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Aberrantly expressed long noncoding RNAs in the eutopic endometria of patients with uterine adenomyosis
European Journal of Obstetrics & Gynecology and Reproductive Biology 199 (2016) 32–37
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Aberrantly expressed long noncoding RNAs in the eutopic endometria of patients with uterine adenomyosis§ Jian Fa Jiang, Ai Jun Sun *, Wei Xue, Yan Deng, Yan Fang Wang Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 October 2015 Received in revised form 7 January 2016 Accepted 29 January 2016
Objective: Adenomyosis is a common gynecologic disease. Alterations in the eutopic endometria might play an important role in the pathogenesis of adenomyosis. Long non-coding RNA (lncRNA) represents key regulators of gene expression. Our goal was to identify differentially expressed long noncoding RNA and messenger RNA (mRNA) in the eutopic endometria of subjects with adenomyosis on a genome-wide scale. Study design: The expression level of lncRNAs and mRNAs in the eutopic endometria from women with adenomyosis and from that of normal control subjects were detected by Affymetrix Human Transcriptome Array 2.0. Bioinformatics analysis was done for further investigation. Three up-regulated and three down-regulated lncRNAs were randomly chosen for validation by quantitative real-time polymerase chain reaction. Results: A total of 165 lncRNAs and 612 mRNAs were aberrantly expressed in the eutopic endometria of subjects with adenomyosis. Pathway analysis indicated that 40 pathways corresponded to up-regulated transcripts and 39 pathways corresponded to downregulated transcripts. A list of genes that might play roles in the pathogenesis of adenomyosis were produced by comparing the difference between coexpression networks. Expression of six chosen lncRNAs was validated by quantitative real-time polymerase chain reaction. Conclusion: This study show for the first time that the lncRNA expression profile is altered in women with adenomyosis and provides new biological foundations for further mechanistic studies in this enigmatic disorder. ß 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Adenomyosis Endometria Long noncoding RNA Microarray
Introduction Adenomyosis is a common gynecologic disease characterized by ectopic endometrial glands and stroma invading the myometrium, associated with adjacent myometrial hypertrophy and hyperplasia [1]. It is clinically characterized by dysmenorrhea, abnormal uterine bleeding, and chronic pelvic pain, and seriously affect the quality of life. However, the pathogenesis of adenomyosis remains unclear [2]. The basal layer of the endometrium invaginates between smooth muscle cell bundles or along lymphatic vessels deep into the myometrium, and constitutes the most widely accepted theory as to the etiology of adenomyosis
§ The work reported was done in Peking Union Medical College Hospital and Gminix Biotechnology Company of Shanghai, People’s Republic of China. * Corresponding author at: Shuaifuyuan no. 1, Dongcheng District, Beijing 100730, China. Tel.: +86 18600045466; fax: +86 01069156039. E-mail address: [email protected] (A.J. Sun).
http://dx.doi.org/10.1016/j.ejogrb.2016.01.033 0301-2115/ß 2016 Elsevier Ireland Ltd. All rights reserved.
[3]. Many studies have shown that a heritable or acquired alteration in the eutopic endometria may play a significant role in the pathogenesis and development of adenomyosis [4,5]. The identification of molecular differences within eutopic endometria of women with adenomyosis is a very important step in our understanding of the pathogenesis of adenomyosis. Previous studies have demonstrated that genetic factors play pivotal roles in the pathogenesis of adenomyosis, partially through epigenetic regulation [4]. Recently, research has shown that long noncoding RNA (lncRNA), defined as RNA molecules >200 nucleotides in length, may function in every aspect of human biology and contribute to the development of various benign or malignant diseases [6]. Functional analysis of lncRNAs has revealed their multi-faceted roles in the regulation of gene expression, from epigenetic modulation in the nucleus to post-transcriptional control in the cytoplasm [7]. Recent studies have shown that lncRNA expression profile is very different in many human conditions, including cancer [8–10], cardiovascular disease [11], metabolic disease [12] and degenerative diseases [13]. But the
J.F. Jiang et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 199 (2016) 32–37
lncRNA expression profile and its potential biological function in adenomyosis is unknown. Our study is the first study of lncRNA expression profile in women with adenomyosis. We firstly constructed the lncRNA and mRNA expression profile in eutopic endometria from adenomyosis patients and from that of normal control subjects. In addition, we conducted functional analysis and constructed co-expression networks to find the potential core genes associated with adenomyosis pathogenesis. Materials and methods Sample collection This study was conducted according to the principles expressed in the Declaration of Helsinki and was approved by the Ethics Review Board of The Peking Union Medical College Hospital, Beijing, China. Written informed consent was obtained from each participant before surgery. The eutopic endometria was obtained from 20 women with adenomyosis during hysterectomy (mean age, 44.7 years). The diagnosis of adenomyosis was confirmed by histopathological examination after operation. Normal endometrial tissues were collected from 16 women found to be free of adenomyosis or endometriosis during surgery (mean age, 44.4 years). Women whose endometria acted as controls were undergoing hysterectomy for symptomatic uterine leiomyomas, none of which were submucosal in location. All subjects in both groups had not received any hormonal treatment within three months of endometrial sampling and had regular menstrual cycles (25–35 days). All endometrial samples were in the proliferative phase according to the menstrual cycle phases and the criteria of endometrial histologic dating [14]. Review of pathology reports revealed no evidence of endometrial hyperplasia or inflammation in any of the samples of two groups. All endometria tissue were snap-frozen using liquid nitrogen after surgical resection and preserved in a 80 8C freezer until use. RNA extraction and quality control assay Total RNA of each frozen endometria was extracted using TRIzol1 Reagent (Life Technologies, California, USA) according to the manufacturer’s protocol. We applied a NanoDrop ND-2000 spectrophotometer to measure the quantity and quality of total RNA, and used standard denaturing agarose gel electrophoresis to test the integrity of total RNA. lncRNA microarray Expression profiling research was performed on RNA from four individuals with adenomyosis as well as four controls. The microarray services were provided by Gminix Biotechnology Company of Shanghai, China. The Affymetrix Human Transcriptome Array 2.0 (Part number: 902162, Affymetrix, California, USA) was used in this study and the Affymetrix array platform was employed for microarray analysis. According to the standard Affymetrix protocol, biotinylated cDNA were produced from 500 ng total RNA by using the Ambion1 Whole Transcript (WT) Expression Kit. Affymetrix Human Transcriptome Array 2.0 array were hybridized with 20 mg cDNA according to Affymetrix recommendations using the Affymetrix labeling and hybridization kits. After washing and staining, genechips were scanned using Affymetrix1 GeneChip Command Console (AGCC) which was installed in GeneChip1 Scanner 3000 7G. After the scan was completed, AGCC saved the image data and computed the probe intensity data. When the probe intensity data
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(.cel file) had been generated, probe summarization was performed in the Expression Console Software, which could be downloaded free from the Affymetrix website. Microarray data analysis We applied random variance model t-test to filter the differentially expressed genes for the experiment group and the control group [15]. After false discovery rate (FDR) analysis and the significant analysis, we applied the P value threshold to identify the differentially expressed genes. P value < 0.05 was considered as significant difference. Hierarchical clustering was carried out to show the different lncRNA and mRNA expression profiles among samples. The microarray data of this research have been uploaded on Gene Expression Omnibus public database (accession number GSE68870). Functional group analysis The Gene Ontology (GO) can organize genes into hierarchical categories [16]. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis is a functional analysis for mRNAs [17]. In this study, we applied GO analysis to find the primary function of the differential expression mRNAs involved and used pathway analysis to find out the significant pathway of the differential genes participating. Fisher’s exact test and chi-square test were used to select the significant GO categories or significant pathway. The threshold of significance was defined by P value and false discovery rate, and the recommended P value cut-off was 0.05. Co-expression network construction To identify the correlation between mRNA and lncRNA, we constructed a lncRNA-mRNA co-expression network for each group. This co-expression network was built based on the normalized signal intensity of the expression in mRNA and lncRNA. For each pair of mRNA-lncRNA, mRNA-mRNA or lncRNA-lncRNA, we firstly calculated the Pearson correlation, and then constructed the network by choosing the significant correlation pairs [18]. In a co-expression network analysis, degree centrality, defined as the number of directly linked neighbors, is the most important and simplest measure of a mRNA or lncRNA centrality within a network that determines the relative significance. Core regulatory factors between two class samples were determined by the degree differences in two co-expression networks [19]. They always exhibit the biggest degree differences. In this study, we recorded the degree as Exp_Degree in endometria of adenomyosis, which was recorded as Con_Degree in the control group. In order to find out core regulatory factors, we performed normalization of the degree [normalized degree(i) = degree(i)/ degree(max)] to exclude other genes’ impact in each co-expression network and calculated the difference value of a gene’s normalized degree (jDiffKj) between the two co-expression networks [20]. LncRNA qRT-PCR Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to verify the expression level of randomly chosen lncRNAs. Total RNA extracted from eutopic endometria of 20 patients with adenomyosis and from the endometria of 16 women without adenomyosis was reverse-transcribed to cDNA using SuperScript III Transcript (Life Technologies, California, USA) according to the manufacturer’s protocol. The expression of three up-regulated lncRNAs and three down-regulated lncRNAs was measured by qRT-PCR, and ACTB was used as an internal control.
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J.F. Jiang et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 199 (2016) 32–37
The following primers were designed using Primer version 5.0: n333955, Forward: 50 -ACCAGTGCAGGCTGCCTATC-30 , Reverse: 50 -GGCAGAATCCAGATGCTCAA-30 ; n337373, Forward: 50 -GCTACCGAGGCTCCAGCTTA-30 , Reverse: 50 -CCCGGTCAACTTCAAGTCCTA-30 ; n338909, Forward: 50 -GGCCTGATGTTCTGCCTGAT-30 , Reverse: 50 CTGCGTCACGTGGATGTAGC-30 ; n341651, Forward: 50 -AGCAAAGGCCATCTTCCAGATA-30 , Reverse: 50 -CAGAGAACTGGCCTGCTAGGA-30 ; n342794, Forward: 50 -TTGTCCGTTCAGCCATGAAG-30 , Reverse: 50 AGCCACACGTTGACCAGAAG-30 ; n387706, Forward: 50 -AGCAGGACGAGCATGGCTAC-30 , Reverse: 50 -TTGGACTGCGTGGCTAGCTT-30 ; ACTB, Forward: 50 -GCCGTGGTGGTGAAGCTGT-30 , Reverse: 50 ACCCACACTGTGCCCATCTA-30 . Reactions were conducted using the 7500 Real-Time PCR System (Applied Biosystems Inc., Foster City, CA). The reaction mixture (20 ml) contained 1ul template cDNA, 0.3 ml SYBR Green I (Life Technologies, California, USA), 1 ml Mg2+, 0.5 ml forward primer, 0.5 ml reverse primer, 0.5 ml dNTPs, 0.2 ml Platinum1 Taq DNA Polymerase, 2 ml PCR buffer, and 14 ml nuclease-free water. The reactions were incubated at 95 8C for 2 min, followed by 40 cycles of 95 8C for 5 s, 60 8C for 40 s. All experiments were completed in triplicate. The expression level of target lncRNA was calculated by the 24Ct method. Analysis using Student’s t-test was performed in SPSS (SPSS, version 20.0, Chicago, IL) and P value <0.05 was defined as significant difference.
Results Differentially expressed lncRNAs and mRNAs In the current study, 165 lncRNAs were found with aberrant expression in the endometria of patients with adenomyosis compared to controls. Among these lncRNAs, 117 were downregulated and 48 were up-regulated (P value <0.05). The top 10 upregulated and down-regulated lncRNAs are listed in Table 1. The hierarchical clustering displayed variations of the expression of lncRNAs among the samples and homogeneity within groups (Fig. 1). From the mRNA expression profiling data, 612 mRNAs were identified as differentially expressed between two groups. Among these mRNAs, 414 were obviously down-regulated and 198 were obviously up-regulated (P value <0.05). The top 10 up-regulated and down-regulated mRNAs were listed in Table 1.
Functional group analysis In the GO analysis, it was found that up-regulated transcripts were highly enriched for axon guidance, signal transduction, small GTPase mediated signal transduction, extracellular matrix organization. The highest enriched GOs of down-regulated coding genes were gene expression, mitotic cell cycle, DNA replication, and RNA metabolic process. KEGG pathway analysis showed that up-regulated coding genes participated in 40 pathways, and the most enriched pathways were MAPK signaling pathway, Focal adhesion, Adherens junction, ECMreceptor interaction, and PI3K-Akt signaling pathway (Fig. 2). With respect to down-regulated transcripts, 39 pathways corresponded, and the most enriched were DNA replication and cell cycle (Fig. 3). Co-expression network construction Based on the normalized signal intensity of coding gene and lncRNA expression, we respectively constructed a mRNA-lncRNA co-expression network for each group. The structure of the coexpression networks of endometria samples with and without adenomyosis was significantly different. The pivotal change of coexpression status of a gene between the two co-expression networks suggests that the expression of the gene very possibly undertakes a critical change in the pathogenesis of adenomyosis. In this study, we have got a list of genes that may play primary roles in the pathogenesis of adenomyosis by calculating the changes of coexpression status of genes (see Table 2). These genes are worthy of further study. LncRNA qRT-PCR Validation The qRT-PCR results demonstrated that the expression of n341651, n342794, and n387706 were obviously increased in the eutopic endometria of adenomyosis compared with the normal endometria (P < 0.05), whereas the expression level of n333955, n337373, and n338909 were significantly decreased (P < 0.05). The results were consistent with the microarray experiment data (Fig. 4). Comments The mechanism of adenomyosis genesis is complicated [3]. Previous research identified alterations at the molecular level
Table 1 The top 10 up-regulated and down-regulated lncRNAs and mRNAs in the endometria identified by microarray test (adenomyosis vs normal). lncRNA Accession number
Regulation
Fold change
mRNA gene symbol
Regulation
Fold change
n342839 n336615 n335092 n387706 n339991 n410159 n335251 n338918 TCONS_l2_00002951 n333546 n333955 n337373 n4541 TCONS_l2_00013366 n334090 n335557 n386477 NR_002960 n338909 TCONS_00022823
Up Up Up Up Up Up Up Up Up Up Down Down Down Down Down Down Down Down Down Down
4.13 3.67 3.54 3.54 3.05 2.7 2.66 2.64 2.5 2.49 0.0032 0.028 0.14 0.22 0.22 0.27 0.31 0.31 0.33 0.33
THBS2 PRUNE2 CD44 ITGA1 A2M SVIL HSPB1 MATN2 RERG COL4A1 HBA1 HBA2 HBB KIAA1210 HIST1H3B HIST1H3F GZMA HIST1H2AB CACNA1D KLRB1
Up Up Up Up Up Up Up Up Up Up Down Down Down Down Down Down Down Down Down Down
5.14 4.22 3.94 3.64 3.06 3.01 2.81 2.79 2.76 2.71 0.02 0.024 0.055 0.18 0.19 0.21 0.22 0.25 0.26 0.27
J.F. Jiang et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 199 (2016) 32–37
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Fig. 1. (A) Hierarchical clustering of 8 specimens according to the 612 aberrantly expressed mRNA. B. Hierarchical clustering of 8 specimens according to the 165 differentially expressed lncRNAs (A1–A4 represent four samples from women with adenomyosis, C1–C4 represent four samples from subjects without adenomyosis).
in the eutopic endometria of women with adenomyosis. A series of molecular and metabolic changes, which include active angiogenesis, increased proliferation, decreased apoptosis, and an impaired cytokine expression play a role in pathogenesis and development of adenomyosis by enhancing the ability of the endometria to infiltrate the junctional zone myometrium [5]. Most of these alterations seem to be in the proliferative phase. Recently, lncRNAs have been confirmed to regulate gene expression at almost every level and play an essential role in cellular proliferation, development and metabolism [21–23]. Given their role in these processes, lncRNAs may be biologically plausible contributors to the
endometria alterations in adenomyosis. The exploration of lncRNA expression profile in the eutopic endometria will help in understanding the pathogenesis of adenomyosis. In this study, we identified the differentially expressed lncRNAs and mRNAs between the experiment group and the control group, including 165 lncRNAs and 612 mRNAs. The two group samples could be clearly differentiated by Hierarchical Clustering. Our results demonstrated that the eutopic endometria in the proliferative phase of women with adenomyosis is altered at the level of gene expression. For mRNAs, another study has obtained the gene expression profile of endometria samples from women with adenomyosis and healthy subjects [24]. The result shown was that they found only 34 genes dysregulated between two groups with
Fig. 2. Pathways corresponding to up-regulated transcripts.
Fig. 3. Pathways corresponded to down-regulated transcripts.
J.F. Jiang et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 199 (2016) 32–37
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Table 2 Potential key genes identified by lncRNA-mRNA co-expression network analysis. lncRNA/mRNA
Biotype
Regulation
Exp_Degree
Con_ Degree
jDiffKj
TLN1 n342794 MYBL1 CCND2 ENST00000406939 n338909 ENST00000435815 PHF5A PRIM1 SCNN1B n341651 ARHGDIA n341158 n387706 RAD51C USP1 PPIL1 n337123 n337373 ENST00000365180
Coding Noncoding Coding Coding Noncoding Noncoding Noncoding Coding Coding Coding Noncoding Coding Noncoding Noncoding Coding Coding Coding Noncoding Noncoding Noncoding
Up Up Down Down Down Down Down Down Down Up Up Up Up Up Down Down Down Down Down Down
25 4 4 6 9 7 5 4 7 7 6 5 5 4 4 4 8 8 3 7
4 18 16 17 19 17 15 14 16 16 15 14 14 13 13 13 16 16 12 15
0.79 0.79 0.68 0.65 0.64 0.61 0.59 0.58 0.56 0.56 0.55 0.54 0.54 0.52 0.52 0.52 0.52 0.52 0.51 0.51
nonparametric tests. Given the design of these two studies, there were several reasons that could explain the discrepancy of the results. Firstly, the diagnosis of adenomyosis was confirmed by histopathological examination, and all types of endometriosis were excluded in both groups in the current study, whereas the previous study only used MRI and TVS to diagnosis adenomyosis, and peritoneal endometriosis and deep infiltrating endometriosis were not excluded in the control group. Secondly, in the previous study, the endometrial samples were collected during the window of implantation, while we included endometria in the proliferative phase. These differences may yield different results as mentioned by Horcajadas et al. [25]. For lncRNAs, one study determined the ectopic and eutopic endometrial lncRNA expression levels in patients with endometriosis. They found nearly 1000 dysregulated lncRNA transcripts in ectopic endometrial tissue [26]. To our knowledge, there is no study to evaluate the differentially expressed lncRNAs in eutopic endometria in women with, versus without, adenomyosis or endometriosis. Among the differentially expressed lncRNAs in this study, few of them have been functionally annotated. In order to further validate the results of microarray, we randomely selected six differentially expressed lncRNAs and detected their expressions level with qRT-PCR. Our results of qRT-PCR demonstrated a strong
consistency with the microarray, thus proving the reliability of our microarray data. Unlike mRNA and highly conserved microRNA, a difficulty in lncRNA study is that only a small fraction of known lncRNAs have been functional annotated [27]. However, the functions of many mRNA have been well described in pathophysiology conditions. The most common way to predict the functions of lncRNA is to construct a correlation between lncRNA and mRNA. The functions of lncRNA could be indirectly determined by the co-expressed mRNA and related biological pathways [28]. By clustering these differentially expressed mRNAs into GO and KEGG pathway annotations, we made a systematic analysis for the functions of the mRNAs. According to GO analysis, we found that the mRNAs displayed abnormal expression in eutopic endometria of adenomyosis enriched for gene expression, DNA replication, and extracellular matrix organization. Meanwhile, the results of pathway analysis showed that the lncRNA may participate in Focal adhesion, PI3K-Akt signaling pathway, oxytocin signaling pathway and VEGF signaling pathway. Some of them are key pathways in the pathogenesis of adenomyosis. VEGF signaling pathway is related with MMPs, which play a important role in the angiogenesis of endometria [5]. Among these pathways, ‘‘PI3K-Akt signaling pathway’’ has been found to be involved in the pathogenesis of adenomyosis. The increased expression of DJ-1 could regulate cell proliferation, migration, and angiogenesis by modulating PI3K/Akt/mTOR signaling pathway [29]. Our results provided new possible signaling pathway for further pathway studies in the pathogenesis of adenomyosis. In the next step, the main problem is how to identify the potential core genes associated with adenomyosis pathogenesis from these differentially expressed genes. Carlson et al. have shown that application of gene co-expression networks can predict the importance of a relative gene for a particular process [19]. In the present study, we have constructed the co-expression network of experimental group and control group respectively, and produced a list of lncRNAs and mRNAs that may closely relate to the pathogenesis of adenomyosis (see Table 2). The function of these lncRNAs and mRNAs, and their association, provide biological foundations for further researches. For example, recent studies have implicated that aberrant expression of Talin1 (TLN1), which is one of the potential core genes associated with adenomyosis pathogenesis found in our study, plays an important role in adhesion, migration, invasion, cellular survival and proliferation [30]. It is possible that TLN1 plays a role in pathogenesis of adenomyosis. However, we need to do further research to support this hypothesis. There are several limitations in our study. The first one is that the sample size is small and further study will have to be conducted with a larger sample size. Another limitation is that we have not provided experimental evidence to demonstrate the functional link between lncRNA and mRNA. Nevertheless, we plan to do further research to illustrate the functional roles of these lncRNAs and mRNAs in the pathogenesis of adenomyosis.
Conclusion
Fig. 4. Comparison between microarray and qRT-PCR results. The height of each column in this graph represents the log-transformed mean fold changes in the expression of lncRNA between adenomyosis group and normal group.
In conclusions, this study is the first time screening of lncRNA expression profiling in the eutopic endometria tissue from women with, versus without, adenomyosis using gene microarrays. Furthermore, this study, for the first time, focuses on the mechanisms of pathogenesis of adenomyosis at lncRNA level. Some of these lncRNAs may play roles in the development of adenomyosis through regulating the transcription of related coding genes. The results are an important resource for further mechanistic studies in this area.
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Conflict of interest The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding statement This work was supported by the National Natural Science Foundation of China (grant numbers 11471024). References [1] Ferenczy A. Pathophysiology of adenomyosis. Hum Reprod Update 1998;4(4): 312–22. [2] Koike N, Tsunemi T, Uekuri C, et al. Pathogenesis and malignant transformation of adenomyosis (review). Oncol Rep 2013;29(3):861–7. [3] Benagiano G, Habiba M, Brosens I. The pathophysiology of uterine adenomyosis: an update. Fertil Steril 2012;98(3):572–9. [4] Benagiano G, Brosens I, Habiba M. Structural and molecular features of the endomyometrium in endometriosis and adenomyosis. Hum Reprod Update 2014;20(3):386–402. [5] Benagiano G, Brosens I. The endometrium in adenomyosis. Womens Health (Lond Engl) 2012;8(3):301–12. [6] Wapinski O, Chang HY. Long noncoding RNAs and human disease. Trends Cell Biol 2011;21(6):354–61. [7] Vance KW, Ponting CP. Transcriptional regulatory functions of nuclear long noncoding RNAs. Trends Genet 2014;30(8):348–55. [8] Wang L, Fu D, Qiu Y, et al. Genome-wide screening and identification of long noncoding RNAs and their interaction with protein coding RNAs in bladder urothelial cell carcinoma. Cancer Lett 2014;349(1):77–86. [9] Yang J, Lin J, Liu T, et al. Analysis of lncRNA expression profiles in non-small cell lung cancers (NSCLC) and their clinical subtypes. Lung Cancer 2014;85(2): 110–5. [10] Malouf GG, Zhang J, Yuan Y, et al. Characterization of long non-coding RNA transcriptome in clear-cell renal cell carcinoma by next-generation deep sequencing. Mol Oncol 2015;9(1):32–43. [11] Wang K, Liu F, Zhou LY, et al. The long noncoding RNA CHRF regulates cardiac hypertrophy by targeting miR-489. Circ Res 2014;114(9):1377–88. [12] Yan B, Tao ZF, Li XM, Zhang H, Yao J, Jiang Q. Aberrant expression of long noncoding RNAs in early diabetic retinopathy. Invest Ophthalmol Vis Sci 2014;55(2):941–51.
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[13] Wan ZY, Song F, Sun Z, et al. Aberrantly expressed long noncoding RNAs in human intervertebral disc degeneration: a microarray related study. Arthritis Res Ther 2014;16(5):465. [14] Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Am J Obstet Gynecol 1975;122(2):262–3. [15] Wright GW, Simon RM. A random variance model for detection of differential gene expression in small microarray experiments. Bioinformatics 2003;19(2): 2448–55. [16] Gene Ontology C. The Gene Ontology (GO) project in 2006. Nucleic Acids Res 2006;34:D322–6. [17] Kanehisa M, Goto S, Kawashima S, Okuno Y, Hattori M. The KEGG resource for deciphering the genome. Nucleic Acids Res 2004;32:D277–80. [18] Prieto C, Risueno A, Fontanillo C, De las Rivas J. Human gene coexpression landscape: confident network derived from tissue transcriptomic profiles. PLoS ONE 2008;3(12):e3911. [19] Carlson MR, Zhang B, Fang Z, Mischel PS, Horvath S, Nelson SF. Gene connectivity, function, and sequence conservation: predictions from modular yeast co-expression networks. BMC Genomics 2006;7:40. [20] Yan Y, Zhang L, Jiang Y, et al. LncRNA and mRNA interaction study based on transcriptome profiles reveals potential core genes in the pathogenesis of human glioblastoma multiforme. J Cancer Res Clin Oncol 2015;141(5):827–38. [21] Grote P, Herrmann BG. Long noncoding RNAs in organogenesis: making the difference. Trends Genet 2015;31(6):329–35. [22] Ng SY, Lin L, Soh BS, Stanton LW. Long noncoding RNAs in development and disease of the central nervous system. Trends Genet 2013;29(8):461–8. [23] Hung T, Wang Y, Lin MF, et al. Extensive and coordinated transcription of noncoding RNAs within cell-cycle promoters. Nat Genet 2011;43(7):621–9. [24] Martinez-Conejero JA, Morgan M, Montesinos M, et al. Adenomyosis does not affect implantation, but is associated with miscarriage in patients undergoing oocyte donation. Fertil Steril 2011;96(4):943–50. [25] Horcajadas JA, Pellicer A, Simo´n C. Wide genomic analysis of human endometrial receptivity: new times, new opportunities. Hum Reprod Update 2007;13(1):77–86. [26] Sun PR, Jia SZ, Lin H, Leng JH, Lang JH. Genome-wide profiling of long noncoding ribonucleic acid expression patterns in ovarian endometriosis by microarray. Fertil Steril 2014;101(4):1038–46. [27] St Laurent G, Wahlestedt C, Kapranov P. The landscape of long noncoding RNA classification. Trends Genet 2015;31(5):239–51. [28] Guttman M, Amit I, Garber M, et al. Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 2009;458(7235):223–7. [29] Guo J, Gao J, Yu X, Luo H, Xiong X, Huang O. Expression of DJ-1 and mTOR in eutopic and ectopic endometria of patients with endometriosis and adenomyosis. Gynecol Obstet Invest 2015;79(3):195–200. [30] Sakamoto S, McCann RO, Dhir R, Kyprianou N. Talin1 promotes tumor invasion and metastasis via focal adhesion signaling and anoikis resistance. Cancer Res 2010;70(5):1885–95.
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European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Aberrantly expressed long noncoding RNAs in the eutopic endometria of patients with uterine adenomyosis§ Jian Fa Jiang, Ai Jun Sun *, Wei Xue, Yan Deng, Yan Fang Wang Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 October 2015 Received in revised form 7 January 2016 Accepted 29 January 2016
Objective: Adenomyosis is a common gynecologic disease. Alterations in the eutopic endometria might play an important role in the pathogenesis of adenomyosis. Long non-coding RNA (lncRNA) represents key regulators of gene expression. Our goal was to identify differentially expressed long noncoding RNA and messenger RNA (mRNA) in the eutopic endometria of subjects with adenomyosis on a genome-wide scale. Study design: The expression level of lncRNAs and mRNAs in the eutopic endometria from women with adenomyosis and from that of normal control subjects were detected by Affymetrix Human Transcriptome Array 2.0. Bioinformatics analysis was done for further investigation. Three up-regulated and three down-regulated lncRNAs were randomly chosen for validation by quantitative real-time polymerase chain reaction. Results: A total of 165 lncRNAs and 612 mRNAs were aberrantly expressed in the eutopic endometria of subjects with adenomyosis. Pathway analysis indicated that 40 pathways corresponded to up-regulated transcripts and 39 pathways corresponded to downregulated transcripts. A list of genes that might play roles in the pathogenesis of adenomyosis were produced by comparing the difference between coexpression networks. Expression of six chosen lncRNAs was validated by quantitative real-time polymerase chain reaction. Conclusion: This study show for the first time that the lncRNA expression profile is altered in women with adenomyosis and provides new biological foundations for further mechanistic studies in this enigmatic disorder. ß 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Adenomyosis Endometria Long noncoding RNA Microarray
Introduction Adenomyosis is a common gynecologic disease characterized by ectopic endometrial glands and stroma invading the myometrium, associated with adjacent myometrial hypertrophy and hyperplasia [1]. It is clinically characterized by dysmenorrhea, abnormal uterine bleeding, and chronic pelvic pain, and seriously affect the quality of life. However, the pathogenesis of adenomyosis remains unclear [2]. The basal layer of the endometrium invaginates between smooth muscle cell bundles or along lymphatic vessels deep into the myometrium, and constitutes the most widely accepted theory as to the etiology of adenomyosis
§ The work reported was done in Peking Union Medical College Hospital and Gminix Biotechnology Company of Shanghai, People’s Republic of China. * Corresponding author at: Shuaifuyuan no. 1, Dongcheng District, Beijing 100730, China. Tel.: +86 18600045466; fax: +86 01069156039. E-mail address: [email protected] (A.J. Sun).
http://dx.doi.org/10.1016/j.ejogrb.2016.01.033 0301-2115/ß 2016 Elsevier Ireland Ltd. All rights reserved.
[3]. Many studies have shown that a heritable or acquired alteration in the eutopic endometria may play a significant role in the pathogenesis and development of adenomyosis [4,5]. The identification of molecular differences within eutopic endometria of women with adenomyosis is a very important step in our understanding of the pathogenesis of adenomyosis. Previous studies have demonstrated that genetic factors play pivotal roles in the pathogenesis of adenomyosis, partially through epigenetic regulation [4]. Recently, research has shown that long noncoding RNA (lncRNA), defined as RNA molecules >200 nucleotides in length, may function in every aspect of human biology and contribute to the development of various benign or malignant diseases [6]. Functional analysis of lncRNAs has revealed their multi-faceted roles in the regulation of gene expression, from epigenetic modulation in the nucleus to post-transcriptional control in the cytoplasm [7]. Recent studies have shown that lncRNA expression profile is very different in many human conditions, including cancer [8–10], cardiovascular disease [11], metabolic disease [12] and degenerative diseases [13]. But the
J.F. Jiang et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 199 (2016) 32–37
lncRNA expression profile and its potential biological function in adenomyosis is unknown. Our study is the first study of lncRNA expression profile in women with adenomyosis. We firstly constructed the lncRNA and mRNA expression profile in eutopic endometria from adenomyosis patients and from that of normal control subjects. In addition, we conducted functional analysis and constructed co-expression networks to find the potential core genes associated with adenomyosis pathogenesis. Materials and methods Sample collection This study was conducted according to the principles expressed in the Declaration of Helsinki and was approved by the Ethics Review Board of The Peking Union Medical College Hospital, Beijing, China. Written informed consent was obtained from each participant before surgery. The eutopic endometria was obtained from 20 women with adenomyosis during hysterectomy (mean age, 44.7 years). The diagnosis of adenomyosis was confirmed by histopathological examination after operation. Normal endometrial tissues were collected from 16 women found to be free of adenomyosis or endometriosis during surgery (mean age, 44.4 years). Women whose endometria acted as controls were undergoing hysterectomy for symptomatic uterine leiomyomas, none of which were submucosal in location. All subjects in both groups had not received any hormonal treatment within three months of endometrial sampling and had regular menstrual cycles (25–35 days). All endometrial samples were in the proliferative phase according to the menstrual cycle phases and the criteria of endometrial histologic dating [14]. Review of pathology reports revealed no evidence of endometrial hyperplasia or inflammation in any of the samples of two groups. All endometria tissue were snap-frozen using liquid nitrogen after surgical resection and preserved in a 80 8C freezer until use. RNA extraction and quality control assay Total RNA of each frozen endometria was extracted using TRIzol1 Reagent (Life Technologies, California, USA) according to the manufacturer’s protocol. We applied a NanoDrop ND-2000 spectrophotometer to measure the quantity and quality of total RNA, and used standard denaturing agarose gel electrophoresis to test the integrity of total RNA. lncRNA microarray Expression profiling research was performed on RNA from four individuals with adenomyosis as well as four controls. The microarray services were provided by Gminix Biotechnology Company of Shanghai, China. The Affymetrix Human Transcriptome Array 2.0 (Part number: 902162, Affymetrix, California, USA) was used in this study and the Affymetrix array platform was employed for microarray analysis. According to the standard Affymetrix protocol, biotinylated cDNA were produced from 500 ng total RNA by using the Ambion1 Whole Transcript (WT) Expression Kit. Affymetrix Human Transcriptome Array 2.0 array were hybridized with 20 mg cDNA according to Affymetrix recommendations using the Affymetrix labeling and hybridization kits. After washing and staining, genechips were scanned using Affymetrix1 GeneChip Command Console (AGCC) which was installed in GeneChip1 Scanner 3000 7G. After the scan was completed, AGCC saved the image data and computed the probe intensity data. When the probe intensity data
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(.cel file) had been generated, probe summarization was performed in the Expression Console Software, which could be downloaded free from the Affymetrix website. Microarray data analysis We applied random variance model t-test to filter the differentially expressed genes for the experiment group and the control group [15]. After false discovery rate (FDR) analysis and the significant analysis, we applied the P value threshold to identify the differentially expressed genes. P value < 0.05 was considered as significant difference. Hierarchical clustering was carried out to show the different lncRNA and mRNA expression profiles among samples. The microarray data of this research have been uploaded on Gene Expression Omnibus public database (accession number GSE68870). Functional group analysis The Gene Ontology (GO) can organize genes into hierarchical categories [16]. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis is a functional analysis for mRNAs [17]. In this study, we applied GO analysis to find the primary function of the differential expression mRNAs involved and used pathway analysis to find out the significant pathway of the differential genes participating. Fisher’s exact test and chi-square test were used to select the significant GO categories or significant pathway. The threshold of significance was defined by P value and false discovery rate, and the recommended P value cut-off was 0.05. Co-expression network construction To identify the correlation between mRNA and lncRNA, we constructed a lncRNA-mRNA co-expression network for each group. This co-expression network was built based on the normalized signal intensity of the expression in mRNA and lncRNA. For each pair of mRNA-lncRNA, mRNA-mRNA or lncRNA-lncRNA, we firstly calculated the Pearson correlation, and then constructed the network by choosing the significant correlation pairs [18]. In a co-expression network analysis, degree centrality, defined as the number of directly linked neighbors, is the most important and simplest measure of a mRNA or lncRNA centrality within a network that determines the relative significance. Core regulatory factors between two class samples were determined by the degree differences in two co-expression networks [19]. They always exhibit the biggest degree differences. In this study, we recorded the degree as Exp_Degree in endometria of adenomyosis, which was recorded as Con_Degree in the control group. In order to find out core regulatory factors, we performed normalization of the degree [normalized degree(i) = degree(i)/ degree(max)] to exclude other genes’ impact in each co-expression network and calculated the difference value of a gene’s normalized degree (jDiffKj) between the two co-expression networks [20]. LncRNA qRT-PCR Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to verify the expression level of randomly chosen lncRNAs. Total RNA extracted from eutopic endometria of 20 patients with adenomyosis and from the endometria of 16 women without adenomyosis was reverse-transcribed to cDNA using SuperScript III Transcript (Life Technologies, California, USA) according to the manufacturer’s protocol. The expression of three up-regulated lncRNAs and three down-regulated lncRNAs was measured by qRT-PCR, and ACTB was used as an internal control.
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The following primers were designed using Primer version 5.0: n333955, Forward: 50 -ACCAGTGCAGGCTGCCTATC-30 , Reverse: 50 -GGCAGAATCCAGATGCTCAA-30 ; n337373, Forward: 50 -GCTACCGAGGCTCCAGCTTA-30 , Reverse: 50 -CCCGGTCAACTTCAAGTCCTA-30 ; n338909, Forward: 50 -GGCCTGATGTTCTGCCTGAT-30 , Reverse: 50 CTGCGTCACGTGGATGTAGC-30 ; n341651, Forward: 50 -AGCAAAGGCCATCTTCCAGATA-30 , Reverse: 50 -CAGAGAACTGGCCTGCTAGGA-30 ; n342794, Forward: 50 -TTGTCCGTTCAGCCATGAAG-30 , Reverse: 50 AGCCACACGTTGACCAGAAG-30 ; n387706, Forward: 50 -AGCAGGACGAGCATGGCTAC-30 , Reverse: 50 -TTGGACTGCGTGGCTAGCTT-30 ; ACTB, Forward: 50 -GCCGTGGTGGTGAAGCTGT-30 , Reverse: 50 ACCCACACTGTGCCCATCTA-30 . Reactions were conducted using the 7500 Real-Time PCR System (Applied Biosystems Inc., Foster City, CA). The reaction mixture (20 ml) contained 1ul template cDNA, 0.3 ml SYBR Green I (Life Technologies, California, USA), 1 ml Mg2+, 0.5 ml forward primer, 0.5 ml reverse primer, 0.5 ml dNTPs, 0.2 ml Platinum1 Taq DNA Polymerase, 2 ml PCR buffer, and 14 ml nuclease-free water. The reactions were incubated at 95 8C for 2 min, followed by 40 cycles of 95 8C for 5 s, 60 8C for 40 s. All experiments were completed in triplicate. The expression level of target lncRNA was calculated by the 24Ct method. Analysis using Student’s t-test was performed in SPSS (SPSS, version 20.0, Chicago, IL) and P value <0.05 was defined as significant difference.
Results Differentially expressed lncRNAs and mRNAs In the current study, 165 lncRNAs were found with aberrant expression in the endometria of patients with adenomyosis compared to controls. Among these lncRNAs, 117 were downregulated and 48 were up-regulated (P value <0.05). The top 10 upregulated and down-regulated lncRNAs are listed in Table 1. The hierarchical clustering displayed variations of the expression of lncRNAs among the samples and homogeneity within groups (Fig. 1). From the mRNA expression profiling data, 612 mRNAs were identified as differentially expressed between two groups. Among these mRNAs, 414 were obviously down-regulated and 198 were obviously up-regulated (P value <0.05). The top 10 up-regulated and down-regulated mRNAs were listed in Table 1.
Functional group analysis In the GO analysis, it was found that up-regulated transcripts were highly enriched for axon guidance, signal transduction, small GTPase mediated signal transduction, extracellular matrix organization. The highest enriched GOs of down-regulated coding genes were gene expression, mitotic cell cycle, DNA replication, and RNA metabolic process. KEGG pathway analysis showed that up-regulated coding genes participated in 40 pathways, and the most enriched pathways were MAPK signaling pathway, Focal adhesion, Adherens junction, ECMreceptor interaction, and PI3K-Akt signaling pathway (Fig. 2). With respect to down-regulated transcripts, 39 pathways corresponded, and the most enriched were DNA replication and cell cycle (Fig. 3). Co-expression network construction Based on the normalized signal intensity of coding gene and lncRNA expression, we respectively constructed a mRNA-lncRNA co-expression network for each group. The structure of the coexpression networks of endometria samples with and without adenomyosis was significantly different. The pivotal change of coexpression status of a gene between the two co-expression networks suggests that the expression of the gene very possibly undertakes a critical change in the pathogenesis of adenomyosis. In this study, we have got a list of genes that may play primary roles in the pathogenesis of adenomyosis by calculating the changes of coexpression status of genes (see Table 2). These genes are worthy of further study. LncRNA qRT-PCR Validation The qRT-PCR results demonstrated that the expression of n341651, n342794, and n387706 were obviously increased in the eutopic endometria of adenomyosis compared with the normal endometria (P < 0.05), whereas the expression level of n333955, n337373, and n338909 were significantly decreased (P < 0.05). The results were consistent with the microarray experiment data (Fig. 4). Comments The mechanism of adenomyosis genesis is complicated [3]. Previous research identified alterations at the molecular level
Table 1 The top 10 up-regulated and down-regulated lncRNAs and mRNAs in the endometria identified by microarray test (adenomyosis vs normal). lncRNA Accession number
Regulation
Fold change
mRNA gene symbol
Regulation
Fold change
n342839 n336615 n335092 n387706 n339991 n410159 n335251 n338918 TCONS_l2_00002951 n333546 n333955 n337373 n4541 TCONS_l2_00013366 n334090 n335557 n386477 NR_002960 n338909 TCONS_00022823
Up Up Up Up Up Up Up Up Up Up Down Down Down Down Down Down Down Down Down Down
4.13 3.67 3.54 3.54 3.05 2.7 2.66 2.64 2.5 2.49 0.0032 0.028 0.14 0.22 0.22 0.27 0.31 0.31 0.33 0.33
THBS2 PRUNE2 CD44 ITGA1 A2M SVIL HSPB1 MATN2 RERG COL4A1 HBA1 HBA2 HBB KIAA1210 HIST1H3B HIST1H3F GZMA HIST1H2AB CACNA1D KLRB1
Up Up Up Up Up Up Up Up Up Up Down Down Down Down Down Down Down Down Down Down
5.14 4.22 3.94 3.64 3.06 3.01 2.81 2.79 2.76 2.71 0.02 0.024 0.055 0.18 0.19 0.21 0.22 0.25 0.26 0.27
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Fig. 1. (A) Hierarchical clustering of 8 specimens according to the 612 aberrantly expressed mRNA. B. Hierarchical clustering of 8 specimens according to the 165 differentially expressed lncRNAs (A1–A4 represent four samples from women with adenomyosis, C1–C4 represent four samples from subjects without adenomyosis).
in the eutopic endometria of women with adenomyosis. A series of molecular and metabolic changes, which include active angiogenesis, increased proliferation, decreased apoptosis, and an impaired cytokine expression play a role in pathogenesis and development of adenomyosis by enhancing the ability of the endometria to infiltrate the junctional zone myometrium [5]. Most of these alterations seem to be in the proliferative phase. Recently, lncRNAs have been confirmed to regulate gene expression at almost every level and play an essential role in cellular proliferation, development and metabolism [21–23]. Given their role in these processes, lncRNAs may be biologically plausible contributors to the
endometria alterations in adenomyosis. The exploration of lncRNA expression profile in the eutopic endometria will help in understanding the pathogenesis of adenomyosis. In this study, we identified the differentially expressed lncRNAs and mRNAs between the experiment group and the control group, including 165 lncRNAs and 612 mRNAs. The two group samples could be clearly differentiated by Hierarchical Clustering. Our results demonstrated that the eutopic endometria in the proliferative phase of women with adenomyosis is altered at the level of gene expression. For mRNAs, another study has obtained the gene expression profile of endometria samples from women with adenomyosis and healthy subjects [24]. The result shown was that they found only 34 genes dysregulated between two groups with
Fig. 2. Pathways corresponding to up-regulated transcripts.
Fig. 3. Pathways corresponded to down-regulated transcripts.
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Table 2 Potential key genes identified by lncRNA-mRNA co-expression network analysis. lncRNA/mRNA
Biotype
Regulation
Exp_Degree
Con_ Degree
jDiffKj
TLN1 n342794 MYBL1 CCND2 ENST00000406939 n338909 ENST00000435815 PHF5A PRIM1 SCNN1B n341651 ARHGDIA n341158 n387706 RAD51C USP1 PPIL1 n337123 n337373 ENST00000365180
Coding Noncoding Coding Coding Noncoding Noncoding Noncoding Coding Coding Coding Noncoding Coding Noncoding Noncoding Coding Coding Coding Noncoding Noncoding Noncoding
Up Up Down Down Down Down Down Down Down Up Up Up Up Up Down Down Down Down Down Down
25 4 4 6 9 7 5 4 7 7 6 5 5 4 4 4 8 8 3 7
4 18 16 17 19 17 15 14 16 16 15 14 14 13 13 13 16 16 12 15
0.79 0.79 0.68 0.65 0.64 0.61 0.59 0.58 0.56 0.56 0.55 0.54 0.54 0.52 0.52 0.52 0.52 0.52 0.51 0.51
nonparametric tests. Given the design of these two studies, there were several reasons that could explain the discrepancy of the results. Firstly, the diagnosis of adenomyosis was confirmed by histopathological examination, and all types of endometriosis were excluded in both groups in the current study, whereas the previous study only used MRI and TVS to diagnosis adenomyosis, and peritoneal endometriosis and deep infiltrating endometriosis were not excluded in the control group. Secondly, in the previous study, the endometrial samples were collected during the window of implantation, while we included endometria in the proliferative phase. These differences may yield different results as mentioned by Horcajadas et al. [25]. For lncRNAs, one study determined the ectopic and eutopic endometrial lncRNA expression levels in patients with endometriosis. They found nearly 1000 dysregulated lncRNA transcripts in ectopic endometrial tissue [26]. To our knowledge, there is no study to evaluate the differentially expressed lncRNAs in eutopic endometria in women with, versus without, adenomyosis or endometriosis. Among the differentially expressed lncRNAs in this study, few of them have been functionally annotated. In order to further validate the results of microarray, we randomely selected six differentially expressed lncRNAs and detected their expressions level with qRT-PCR. Our results of qRT-PCR demonstrated a strong
consistency with the microarray, thus proving the reliability of our microarray data. Unlike mRNA and highly conserved microRNA, a difficulty in lncRNA study is that only a small fraction of known lncRNAs have been functional annotated [27]. However, the functions of many mRNA have been well described in pathophysiology conditions. The most common way to predict the functions of lncRNA is to construct a correlation between lncRNA and mRNA. The functions of lncRNA could be indirectly determined by the co-expressed mRNA and related biological pathways [28]. By clustering these differentially expressed mRNAs into GO and KEGG pathway annotations, we made a systematic analysis for the functions of the mRNAs. According to GO analysis, we found that the mRNAs displayed abnormal expression in eutopic endometria of adenomyosis enriched for gene expression, DNA replication, and extracellular matrix organization. Meanwhile, the results of pathway analysis showed that the lncRNA may participate in Focal adhesion, PI3K-Akt signaling pathway, oxytocin signaling pathway and VEGF signaling pathway. Some of them are key pathways in the pathogenesis of adenomyosis. VEGF signaling pathway is related with MMPs, which play a important role in the angiogenesis of endometria [5]. Among these pathways, ‘‘PI3K-Akt signaling pathway’’ has been found to be involved in the pathogenesis of adenomyosis. The increased expression of DJ-1 could regulate cell proliferation, migration, and angiogenesis by modulating PI3K/Akt/mTOR signaling pathway [29]. Our results provided new possible signaling pathway for further pathway studies in the pathogenesis of adenomyosis. In the next step, the main problem is how to identify the potential core genes associated with adenomyosis pathogenesis from these differentially expressed genes. Carlson et al. have shown that application of gene co-expression networks can predict the importance of a relative gene for a particular process [19]. In the present study, we have constructed the co-expression network of experimental group and control group respectively, and produced a list of lncRNAs and mRNAs that may closely relate to the pathogenesis of adenomyosis (see Table 2). The function of these lncRNAs and mRNAs, and their association, provide biological foundations for further researches. For example, recent studies have implicated that aberrant expression of Talin1 (TLN1), which is one of the potential core genes associated with adenomyosis pathogenesis found in our study, plays an important role in adhesion, migration, invasion, cellular survival and proliferation [30]. It is possible that TLN1 plays a role in pathogenesis of adenomyosis. However, we need to do further research to support this hypothesis. There are several limitations in our study. The first one is that the sample size is small and further study will have to be conducted with a larger sample size. Another limitation is that we have not provided experimental evidence to demonstrate the functional link between lncRNA and mRNA. Nevertheless, we plan to do further research to illustrate the functional roles of these lncRNAs and mRNAs in the pathogenesis of adenomyosis.
Conclusion
Fig. 4. Comparison between microarray and qRT-PCR results. The height of each column in this graph represents the log-transformed mean fold changes in the expression of lncRNA between adenomyosis group and normal group.
In conclusions, this study is the first time screening of lncRNA expression profiling in the eutopic endometria tissue from women with, versus without, adenomyosis using gene microarrays. Furthermore, this study, for the first time, focuses on the mechanisms of pathogenesis of adenomyosis at lncRNA level. Some of these lncRNAs may play roles in the development of adenomyosis through regulating the transcription of related coding genes. The results are an important resource for further mechanistic studies in this area.
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Conflict of interest The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding statement This work was supported by the National Natural Science Foundation of China (grant numbers 11471024). References [1] Ferenczy A. Pathophysiology of adenomyosis. Hum Reprod Update 1998;4(4): 312–22. [2] Koike N, Tsunemi T, Uekuri C, et al. Pathogenesis and malignant transformation of adenomyosis (review). Oncol Rep 2013;29(3):861–7. [3] Benagiano G, Habiba M, Brosens I. The pathophysiology of uterine adenomyosis: an update. Fertil Steril 2012;98(3):572–9. [4] Benagiano G, Brosens I, Habiba M. Structural and molecular features of the endomyometrium in endometriosis and adenomyosis. Hum Reprod Update 2014;20(3):386–402. [5] Benagiano G, Brosens I. The endometrium in adenomyosis. Womens Health (Lond Engl) 2012;8(3):301–12. [6] Wapinski O, Chang HY. Long noncoding RNAs and human disease. Trends Cell Biol 2011;21(6):354–61. [7] Vance KW, Ponting CP. Transcriptional regulatory functions of nuclear long noncoding RNAs. Trends Genet 2014;30(8):348–55. [8] Wang L, Fu D, Qiu Y, et al. Genome-wide screening and identification of long noncoding RNAs and their interaction with protein coding RNAs in bladder urothelial cell carcinoma. Cancer Lett 2014;349(1):77–86. [9] Yang J, Lin J, Liu T, et al. Analysis of lncRNA expression profiles in non-small cell lung cancers (NSCLC) and their clinical subtypes. Lung Cancer 2014;85(2): 110–5. [10] Malouf GG, Zhang J, Yuan Y, et al. Characterization of long non-coding RNA transcriptome in clear-cell renal cell carcinoma by next-generation deep sequencing. Mol Oncol 2015;9(1):32–43. [11] Wang K, Liu F, Zhou LY, et al. The long noncoding RNA CHRF regulates cardiac hypertrophy by targeting miR-489. Circ Res 2014;114(9):1377–88. [12] Yan B, Tao ZF, Li XM, Zhang H, Yao J, Jiang Q. Aberrant expression of long noncoding RNAs in early diabetic retinopathy. Invest Ophthalmol Vis Sci 2014;55(2):941–51.
37
[13] Wan ZY, Song F, Sun Z, et al. Aberrantly expressed long noncoding RNAs in human intervertebral disc degeneration: a microarray related study. Arthritis Res Ther 2014;16(5):465. [14] Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Am J Obstet Gynecol 1975;122(2):262–3. [15] Wright GW, Simon RM. A random variance model for detection of differential gene expression in small microarray experiments. Bioinformatics 2003;19(2): 2448–55. [16] Gene Ontology C. The Gene Ontology (GO) project in 2006. Nucleic Acids Res 2006;34:D322–6. [17] Kanehisa M, Goto S, Kawashima S, Okuno Y, Hattori M. The KEGG resource for deciphering the genome. Nucleic Acids Res 2004;32:D277–80. [18] Prieto C, Risueno A, Fontanillo C, De las Rivas J. Human gene coexpression landscape: confident network derived from tissue transcriptomic profiles. PLoS ONE 2008;3(12):e3911. [19] Carlson MR, Zhang B, Fang Z, Mischel PS, Horvath S, Nelson SF. Gene connectivity, function, and sequence conservation: predictions from modular yeast co-expression networks. BMC Genomics 2006;7:40. [20] Yan Y, Zhang L, Jiang Y, et al. LncRNA and mRNA interaction study based on transcriptome profiles reveals potential core genes in the pathogenesis of human glioblastoma multiforme. J Cancer Res Clin Oncol 2015;141(5):827–38. [21] Grote P, Herrmann BG. Long noncoding RNAs in organogenesis: making the difference. Trends Genet 2015;31(6):329–35. [22] Ng SY, Lin L, Soh BS, Stanton LW. Long noncoding RNAs in development and disease of the central nervous system. Trends Genet 2013;29(8):461–8. [23] Hung T, Wang Y, Lin MF, et al. Extensive and coordinated transcription of noncoding RNAs within cell-cycle promoters. Nat Genet 2011;43(7):621–9. [24] Martinez-Conejero JA, Morgan M, Montesinos M, et al. Adenomyosis does not affect implantation, but is associated with miscarriage in patients undergoing oocyte donation. Fertil Steril 2011;96(4):943–50. [25] Horcajadas JA, Pellicer A, Simo´n C. Wide genomic analysis of human endometrial receptivity: new times, new opportunities. Hum Reprod Update 2007;13(1):77–86. [26] Sun PR, Jia SZ, Lin H, Leng JH, Lang JH. Genome-wide profiling of long noncoding ribonucleic acid expression patterns in ovarian endometriosis by microarray. Fertil Steril 2014;101(4):1038–46. [27] St Laurent G, Wahlestedt C, Kapranov P. The landscape of long noncoding RNA classification. Trends Genet 2015;31(5):239–51. [28] Guttman M, Amit I, Garber M, et al. Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 2009;458(7235):223–7. [29] Guo J, Gao J, Yu X, Luo H, Xiong X, Huang O. Expression of DJ-1 and mTOR in eutopic and ectopic endometria of patients with endometriosis and adenomyosis. Gynecol Obstet Invest 2015;79(3):195–200. [30] Sakamoto S, McCann RO, Dhir R, Kyprianou N. Talin1 promotes tumor invasion and metastasis via focal adhesion signaling and anoikis resistance. Cancer Res 2010;70(5):1885–95.