up-regulated and 222 genes were down-regulated. These differentially
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expressed genes cover various GO terms through multiple signalling pathways. Among 169 GO terms exerted by the 225 up-regulated genes, there were six
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with higher ES (Higher ES indicates that the corresponding GO term and signalling pathway are more statistically significant.) and higher frequencies of occurrence. The GO term “respiratory electron transport chain” had the highest ES (ES = 4.30). There were eight signalling pathways that showed statistical significance, in which up-regulated genes were involved. The pathway of folate biosynthesis was the most statistically significant (ES = 3.12). However, the GO terms exerted by these up-regulated genes such as the electron transport chain and cell respiration were not strongly associated with the occurrence and development of ovarian endometriomas. 15
ACCEPTED MANUSCRIPT Among the 480 GO terms exerted by 222 down-regulated genes, 11 functions were determined to be strongly associated with ovarian
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endometriomas, including vasculature development, immune responses to
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inflammation, chemotaxis, cell adhesion, and epithelium development. Among these, vasculature development had the smallest FDR (FDR = 0.0142, FDR is the absolute fold change of error rate, which has the same meaning as
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the P value. A smaller FDR means a higher statistical significance.). On the
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other hand, the highest ES (ES = 3.73) was found in vasculature development, indicating the GO term of vasculature development had the highest level of
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statistical significance. There were 13 signalling pathways with statistical
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significance, in which down-regulated genes were involved. Among these, the
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MAPK pathway was the most statistically significant (FDR = 0.0920, ES = 3.44). Among the down-regulated genes involved in 11 GO terms, 15
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down-regulated genes not only showed GO terms that were strongly associated with the pathogenesis of ovarian endometriomas, but also exerted their functions through 13 signalling pathways. These overlapped both in terms of GO terms and signalling pathways. Among these, six genes were excluded because they had higher frequencies of occurrence in GO terms and signalling pathways, and their sites were close to the front of the signalling pathways. These six genes were IL6, CD36, JUNB, B4GALT1, HES1, and NR4A1. Each gene was involved in one or more GO terms, and played a role through one or more signalling pathways. 16
ACCEPTED MANUSCRIPT IL-6 was the gene with the highest level of statistical significance among the six genes. It has been widely studied in the research of the pathogenic ovarian
endometriomas
[22].
As
an
T
of
endometrium
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mechanism
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immunoregulatory gene, IL-6 promotes the metastasis, localization, and invasive growth of ectopic endometrium. Some studies have shown that the IL-6 level secreted by the ectopic endometrial stromal cells of the patients
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with EMs was six-fold higher than that secreted by the normal endometrium
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[23,24]. In this study, the ovarian endometriosis-associated GO terms, in which IL-6 was involved, included inflammatory response, chemotaxis,
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epithelium development, and vasculature development. The five other genes
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identified in this study were rarely reported genes in the field of ovarian
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endometriosis research. CD36 is commonly reported in the studies of atherosclerosis and tumour diseases, with the biological effects of
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inflammation, angiogenesis, tumour, and lipid metabolism [25-27]. JUNB is often reported in liver cancer [28] and has been extensively studied in cancer research; it also plays an important role in tumour neo-vascularisation, angiogenesis, tumour formation, metastasis, and invasion [29,30]. B4GALT1 is among the glycosyltransferases that have been extensively studied [31]. It imparts an adhesion molecule-like effect. HES1, which is involved in cell differentiation [32], is reported in the researches on promoting neural stem cell proliferation [33]. NR4A1 is an immediate early gene product found in pheochromocytoma cells and fibroblasts [34], and functions in proliferation 17
ACCEPTED MANUSCRIPT and differentiation [35]. However, in this study, CD36 and JUNB were involved in cell adhesion and vasculature development, respectively. They
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have also been associated with angiogenesis in ovarian endometriomas and
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tumour-like growth. Both B4GALT1 and HES1 are involved in epithelium growth, cell adhesion, and vasculature development. NR4A1 plays a role in chemotaxis and angiogenesis. These results suggest that these gene
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expressions in the serum from the patients with ovarian endometriomas may
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be related to adhesion, differentiation, chemotaxis, and vascularization in the growth process of ovarian endometriomas. After alcohol sclerotherapy, the
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expressions of these genes were down-regulated, and their corresponding GO
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terms were also inhibited, supporting an identification of potential
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EMs-related gene network. Although we do not provide direct evidence for these gene functions in disease development and progression of EMs, the
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expression levels of these genes in the serum from the patients may be useful as a biomarker for predicting the prognosis in disease recurrence, progression and therapeutic efficacy.
Conclusions In the present study, we identified differentially expressed gene profiles before and after the ethanol sclerotherapy in the serum from the patients with ovarian endometriomas. Notably, the expressions of IL6, CD36, JUNB, B4GALT1, HES1, and NR4A1, which are strongly associated with the 18
ACCEPTED MANUSCRIPT pathogenesis of ovarian endometriomas, were significantly down-regulated after ethanol sclerotherapy. This identification may help us in understanding
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EMs pathogenesis and provide potential biomarkers for clinical application.
Acknowledgements
The sponsors of this study were the National Natural Science Foundation of
Innovation
Research
Program
Fund
of
China
(grant
no.
MA
Bureau
NU
China (grant no. 81271646 to X. Q. Dong), Harbin Science and Technology
2012RFXXS057) and Heilongjiang Key Technologies R&D Program funds of
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[34] Hazel TG, Nathans D, Lau LF. A gene inducible by serum growth factors encodes a member of the steroid and thyroid hormone receptor superfamily. Proc Natl Acad Sci 1988; 85: 8444-8. [35] Yu L, Su YS, Zhao J, Wang H, Li W. Repression of NR4A1 by a chromatin modifier promotes docetaxel resistance in PC-3 human prostate cancer cells. FEBS Lett 2013; 587: 2542-51. 24
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Figure Legends
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Fig.1. “Volcano plot” of differentially expressed genes
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Volcano Plots are useful tools for visualizing differential expression between two different conditions (fold-change values and P-values). Fold change stands for Multiple of postoperative vs preoperative. The upper-left
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region bounded by horizontal line y = a and vertical line x = -1 represents
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down-regulated genes; the up-right region bounded by horizontal line y = a
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and vertical line x = 1 represented up-regulated genes.
Terms
stands
for
gene
ontology
of
up-regulated
genes,
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GO
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Fig.2. Main GO terms exerted by up-regulated genes
Enrichment_Score stands for the Enrichment Score value of the GO terms of
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up-regulated genes. The vertical axis represents the full names of six GO terms; the horizontal axis represents the ES of the GO terms.
Fig.3. Signaling pathways in which the up-regulated genes were involved Signaling pathways stands for the name of the signalling pathways of up-regulated genes, Enrichment_Score stands for the Enrichment Score value of the signalling pathways of up-regulated genes. The vertical axis represents the full names of eight signalling pathways; the horizontal axis represents the ES of the signalling pathways. 25
ACCEPTED MANUSCRIPT
Fig.4. Ovarian endometriosis-associated GO terms exerted by down-regulated
stands
for gene
ontology of
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terms
down-regulated
genes,
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GO
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genes
Enrichment_Score stands for the Enrichment Score value of the GO terms of down-regulated genes, it equals "-log10 (P-value)". The vertical axis
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represents the full names of 11 GO terms; the horizontal axis represents the
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ES of the GO terms.
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Fig.5. Signalling pathways in which the down-regulated genes were involved
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Signalling pathways stands for the name of the signalling pathways of
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down-regulated genes, Enrichment_Score stands for the Enrichment Score value of the signalling pathways of down-regulated genes, it equals "-log10
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(P-value)". The vertical axis represents the full names of 13 signalling pathways; the horizontal axis represents the ES of the signalling pathways.
Fig.6. Comparison of fold changes in differentially expressed genes between microarray and qPCR The horizontal axis represents genes; the vertical axis represents fold changes; the dark column represents microarray analysis; the light-coloured column represents qPCR.
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Figure 6
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ACCEPTED MANUSCRIPT
B4GALT1 HES1 NR4A1 GAPDH
95
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Product size (bp)
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US MA N
JUNB
Annealing Tm (℃) 60
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CD36
F:5’TGACAAACAAATTCGGTACATCC3’ R:5’GTGCCTCTTTGCTGCTTTCA3’ F:5’ TGGTTCCGTACCCTGTTACTACC3’ R:5’ CGTCGGATTCAAATACAGCATAG3’ F:5’ GGAACAGCCCTTCTACCACGA3’ R:5’ GGCTCGGTTTCAGGAGTTTG3’ F:5’ATGTTTGCCTGGTCCGTGAG3’ R:5’GCCAGCACAGCATCTCCTTTA3’ F:5’GCCAGTTTGCTTTCCTCATTC3’ R:5’ TTCTCTCCCAGTATTCAAGTTCCT3’ F:5’GCAAGTGGGCGGAGAAGAT3’ R:5’CCTCGCCTGGCTTAGACCT3’ F:5’GGGAAACTGTGGCGTGAT3’ R:5’GAGTGGGTGTCGCTGTTGA3’
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IL6
Primer sequences
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Gene
CR
Table 1. Primer sequences and PCR parameters for different selected genes.
60
282
60
101
60
235
60
253
60
126
60
299
"Gene" stands for the name of genes; “Primer sequences” stands for the base sequence of primers; "Annealing Tm" stands for annealing temperature, the unit is ℃; "Product size" stands for the length of the fragment after the primer for amplification; F: Forward primer, R: Reversed primer.
33
ACCEPTED MANUSCRIPT
Terms vasculature development
GO:0031349
0.0174
GO:0070741 GO:0060429
positive regulation of 0.0003 defense response response to interleukin-6 0.0091 epithelium development 0.0126
GO:1900046 GO:0006935
regulation of hemostasis chemotaxis
0.0160 0.0230
0.2084 0.2560
GO:0007155
cell adhesion
0.0246
GO:0006954
inflammatory response
0.0335
0.3338
GO:0050727
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FDR 0.0142
regulation of 0.0398 inflammatory response platelet activation 0.0399 epidermal growth factor 0.0493 receptor signaling pathway
0.3748
IL6, FOXA2 NKX2-5, B4GALT1, DVL1, ERRFI1, FOXA2, IL6, SPDEF, SOCS3, HES1, ETV5 THBD, FOXA2, CD36 IL6, RHOB, CACNB1, DVL1, NR4A1, HOXB9, CXCL2, CYR61, FOSL1, CMTM1 CD36, TNFRSF12A, BCAM, CYR61, TYRO3, KIFC3, FOXA2, RHOB, B4GALT1, HES1, NEDD9, PTPRS, LY6D, NPNT B4GALT1, IL6, OSM, TYRO3, IER3, FOS, CXCL2, IRAK2 IL6, OSM, TYRO3, IER3
0.3748 0.4416
THBD, TYRO3, RHOB, CD36, IL6 ERRFI1, NR4A1, TRIB3, RICTOR
CR
US
MA N
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0.1545 0.1834
Genes VHL, SH2D2A, TNFRSF12A, NKX2-5, JUNB, CYR61, NR4A1, IL6, RHOB, HES1, B4GALT1, ERRFI1, SOCS3 CD36, DUSP4, ELK1, FOS, IRAK2, IL6, OSM, TYRO3
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GO.ID GO:0001944
GO:0030168 GO:0007173
0.2697
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P-value 0.0002
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Table 2. The GO terms associated with ovarian endometriomas of down-regulated genes.
"GO" stands for gene ontology, "GO.ID" stands for the ID of gene ontology term; "Terms" stands for the name of gene ontology term; "Pvalue" stands for the significance testing value of the GOID, P-value cut-off: 0.05; "FDR" stands for the false discovery rate of the GOID, using Benjamini & Hochberg (1995) method; "Genes" stands for the DE genes associated with the GOID. 34
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hsa04115 hsa05164 hsa04380
MAPK signaling pathway
0.0004
0.0920
Glycosphingolipid biosynthesis-lacto and neolacto series HTLV-I infection
0.0023
0.2718
CACNB1, DDIT3, DUSP2, DUSP4, DUSP5, ELK1, FOS, GADD45A, HSPA1A, NR4A1 B4GALT1, FUT1, FUT3
0.0053
0.2718
Adipocytokine signaling pathway Prion diseases Herpes simplex infection Legionellosis Dorso-ventral axis formation Maturity onset diabetes of the young Protein processing in endoplasmic reticulum p53 signaling pathway Influenza A Osteoclast differentiation
0.0054 0.0054 0.0107 0.0188 0.0250 0.0270 0.0277 0.0326 0.0330 0.0425
US
CR
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Genes
0.2718 0.2718 0.4493 0.6727 0.6900 0.6900 0.6900
DVL1, ELK1, ETS2, FOS, FOSL1, HLA-C, IL6, MYBL1 CD36, PPARA, PRKAB1, SOCS3 ELK1, HSPA1A, IL6 FOS, GTF2IRD1, HLA-C, IL6, PER1, SOCS3 CXCL2, HSPA1A, IL6 CPEB1, ETS2 FOXA2, HES1 DDIT3, DNAJB1, ERN1, HSPA1A, PPP1R15A
0.6900 0.6900 0.8200
GADD45A, PMAIP1, TNFRSF10B DNAJB1, HSPA1A, IL6, SOCS3, TNFRSF10B FOS, FOSL1, JUNB, SOCS3
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hsa04920 hsa05020 hsa05168 hsa05134 hsa04320 hsa04950 hsa04141
FDR
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hsa05166
P-value
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hsa00601
Definition
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Pathway ID hsa04010
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Table 3. The 13 significant signalling pathways in which the down-regulated genes were involved.
"Pathway ID" stands for Pathway identifiers used in KEGG; "Definition" stands for the definition of the Pathway ID; P-value cut-off: 0.05; "FDR" stands for Absolute Fold Change, the absolute ratio (no log scale) of normalized intensities between two conditions; "Genes" stands for the DE genes associated with the Pathway ID.
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Gene_ name IL6
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Table 4. The down-regulated genes that had overlaps in both signalling pathways and GO terms.
Description
Signalling pathways
GO terms
0.0006
interleukin 6 (interferon, beta 2)
CD36
0.0352
JUNB B4GAL T1
0.0291 0.0164
HES1
0.0217
NR4A1
0.0024
CD36 molecule (thrombospondin receptor) jun B proto-oncogene UDP-Gal: betaGlcNAc beta 1,4galactosyltransferase, polypeptide 1 hairy and enhancer of split 1, (Drosophila) nuclear receptor subfamily 4, group A, member 1
HTLV-I infection, Prion diseases, vasculature development, positive regulation of defense Herpes simplex infection, response, response to interleukin-6, epithelium Legionellosis, Influenza A development,chemotaxis, inflammatory response, regulation of inflammatory response, platelet activation Adipocytokine signaling pathway positive regulation of defense response, regulation of hemostasis, cell adhesion, platelet activation Osteoclast differentiation vasculature development Glycosphingolipid vasculature development, epithelium development, cell biosynthesis-lacto and neolacto adhesion, inflammatory response series Maturity onset diabetes of the vasculature development, epithelium development, cell young adhesion MAPK signaling pathway vasculature development, chemotaxis, epidermal growth factor receptor signaling pathway
AC
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TE D
MA N
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CR
P-value
P-value calculated from t-test, P-value cut-off: 0.05; "Description" stands for the full name of gene; "Signalling pathways" stands for the name of the pathways; "GO terms" stands for gene ontology.
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ACCEPTED MANUSCRIPT Highlights This article combines the clinical research and basic research, our aim is to
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serve the clinical treatment by combining basic research. Ovarian endometriosis,
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which is the most common type of endometriosis, is a refractory gynaecological disease. Ultrasound-guided interventional therapies have been used in the treatment of ovarian endometriosis in recent year. The paper was combine
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ultrasound-guided interventional therapy with NimbleGen human gene
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expression microarrays analysis to detect the differentially expressed genes in the sera before and after the therapy in patients with ovarian endometriomas to
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evalue the effect of the therapy from at the molecular level. And there is rare
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report on it recently. We believe that this study has a high clinical value.
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