TY - JOUR
AU - Chang,, Xiaohong
AB - Abstract Endometriosis (EM) is a mysterious and complicated disease that has been found to be multifactorial. Recent studies demonstrated that long noncoding RNAs (lncRNAs) play an important role in the pathogenesis of EM. However, the functional and biological mechanisms of lncRNAs in EM remain unknown. Here, we performed microarray analyses to compare the lncRNA expression profiles of four paired ectopic endometrial (EC) tissues and eutopic endometrial (EU) tissues from patients with ovarian EM. A novel lncRNA, CCDC144NL-AS1, was identified as being potentially functional. CCDC144NL-AS1 expression was upregulated in EC tissues compared to EU and normal endometrial (NE) tissues. Its expression was higher in EC tissues than in EU tissues in 86.7% (26/30) of patients with EM. Despite the lack of a significant increase according to revised American Fertility Society (rAFS) stages, approximately 60% of stage VI EM cases exhibited higher CCDC144NL-AS1 levels, many more than in the stage II–III cases. Subcellular fractionation demonstrated that CCDC144NL-AS1 was localized in the cytoplasm and nucleus of the human EM-derived immortalized endometrial stromal cell line hEM15A. CCDC144NL-AS1 depletion suppressed the migration and invasion of hEM15A cells, but exerted no effects on cell adhesion, proliferation, apoptosis, or cell cycle. Knockdown of CCDC144NL-AS1 dramatically altered the distribution of cytoskeletal filamentous actin (F-actin) stress fibers compared to the negative control group treatment. Western blot analysis revealed that knockdown of CCDC144NL-AS1 attenuated the protein levels of vimentin filaments and MMP-9, but not N-cadherin or β-catenin. Collectively, our results suggest that CCDC144NL-AS1 might be involved in the pathogenesis of EM and provide a novel target for ovarian EM. Introduction Endometriosis (EM) is an ambiguous gynecological disorder that is characterized by the extrauterine presence of endometrium-like tissue, predominantly on the ovaries and pelvic peritoneum. This debilitating and excruciating disease affects 6–10% of women of childbearing age [1], with symptoms of pelvic mass, infertility, and chronic pain (dysmenorrhea, dyspareunia, and dyschezia). Increasing evidence has indicated that the cause of EM is multifactorial and that EM is not simply a local disorder, but is instead a complicated, chronic systemic process [2]. The gold standard for the diagnosis of EM is laparoscopic surgery, which is invasive and expensive. Deficiencies in effective biomarkers may be a significant reason for the lagging diagnosis of this costly disease [3]. Therefore, the mysterious etiologies of EM must be elucidated to facilitate its prevention, diagnosis, and treatment. Over the past few years, genome-wide analyses have identified that only 1% of mammalian genes carry protein-coding potential, and most of the remaining genetic material is transcribed into noncoding RNAs which were long been regarded as genomic “dark matter” or transcriptional noise [4]. Noncoding RNAs larger than 200 nt are generally termed long noncoding RNAs (lncRNAs), which are involved in the pathogenesis and development of different diseases, including EM [5–8]. H19, the first reported lncRNA, plays a critical role in many diseases, including colon cancer [9], abdominal aortic aneurysm [10], cholestatic liver injury [11], and inflammation [12]. Ghazal et al. reported that H19 was significantly decreased in the endometrium of EM patients compared to healthy counterparts, and perturbation of the H19/Let-7/IGF1R regulatory pathway may be instrumental in EM-associated infertility [8]. However, many lncRNAs have not been functionally characterized, and the roles that lncRNAs play in EM are poorly understood. EM is a benign disease that exhibits many malignant characteristics, such as dissemination, implantation, and metastasis, which result from undiscovered pathogenic mechanisms. Many cell migration and invasion-related molecules and processes are associated with the progression of EM. Epithelial-to-mesenchymal transition (EMT) plays a vital role in cell invasion and metastasis. EMT is the process by which epithelial cells lose the polarized organization of the cytoskeleton and cell junctions and acquire mesenchymal phenotypes, such as high motility. These alterations are prerequisites for the initial formation of endometriotic lesions [13]. Degradation of the extracellular matrix (ECM) and basement membrane is thought to be a key step in cell migration and invasion. Matrix metalloproteinases (MMPs) are proteolytic enzymes that facilitate the degradation process and are implicated in EM [14, 15]. Cytoskeletal systems regulate cell migration. A genome-wide association study showed that CDC42, which is known to play role in regulating filamentous actin (F-actin) in the cytoskeleton, was one of the top four genes associated with EM [16]. In the present study, we performed microarrays to identify lncRNA expression profiles in four paired ectopic endometrial (EC) and eutopic endometrial (EU) samples from EM patients. The differentially expressed lncRNAs were randomly selected and validated by quantitative reverse transcription polymerase chain reaction (qPCR). We found that the lncRNA CCDC144NL-AS1 was significantly increased in ectopic tissues. We examined the role of CCDC144NL-AS1 in regulating cytoskeleton reorganization, cell migration, and invasion in a human EM-derived immortalized endometrium stromal cell line, hEM15A, using small-interfering RNA (siRNA) technology. Western blotting was also performed to investigate the effects of CCDC144NL-AS1 knockdown on migration-related proteins. Materials and methods Ethical approval The Medical Ethics Committee of Peking University People's Hospital approved the study protocol. Informed consent was obtained from each patient prior to sampling. Patients and samples The samples and clinical profiles (Table 1) were collected from the Department of Obstetrics and Gynecology of Peking University People's Hospital (Beijing, China) from July 2014 to October 2017. Paired EC and EU samples were obtained from 34 patients with histologically confirmed stage II to IV ovarian EM (IV) during hysteroscopy combined with laparoscopy. Paired samples from four patients (two in the proliferative phase and two in the secretory phase) were used in the microarray analysis, and 30 other paired samples were used for qPCR validation. Simultaneously, 27 normal endometrium (NE) specimens were collected from women who were laparoscopically and/or hysteroscopically proven to be free of EM. The harvested specimens were divided into two parts: one-half were snap-frozen in liquid nitrogen and the other half were used for histological examination. All study participants had regular menstrual cycles and did not receive hormone therapy for at least 3 months prior to surgery. Women with polycystic ovarian syndrome, a co-existing inflammatory disease, or malignancy were excluded from the study. Table 1. Clinical features of study subjects. Study subjects Controls EM na 61 27 34 Age (years) 61 39.9 ± 8.4 32.2 ± 5.2 Gravidity   0 59 5 (20.0) 22 (64.7)   ≥1 20 (80.0) 12 (35.3) Parity   0 61 11 (40.7) 27 (79.4)   ≥1 16 (59.3) 7 (20.6) Cycle phase  Proliferative 59 18 (72.0) 24 (70.6)  Secretory 7 (28.0) 10 (29.4) Study subjects Controls EM na 61 27 34 Age (years) 61 39.9 ± 8.4 32.2 ± 5.2 Gravidity   0 59 5 (20.0) 22 (64.7)   ≥1 20 (80.0) 12 (35.3) Parity   0 61 11 (40.7) 27 (79.4)   ≥1 16 (59.3) 7 (20.6) Cycle phase  Proliferative 59 18 (72.0) 24 (70.6)  Secretory 7 (28.0) 10 (29.4) Age is presented as mean ± SD; the other variables are presented as the number (%). an means the number of cases with complete clinical data. View Large Table 1. Clinical features of study subjects. Study subjects Controls EM na 61 27 34 Age (years) 61 39.9 ± 8.4 32.2 ± 5.2 Gravidity   0 59 5 (20.0) 22 (64.7)   ≥1 20 (80.0) 12 (35.3) Parity   0 61 11 (40.7) 27 (79.4)   ≥1 16 (59.3) 7 (20.6) Cycle phase  Proliferative 59 18 (72.0) 24 (70.6)  Secretory 7 (28.0) 10 (29.4) Study subjects Controls EM na 61 27 34 Age (years) 61 39.9 ± 8.4 32.2 ± 5.2 Gravidity   0 59 5 (20.0) 22 (64.7)   ≥1 20 (80.0) 12 (35.3) Parity   0 61 11 (40.7) 27 (79.4)   ≥1 16 (59.3) 7 (20.6) Cycle phase  Proliferative 59 18 (72.0) 24 (70.6)  Secretory 7 (28.0) 10 (29.4) Age is presented as mean ± SD; the other variables are presented as the number (%). an means the number of cases with complete clinical data. View Large Long noncoding RNA microarrays and bioinformatic analyses The original microarray cohort included four patients (two in proliferative phase and two in secretory phase) without other estrogen-dependent diseases. The Agilent human lncRNA Array V4.0 was designed with four identical arrays per slide (4 × 180K format). Each array contained probes for approximately 41,000 human lncRNAs, derived from authoritative databases, such as Gencode, Ensembl, RefSeq, and the Runsheng Chen laboratory database (Institute of Biophysics, Chinese Academy of Science). After quantile normalization, normalized expression values were log2 transformed. Long noncoding RNAs were determined to be differentially expressed with absolute values of log2 fold change (|logFC|) ≥ 1.5 in paired EC and EU samples among all patients. A heatmap of differentially expressed lncRNAs was generated with the gplots package in the R language after transformation to Z-score values. CapitalBio Corporation (Beijing, China) performed microarray profiling. The raw microarray data are available in the NCBI GEO Archive (accession number is GSE86534). Cell culture and cell transfection The hEM15A cell line was cultured as previously described [17, 18]. The purity of hEM15As was examined via separate immunofluorescence staining for the stromal marker vimentin, epithelial marker pan cytokeratin, and fibroblast antigen protein. The secondary antibodies were Alexa Fluor 488-conjugated goat anti-mouse antibodies and Alexa Fluor 594-conjugated goat anti-rabbit antibodies. Detailed information about the antibodies is listed in Supplementary Table S1. Cells were cultured in six-well plates and transfected with 60 nM siRNA oligonucleotides using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). The following siRNA sequences were used: CCDC144NL-AS1 siRNA 1, 5′-GGAAUUGGUGAUUGGCUUUTT-3′; and CCDC144NL-AS1 siRNA 2, CCUGUACAUCCUUACCUAUTT. SiRNAs against CCDC144NL-AS1 and a scrambled negative control (NC) were designed and synthesized by GenePharma (Suzhou, China). RNA isolation and quantitative real-time PCR Total RNA from frozen tissues and cells was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) and reverse transcribed using FastQuant RT Super Mix (Tiangen, Beijing, China). Quantitative PCR was performed using Power SYBR ® Green PCR Master Mix (Applied Biosystems, Austin, TX, USA) and a Bio-Rad CFX Connect Real-Time PCR System (Hercules, CA, USA). The PCR cycling program was as follows: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. We selected four candidate housekeeping genes (YWHAZ, CYC1, β-actin, and GAPDH) showing relative stability in human tissues or endometrium [19, 20], and tested their expression stability in paired EC and EU tissues (n = 4) from EM patients with Bio-Rad CFX Maestro software, which is based on GeNorm, a widely accepted algorithm for calculating ideal reference genes. GAPDH was stably expressed in EC and EU tissues with the lowest M-value (0.297) and the highest stability value (1.213) (Supplementary Figure S1). However, CYC1 was expressed at too low a level to be included in the analyses. The stability value was transformed from Ln(1/Average M-value). We selected GAPDH as the housekeeping gene. The specific primers used for amplification were synthesized by Sangon Biotech (Beijing, China) and are listed in Supplementary Table S2. The housekeeping gene GAPDH was used to permit semiquantitative comparisons of transcription rates using the 2−ΔΔCt method. All reactions were performed in triplicate. Subcellular fractionation Separation of cytosolic and nuclear fractions was performed using the Minute TM Kit (Invent Biotechnologies, Berkshire, Plymouth, MN, USA) according to the manufacturer's recommendations, and RiboLock RNase Inhibitor (Thermo Scientific, Waltham, MA, USA) was used to inhibit RNA degradation. GAPDH, β-actin, U1, and U6 were used as fractionation indicators for qPCR. Primer sequences are listed in Supplementary Table S2. Cell proliferation assay Cell proliferation assays were carried out using the Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan) according to the manufacturer's protocol. Cells were transfected with siRNAs and plated in 96-well plates at a density of approximately 3000 cells in 100 μl of medium per well. The absorbance of reduced water-soluble tetrazolium salt at 450 nm was assessed at 0, 24, 48, 72, and 96 h. Each experiment was tested in five replicates. Wound healing assay Wound healing assays were performed to evaluate the migration ability of cells. Cells were seeded into six-well dishes and cultured until 80–90% confluence. A sterilized pipette tip was used to create scratch wounds across the cell monolayer, and the wells were washed with PBS to remove floating cells and debris. Wound healing was recorded photographically in the same position using an inverted phase-contrast microscope (LEICA DMIL LED, Wetzlar, Hesse-Darmstadt, Germany) at 0, 24, and 48 h. Each time point was normalized to the 0-h image area, and results are reported as the percent area closed. A total of five areas were randomly selected in each well, and experiments were performed at least three times. Cell migration and invasion assays Cell migration and invasion assays were carried out in 24-well transwell chambers (Coring Costar Corporation, Corning, NY, USA). After 24 h of transfection, cells (2 × 104) were suspended in 200 μl of serum-free medium and loaded on the upper chambers of inserts for migration assays (without Matrigel) and invasion assays (with Matrigel). Medium supplemented with 20% FBS was placed in the lower chambers and used as a chemoattractant. After 24 h of incubation at 37°C, the inserts were removed and noninvading cells were carefully removed with a cotton wool swab. The migrated or invasive cells below the membrane were fixed with methanol for 20 min and stained with 0.1% crystal violet for 20 min. We selected five random fields per well for imaging and quantification of the visible cells that migrated to the lower side of the membrane under an inverted phase-contrast microscope (LEICA DMIL LED, Wetzlar, Hesse-Darmstadt, Germany). Each experiment was repeated at least three times. Cell adhesion assay Endometrial cells adhesion to the vascular endothelium is an important step in the progression of EM. We investigated whether siRNA transfection would affect the ability of hEM15A cells adhere to human umbilical vein endothelial cells (HUVECs) [21, 22]. HUVECs were grown in DMEM medium containing 10% FBS in six-well plates to 90–95% confluence. hEM15A cells were labeled with 3 μM CellTracker™ Red CMPTX dyes (Life Technologies, Eugene, Oregon, USA) in PBS for 30 min under growth conditions. After washing twice with PBS, the labeled hEM15A cells were seeded in six-well plates and transfected for 48 h. The labeled hEM15A cells were co-cultured with HUVECs at a density of 2 × 104 cells per well for 30 min or 60 min in an incubator. Nonadherent cells were washed three times with PBS, and images of 10 random fields were captured using a fluorescence microscope (LEICA DMIL LED, Wetzlar, Hesse-Darmstadt, Germany) to measure the number of adherent cells. The experiments were repeated at least three times. Flow cytometric analysis After 48 h of transfection, apoptotic cells were analyzed using an Annexin V/PI double staining kit (Lianke Biotech, Hangzhou, China). For cell cycle analysis, the BD Cycletest™ Plus DNA Kit (BD Biosciences, San Jose, CA, USA) was used according to the manufacturer's instructions. Fluorescence-activated cells were quantified using a flow cytometer. Each experiment was performed in triplicate. Confocal immunofluorescence microscopy Cells were cultured and transfected on glass coverslips (35-mm, Nest, Wuxi, China), fixed with 4% paraformaldehyde for 20 min, and permeabilized with 0.2% Triton X-100 for 10 min. Cells were then blocked with 1% bovine serum albumin (Solarbio, Beijing, China) for 30 min, and actin filaments were stained using rhodamine phalloidin (Life Technologies, Eugene, Oregon, USA). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Solarbio, Beijing, China). Finally, cells were covered with 90% glycerol and analyzed under a confocal microscope (LEICA TCS SP8, Wetzlar, Hesse-Darmstadt, Germany). Images for comparisons were acquired with identical laser intensity, exposure time, and filter. The area of F-actin was quantitated by Image J software (NIH). Images of at least five random fields were analyzed in each sample, and experiments were repeated in samples from three different individuals. Western blotting After 48 h of transfection, cells were washed with ice-cold PBS and lysed using RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with PMSF (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer's instructions. Protein concentrations were determined by Bradford assay (Bio-rad, Hercules, CA, USA). Equal amounts of protein harvested from different cell lysates were loaded on 10% SDS-PAGE gels using a Bio-Rad system and transferred to PVDF membranes (Millipore Corporation, Bedford, MA, USA). The membranes were blocked for 1 h at room temperature and incubated overnight at 4°C with the indicated primary antibodies: N-cadherin, β-catenin, vimentin, MMP9, and GAPDH. Detailed information about the antibodies is listed in Supplementary Table S1. The membranes were then washed with 0.05% Tween 20 in TBS and incubated with a goat anti-rabbit peroxidase-conjugated secondary antibody (ZB-2301, ZSGB-BIO, Beijing, China) for 1 h at 37°C. All protein bands were scanned using a Bio-Rad ChemiDocTM XRS + system (Hercules, CA, USA) and GAPDH was used for a loading control for the western blot analysis. Each experiment was repeated at least three times. Statistical analysis The data are expressed as the mean ± standard deviation (SD) from at least three independent experiments. Statistical analysis was performed using SPSS 17.0 software (Chicago, IL, USA). Normality was tested using the Kolmogorov–Smirnov test. One-sample t-tests were used to compare the qPCR results with the microarray data for the six randomly selected lncRNAs. Comparisons between multiple groups were assessed using a one-way or two-way (two independent variables) analysis of variance (ANOVA) test. A Fisher exact test was used to evaluate the relationships between clinical characteristics and lncRNA CCDC144NL-AS1 expression. P-values of 0.05 or less were considered statistically significant. Results Differentially expressed long noncoding RNAs identified by microarray analyses The microarray analyses identified 755 lncRNAs and 686 lncRNAs that were upregulated and downregulated, respectively, in EC compared to EU. The most significantly upregulated lncRNA was uc.343- (logFC 5.80), and LINC00617 was the most significantly downregulated lncRNA (logFC 5.85). A heatmap demonstrated that differentially expressed lncRNAs were clearly self-segregated into EC and EU clusters (Figure 1A). The distribution pattern of dysregulated lncRNAs was presented in a scatter plot (Figure 1B). A volcano plot was used for assessing the statistical significance of differentially expressed lncRNAs between EC and EU (Figure 1C). Supplementary Table S3 lists the top 20 dysregulated lncRNAs. Figure 1. View largeDownload slide Dysregulated lncRNAs between paired ectopic endometrium and eutopic endometrium tissues in endometriosis. (A) Heatmap showing differentially expressed lncRNA profiles from paired ectopic endometrium (EC) and eutopic endometrium (EU) tissues in endometriosis patients. Red and green indicate expression at relatively high and low levels, respectively. (B) Scatter plot. (C) Volcano plot. (D) The qPCR results for six chosen dysregulated lncRNAs were consistent with the microarray results. The data were expressed as the mean ± SD from at least three independent experiments. The fold change was positive when the expression of an lncRNA was upregulated in EC tissues compared with EU tissues, and negative when it was downregulated. 1–4 indicate the numbers of the patients for the microarray. DE, differentially expressed. Figure 1. View largeDownload slide Dysregulated lncRNAs between paired ectopic endometrium and eutopic endometrium tissues in endometriosis. (A) Heatmap showing differentially expressed lncRNA profiles from paired ectopic endometrium (EC) and eutopic endometrium (EU) tissues in endometriosis patients. Red and green indicate expression at relatively high and low levels, respectively. (B) Scatter plot. (C) Volcano plot. (D) The qPCR results for six chosen dysregulated lncRNAs were consistent with the microarray results. The data were expressed as the mean ± SD from at least three independent experiments. The fold change was positive when the expression of an lncRNA was upregulated in EC tissues compared with EU tissues, and negative when it was downregulated. 1–4 indicate the numbers of the patients for the microarray. DE, differentially expressed. Quantitative real-time PCR validation To validate the microarray data, we randomly selected two upregulated lncRNAs (AC022034.2–201 and CCDC144NL-AS1) and four downregulated lncRNAs (LINC00617/TUNAR-203, LINC01764, LINC01541, and UCA1). We used qPCR to verify the expression levels of these lncRNAs in another 30 paired EC and EU samples. The qPCR results showed the same trends of up- or downregulation as the microarray data (Figure 1D). The statistical analyses indicated no significant difference between the microarray data (black column in Figure 1D) and the qPCR results (gray column in Figure 1D) for AC022034.2–201 (P = 0.172), CCDC144NL-AS1 (P = 0.626), LINC00617 (P = 0.939), LINC01764 (P = 0.055), and UCA1 (P = 0.391). However, a significant difference was found for LINC01541 (P = 0.014), despite the fact that the expression tendency was consistent with the microarray data, which may have resulted from the relatively small sample size. CCDC144NL-AS1 was frequently upregulated in ectopic tissues The lncRNA CCDC144NL-AS1 is an antisense lncRNA located on human chromosome 17: 20 868 433–20 905 230 (Figure 2A). CCDC144NL-AS1 was included in NONCODE v5.0 [NONCODE: NONHSAG073025], which is a publicly authoritative and comprehensive ncRNA database. To confirm that CCDC144NL-AS1 was indeed a noncoding RNA, we assessed the protein-coding potential of CCDC144NL-AS1 using the Coding Potential Calculator (CPC) algorithm [23] and Coding Potential Assessment Tool (CPAT) [24] (Figure 2B and C). The CPC algorithm discriminates protein coding genes (positive score) from noncoding transcripts (negative score), and CCDC144NL-AS1 exhibited a CPC score of –1.276. CPAT revealed that the coding probability of CCDC144NL-AS1was 0.025, indicating that it had no coding label. As noted above, CCDC144NL-AS1 was upregulated in EC tissues compared to EU tissues (P < 0.0001; Figures 1D and 2D), and we found that its expression was also significantly higher in EC tissues than in NE tissues (P < 0.0001; Figure 2D). Based on the qPCR validation results, we found that the expression levels of the lncRNA CCDC144NL-AS1 were higher in EC tissues than in EU tissues in 86.7% (26/30) of EM patients. To investigate whether the expression of lncRNA CCDC144NL-AS1 was associated with clinicopathological features, we divided the 30 EM patients into a high-expression group and low-expression group according to the average expression level of CCDC144NL-AS1. Approximately 60.0% (9/15) of the stage IV EM patients exhibited high CCDC144NL-AS1 levels, which was a greater percentage than that of the stage II–III patients (26.7%, 4/15; P = 0.139). However, this difference was not statistically significant, which may be the result of the relatively small sample size. The expression of CCDC144NL-AS1 was not correlated with age, infertility, dysmenorrhea, unilateral or bilateral cyst, relapse condition, or menstrual cycle phase (Table 2). We further determined that CCDC144NL-AS1 was expressed in the hEM15A cell line. Subcellular fractionation of hEM15A cells suggested that CCDC144NL-AS1 was localized in the cytoplasm and nucleus (Figure 2E). Figure 2. View largeDownload slide Characterization of CCDC144NL-AS1 in endometriosis. (A) Genome browser view of the CCDC144NL-AS1 locus. (B) Coding Potential Calculator (CPC) and Coding Potential Assessment Tool (CPAT) analyses revealed that CCDC144NL-AS1 shows no probability of protein coding. (C) The expression of CCDC144NL-AS1 was upregulated in ectopic endometrium (EC) tissues compared to eutopic endometrium (EU) and normal endometrium (NE) tissues (both Ps < 0.001). (D) Subcellular localization of CCDC144NL-AS1 in hEM15A cells. β-actin, GAPDH, U1, and U6 were used as nuclear and cytoplasmic controls. The results are presented as the percentage of β-actin, GAPDH, U1, and U6, and CCDC144NL-AS1 levels and the total levels for each were taken as 100%. Data were analyzed with one-way ANOVA test. ****P < 0.0001. Figure 2. View largeDownload slide Characterization of CCDC144NL-AS1 in endometriosis. (A) Genome browser view of the CCDC144NL-AS1 locus. (B) Coding Potential Calculator (CPC) and Coding Potential Assessment Tool (CPAT) analyses revealed that CCDC144NL-AS1 shows no probability of protein coding. (C) The expression of CCDC144NL-AS1 was upregulated in ectopic endometrium (EC) tissues compared to eutopic endometrium (EU) and normal endometrium (NE) tissues (both Ps < 0.001). (D) Subcellular localization of CCDC144NL-AS1 in hEM15A cells. β-actin, GAPDH, U1, and U6 were used as nuclear and cytoplasmic controls. The results are presented as the percentage of β-actin, GAPDH, U1, and U6, and CCDC144NL-AS1 levels and the total levels for each were taken as 100%. Data were analyzed with one-way ANOVA test. ****P < 0.0001. Table 2. Clinical features and lncRNA CCDC144NL-AS1 expression in EM patients. lncC expressiona Clinical features Total case Low (%) High (%) P valueb Age 0.443  <35 years 21 13(61.9) 8(38.1)   ≥35 years 9 4(44.4) 5(55.6) rAFS stage 0.139  II-III 15 11(73.3) 4(26.7)  VI 15 6(40.0) 9(60.0) Infertility 1.000  No 5 3(60.0) 2(40.0)  Yes 21 12(57.1) 9(42.9) Dysmenorrhea 1.000  No 11 6(54.5) 5(45.6)  Yes 19 11(57.9) 8(42.1) Ovarian endometriotic cyst 0.460  Unilateral 17 11(64.7) 6(35.3)  Bilateral 13 6(46.1) 7(53.8) EM relapse 0.604  No 21 11(52.4) 10(47.6)  Yes 4 3(75) 1(25) Menstrual cycle 1.000  Proliferative phase 22 14(63.6) 8(36.4)  Secretory phase 8 5(62.5) 3(37.5) lncC expressiona Clinical features Total case Low (%) High (%) P valueb Age 0.443  <35 years 21 13(61.9) 8(38.1)   ≥35 years 9 4(44.4) 5(55.6) rAFS stage 0.139  II-III 15 11(73.3) 4(26.7)  VI 15 6(40.0) 9(60.0) Infertility 1.000  No 5 3(60.0) 2(40.0)  Yes 21 12(57.1) 9(42.9) Dysmenorrhea 1.000  No 11 6(54.5) 5(45.6)  Yes 19 11(57.9) 8(42.1) Ovarian endometriotic cyst 0.460  Unilateral 17 11(64.7) 6(35.3)  Bilateral 13 6(46.1) 7(53.8) EM relapse 0.604  No 21 11(52.4) 10(47.6)  Yes 4 3(75) 1(25) Menstrual cycle 1.000  Proliferative phase 22 14(63.6) 8(36.4)  Secretory phase 8 5(62.5) 3(37.5) aLncC expression was divided into low and high levels according to the average expression level. bBy the Fisher exact test. rAFS, revised American Fertility Society. View Large Table 2. Clinical features and lncRNA CCDC144NL-AS1 expression in EM patients. lncC expressiona Clinical features Total case Low (%) High (%) P valueb Age 0.443  <35 years 21 13(61.9) 8(38.1)   ≥35 years 9 4(44.4) 5(55.6) rAFS stage 0.139  II-III 15 11(73.3) 4(26.7)  VI 15 6(40.0) 9(60.0) Infertility 1.000  No 5 3(60.0) 2(40.0)  Yes 21 12(57.1) 9(42.9) Dysmenorrhea 1.000  No 11 6(54.5) 5(45.6)  Yes 19 11(57.9) 8(42.1) Ovarian endometriotic cyst 0.460  Unilateral 17 11(64.7) 6(35.3)  Bilateral 13 6(46.1) 7(53.8) EM relapse 0.604  No 21 11(52.4) 10(47.6)  Yes 4 3(75) 1(25) Menstrual cycle 1.000  Proliferative phase 22 14(63.6) 8(36.4)  Secretory phase 8 5(62.5) 3(37.5) lncC expressiona Clinical features Total case Low (%) High (%) P valueb Age 0.443  <35 years 21 13(61.9) 8(38.1)   ≥35 years 9 4(44.4) 5(55.6) rAFS stage 0.139  II-III 15 11(73.3) 4(26.7)  VI 15 6(40.0) 9(60.0) Infertility 1.000  No 5 3(60.0) 2(40.0)  Yes 21 12(57.1) 9(42.9) Dysmenorrhea 1.000  No 11 6(54.5) 5(45.6)  Yes 19 11(57.9) 8(42.1) Ovarian endometriotic cyst 0.460  Unilateral 17 11(64.7) 6(35.3)  Bilateral 13 6(46.1) 7(53.8) EM relapse 0.604  No 21 11(52.4) 10(47.6)  Yes 4 3(75) 1(25) Menstrual cycle 1.000  Proliferative phase 22 14(63.6) 8(36.4)  Secretory phase 8 5(62.5) 3(37.5) aLncC expression was divided into low and high levels according to the average expression level. bBy the Fisher exact test. rAFS, revised American Fertility Society. View Large CCDC144NL-AS1 knockdown attenuated cell migration and invasion in endometriosis To characterize the hEM15A cell line, we performed immunofluorescence staining. The results revealed that the immortalized stromal cells were vimentin+/pan cytokeratin-/fibroblast antigen protein- (Supplementary Figure S2). To elucidate the role of CCDC144NL-AS1 in EM, siRNAs were designed to quench its expression in hEM15A cells. Significantly decreased CCDC144NL-AS1 levels were detected at 24 h (NC vs siRNA1, P = 0.0016; NC vs siRNA2, P = 0.0008) and 48 h (NC vs siRNA1, P = 0.0013; NC vs siRNA2, P = 0.0006) after transient transfection with siRNAs. There was no difference in the knockdown efficiency at 24 and 48 h (NC, P > 0.9999; siRNA1, P = 0.9986; siRNA2, P = 0.9299) (Figure 3A). The effect of CCDC144NL-AS1 on cell migration and invasion was determined using wound healing and transwell assays. As shown in Figure 3B and C, the wound healing assay at 48 h showed decreased motility of siCCDC144NL-AS1 cells (NC vs siRNA1, P = 0.01; NC vs siRNA2, P = 0.003). Transwell assays revealed that the silencing of CCDC144NL-AS1 inhibited the migration (NC vs siRNA1, P = 0.03; NC vs siRNA2; P = 0.008) and invasion (NC vs siRNA1, P = 0.0001; NC vs siRNA2; P = 0.0001) of hEM15A cells in vitro (Figure 3D, E, and F). Cytoskeleton dynamics are involved in cell motility and migration. Therefore, we examined the role of CCDC144NL-AS1 in regulating F-actin cytoskeleton. Decreased areas of F-actin throughout siCCDC144NL-AS1 cells were observed under confocal immunofluorescence microscopy compared to negative controls (NC vs siRNA1, P = 0.0001; NC vs siRNA2, P = 0.0001; Figure 3G and H). Figure 3. View largeDownload slide Knockdown of CCDC144NL-AS1 suppressed cell migration and invasion, and induced cytoskeleton remodeling in hEM15A cells. (A) Relative CCDC144NL-AS1 RNA levels in hEM15A cells transfected with the indicated siRNAs at 24 and 48 h analyzed using qPCR. (B and C) Representative images and quantification of cell motility changes in wound healing assays at 0, 24, and 48 h. Magnification ×40; scale bar, 100 μm. (D–F) Representative images and quantification of transwell migration (without matrigel) and invasion (with matrigel) assays 24 h after CCDC144NL-AS1 knockdown. Magnification ×100; scale bar, 100 μm. (G) Transfected hEM15A cells were triple-stained using CCDC144NL-AS1siRNAs-FAM (green), rhodamine phalloidin (F-actin, red), and 4′,6-diamidino-2-phenylindole (DAPI, blue) 48 h after transfection. (H) The area of F-actin was quantitated in images of at least five random fields from each sample, and three independent experiments were performed. Scale bar, 20 μm. The results are represented as the mean ± SD from at least three independent experiments. Data were analyzed with one-way or two-way (two independent variables) ANOVA test.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Figure 3. View largeDownload slide Knockdown of CCDC144NL-AS1 suppressed cell migration and invasion, and induced cytoskeleton remodeling in hEM15A cells. (A) Relative CCDC144NL-AS1 RNA levels in hEM15A cells transfected with the indicated siRNAs at 24 and 48 h analyzed using qPCR. (B and C) Representative images and quantification of cell motility changes in wound healing assays at 0, 24, and 48 h. Magnification ×40; scale bar, 100 μm. (D–F) Representative images and quantification of transwell migration (without matrigel) and invasion (with matrigel) assays 24 h after CCDC144NL-AS1 knockdown. Magnification ×100; scale bar, 100 μm. (G) Transfected hEM15A cells were triple-stained using CCDC144NL-AS1siRNAs-FAM (green), rhodamine phalloidin (F-actin, red), and 4′,6-diamidino-2-phenylindole (DAPI, blue) 48 h after transfection. (H) The area of F-actin was quantitated in images of at least five random fields from each sample, and three independent experiments were performed. Scale bar, 20 μm. The results are represented as the mean ± SD from at least three independent experiments. Data were analyzed with one-way or two-way (two independent variables) ANOVA test.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Influence of CCDC144NL-AS1 knockdown on other cell phenotypes in endometriosis We also determined whether CCDC144NL-AS1 depletion affected cell adhesion, proliferation, apoptosis, and cell cycle. The interaction between endometrial cells and vascular endothelium cells occurs throughout the vascularization process, which is a major hallmark in the pathogenesis of EM [25]. We investigated whether CCDC144NL-AS1 affected hEM15A cell adhesion to HUVECs. Our results showed that the number of cells adhering to HUVECs was not different between siCCDC144NL-AS1 cell and negative control cells at 30 min (NC vs siRNA1, P = 0.4223; NC vs siRNA2, P = 0.8743) and 60 min (NC vs siRNA1, P = 0.7442; NC vs siRNA2, P = 0.9260) (Figure 4A and B). The CCK-8 assay showed that knockdown of CCDC144NL-AS1 had no effect on the proliferation of hEM15A cells (NC vs siRNA1, P = 0.8981; NC vs siRNA2, P = 0.9889; Figure 4C). Flow cytometry analysis demonstrated that the inhibition of CCDC144NL-AS1 did not affect cell apoptosis (Figure 4D and E) or cell cycle (Figure 4F and G). Figure 4. View largeDownload slide Influence of CCDC144NL-AS1 on cell proliferation, adhesion, apoptosis, and cell cycle in hEM15A cells. (A and B) Representative images and quantification of the adhesion assay for hEM15A cells and HUVECs. Adherent hEM15A cells were photographed. Magnification ×100; scale bar, 200 μm. (C) Cell proliferation using the CCK-8 assay. Absorbance levels of reduced water-soluble tetrazolium salt measured at 450 nm from cells transfected with the indicated siRNAs after transfection at the designated time points. (D and E) Annexin V/PI staining assessment of apoptosis in hEM15A cells after siRNA transfection at 48 h. (F and G) Cell cycle analysis to evaluate the relative cell numbers in each cell-cycle phase after propidium iodide staining of siCCDC144NL-AS1 cells. The results are represented as the mean ± SD from at least three independent experiments. Data were analyzed with the one-way or two-way (two independent variables) ANOVA test. Figure 4. View largeDownload slide Influence of CCDC144NL-AS1 on cell proliferation, adhesion, apoptosis, and cell cycle in hEM15A cells. (A and B) Representative images and quantification of the adhesion assay for hEM15A cells and HUVECs. Adherent hEM15A cells were photographed. Magnification ×100; scale bar, 200 μm. (C) Cell proliferation using the CCK-8 assay. Absorbance levels of reduced water-soluble tetrazolium salt measured at 450 nm from cells transfected with the indicated siRNAs after transfection at the designated time points. (D and E) Annexin V/PI staining assessment of apoptosis in hEM15A cells after siRNA transfection at 48 h. (F and G) Cell cycle analysis to evaluate the relative cell numbers in each cell-cycle phase after propidium iodide staining of siCCDC144NL-AS1 cells. The results are represented as the mean ± SD from at least three independent experiments. Data were analyzed with the one-way or two-way (two independent variables) ANOVA test. CCDC144NL-AS1 knockdown suppressed vimentin and MMP-9 expression Many cell migration and invasion-related molecules and processes, such as MMPs and EMT, are associated with the progression of EM. We evaluated the effects of CCDC144NL-AS1 on EMT regulation and MMP-9, which is the largest gelatinase in the MMP family, to elucidate its role in cell migration and invasion. Western blot analysis showed that the expression of vimentin (NC vs siRNA1, P = 0.0007; NC vs siRNA2, P = 0.0002) and MMP-9 (NC vs siRNA1, P = 0.0009; NC vs siRNA2, P = 0.0001) decreased after CCDC144NL-AS1 knockdown, and there was no significant difference in N-cadherin (NC vs siRNA1, P = 0.8378; NC vs siRNA2, P = 0.7978) or β-catenin (NC vs siRNA1, P = 0.9992; NC vs siRNA2, P = 0.7340) (Figure 5A–E). Figure 5. View largeDownload slide Effects of CCDC144NL-AS1 on the protein expression of migration-related molecules in hEM15A cells. (A) Representative data from western blot analyses for N-cadherin, MMP-9, β-catenin, and vimentin. Quantification of N-cadherin (B), MMP-9 (C), β-catenin (D), and vimentin (E) protein levels in negative control and siRNA treatment groups. GAPDH was used for normalization of protein expression. Results are presented as the mean ± SD, n = 3. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Figure 5. View largeDownload slide Effects of CCDC144NL-AS1 on the protein expression of migration-related molecules in hEM15A cells. (A) Representative data from western blot analyses for N-cadherin, MMP-9, β-catenin, and vimentin. Quantification of N-cadherin (B), MMP-9 (C), β-catenin (D), and vimentin (E) protein levels in negative control and siRNA treatment groups. GAPDH was used for normalization of protein expression. Results are presented as the mean ± SD, n = 3. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Discussion The pathogenesis of EM is not clear. Despite growing evidence indicating that lncRNAs participate in the occurrence and development of EM, the function of most lncRNAs is not well understood. In this study, we performed microarray analyses to compare lncRNA expression patterns in paired EC and EU tissues from EM patients, and found that 1441 lncRNA transcripts were dysregulated. We randomly chose six of the dysregulated lncRNAs for validation using qPCR. We further identified a novel lncRNA, CDC144NL-AS1, which was significantly expressed higher in EC tissues than in EU and NE tissues. A higher percentage of stage IV EM patients than stage II-III EM patients exhibited high CCDC144NL-AS1 levels, although this difference was not significant (60.0% vs 26.7%; P = 0.139). To the best of our knowledge, only one report related to CDC144NL-AS1 has been published. Guo et al. used data from The Cancer Genome Atlas and integrated a miRNA-lncRNA signature, including CDC144NL-AS1 [26]. They found that this lncRNA was associated with prognostic outcomes of ovarian patients with wild-type BRCA1/2. However, the possible functions and mechanisms of CDC144NL-AS1 have not yet been reported. One of the major reasons for the current delay fundamental theoretical research on EM is the lack of publicly available model cell lines. We previously established hEM15A, the first human EM-derived immortalized eutopic endometrial stromal cell (ESC) line, which is a useful tool for in vitro studies of EM [17, 18]. We found that CCDC144NL-AS1 was expressed in the hEM15A cell line, and subcellular fractionation showed that it was localized in the cytoplasm and nucleus (Figure 2E). Therefore, we attenuated CCDC144NL-AS1 expression in hEM15A cells using siRNA technology. The results demonstrated that CCDC144NL-AS1 depletion suppressed the migration and invasion of hEM15A cells (Figure 3B–F), but exerted no significant effects on cell proliferation, adhesion, apoptosis, or cell cycle (Figure 4). Although EM is a common benign disorder, it shares many features with malignancies such as migration and invasion. Emerging studies have demonstrated that several molecules, factors, and pathways affected EM-associated migration and invasion. Chloride channel-3 (ClC-3), a member of the voltage-gated Cl¯ channel superfamily, was found to be significantly upregulated in EC tissues and to be involved in the migration and invasion of ESCs via regulating MMP-9 [27]. Environmental factors have emerged as additional important players in the pathogenesis of EM in recent decades. Hu et al. reported that polychlorinated biphenyl 104, which is a hazardous environmental contaminant, promoted the migration and invasion of ESCs via the induction of MMP-3 and MMP-10 expression [28]. The RhoA/RhoC/ROCK1 pathway is known to modulate the cytoskeleton distribution, and to participate in the pathogenesis of EM [29]. Therefore, we hypothesized that overexpression of CCDC144NL-AS1 in EC tissues may also exert similar biological effects on cell migration and invasion via certain molecular mechanism(s). Dynamic alterations of the cytoskeleton are involved in cell motility and migration. The cytoskeletal systems in mammalian cells include three components: the actin cytoskeleton, the intermediate filament network, and microtubules. Each system regulates all or part of the migration process [30]. F-actin is present throughout the cell body and interacts with myosin to generate traction force. Interestingly, our results revealed that blocking the expression of CCDC144NL-AS1 obviously affected the morphology of the actin cytoskeleton compared to the negative control treatment. More specifically, F-actin appeared reduced throughout hEM15A cells and exhibited a tendency to accumulate at the edge of cells (Figure 3G and H). There is a wealth of evidence suggesting relationships between the dynamics of cytoskeletal systems and EMT processes. EMT biomarkers, including N-cadherin, β-catenin, and vimentin, are also recognized as migration-related molecules. MMPs are related to the EMT process because they facilitate ECM degradation, regulate the cytoskeleton, and enhance migration and invasion capacity. In this study, western blot analysis showed that knockdown of CCDC144NL-AS1 attenuated the expression of vimentin and MMP-9, but did not affect that of N-cadherin or β-catenin. Vimentin is a major and conserved type III intermediate filament protein that is found in various types of mesenchymal cells, including hEM15A cells [31], and plays a crucial role in cell adhesion, migration, invasion, and cell signal transduction [32, 33]. Recent advances have shown that lncRNA-Dreh interacted with vimentin to inhibit its expression, and altered the cytoskeleton structure to suppress metastasis in hepatocellular carcinoma [34]. Zeng et al. reported that the lncRNA LINC00675 combined with vimentin to increase its phosphorylation and cause the collapse of vimentin filaments in gastric cancer cells, which reduced cell metastasis [35]. MMP-9, also known as the largest gelatinase in the MMP family, was elevated in ectopic tissues, plasma, and peritoneal fluid in patients with EM [36]. Yang et al. found that the estrogen-induced activation of osteopontin increased MMP-9 expression and may be related to the migration of endometrial epithelial cells in EM patients [15]. Therefore, we hypothesized that the lncRNA CCDC144NL-AS1 may also interact with vimentin and MMP-9 either directly and indirectly and regulate cytoskeleton structure and cell motility. The subcellular localization of an lncRNA transcript determines its function. For instance, HOTAIR, a well-studied lncRNA, primarily performs its function in two patterns. HOTAIR in the nucleus alters histone H3 lysine 27 methylation and reprograms the chromatin state to promote cancer metastasis [37]. In addition, HOTAIR acts as competitive endogenous RNA in the cytoplasm and promotes tumourigenesis of carcinoma cells via inhibiting microRNAs (miRNAs), such as miR-217 [38]. The lncRNA CCDC144NL-AS1 is an antisense lncRNA located on human chromosome 17 (Figure 2A). Antisense transcripts are frequently functional and play various biological roles via diverse transcriptional and post-transcriptional gene regulatory mechanisms [39]. The present study showed that CCDC144NL-AS1 localized in the cytoplasm and nuclei (Figure 2E), which suggested that it may exert its function though multiple mechanisms. Taken together, the results of this study, for the first time, offer clinical and experimental evidence that elevated expression of the lncRNA CDC144NL-AS1 may facilitate the development of EM by regulating F-actin and vimentin, which promotes the migration and invasion of hEM15A cells. Hence, the lncRNA CDC144NL-AS1 may be a potential therapeutic target for EM. Supplementary data Supplementary Figure S1. Stability ranking of housekeeping genes tested from paired eutopic and ectopic endometrium tissues. The M-value was determined using the geNorm program. The expression stability value was transformed from Ln(1/Average M-value). Genes are ranked from left to right in order of the expression stability value. Green and yellow indicate ideal and acceptable housekeeping genes, respectively. Supplementary Figure S2. Characterization of the hEM15A cell line. Immunofluorescence staining for vimentin (A, green), pan cytokeratin (B, green), and fibroblast antigen protein (C, red) in hEM15A cells. The cell nuclei were labeled with DAPI (A, B, and C, blue). Magnification ×400; scale bar, 100 μm. Supplementary Table S1. Antibodies used in the study. Supplementary Table S2. Primers for quantitative real-time PCR. Supplementary Table S3. Top 20 dysregulated lncRNAs in ectopic endometrium tissues compared with paired eutopic endometrium tissues from endometriosis patients. Acknowledgments We appreciate the candidates who were enrolled in this study and the many gynecologists, nurses, and operating room staff, whose efforts in collecting the samples greatly contributed to this research. We gratefully acknowledge the assistance of Xin Yu in the flow cytometric experiments and data analyses. We also thank Suge Gao for her valuable contributions to preparing the experimental materials and equipment. Conflict of Interest: The authors have declared that no conflict of interest exists. Notes Edited by Dr. Peter J. Hansen Footnotes † Grant support: This work was supported by the Beijing Natural Science Foundation (No. 7182173) and National Natural Science Foundation of China (No. 81671431). References 1. Giudice LC . Clinical practice . Endometriosis . N Engl J Med 2010 ; 362 ( 25 ): 2389 – 2398 . 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TI - Knockdown of long noncoding RNA CCDC144NL-AS1 attenuates migration and invasion phenotypes in endometrial stromal cells from endometriosis
JO - Biology of Reproduction
DO - 10.1093/biolre/ioy252
DA - 2019-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/knockdown-of-long-noncoding-rna-ccdc144nl-as1-attenuates-migration-and-uXAvf6IAoA
SP - 939
VL - 100
IS - 4
DP - DeepDyve
ER -