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The long non-coding RNA PCSEAT exhibits an oncogenic property in prostate cancer and functions as a competing endogenous RNA that associates with EZH2
Biochemical and Biophysical Research Communications xxx (2018) 1e7
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The long non-coding RNA PCSEAT exhibits an oncogenic property in prostate cancer and functions as a competing endogenous RNA that associates with EZH2 Xiaohui Yang a, b, Liang Wang c, Rong Li b, Yuhui Zhao c, Yinmin Gu b, Siying Liu b, Tianyou Cheng d, Kuohsiang Huang b, Yi Yuan e, Dalong Song e, Shan Gao b, d, e, * a
Laboratory for Noncoding RNA & Cancer, School of Life Sciences, Shanghai University, Shanghai, 200444, China CAS Key Laboratory of Bio-medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163, China c CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China d Shanxi Academy of Advanced Research and Innovation, Taiyuan, 030032, China e Medical College, Guizhou University, Guiyang, 550025, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 May 2018 Accepted 22 May 2018 Available online xxx
Prostate cancer (PCa) is the most common malignancy and the leading cause of cancer deaths in males. Recent studies demonstrate that long non-coding RNAs (lncRNAs) are involved in many aspects of PCa. However, their biological roles in PCa remain imperfectly understood. Here, we characterized an lncRNA, PCa specific expression and EZH2-associated transcript (PCSEAT, annotated as PRCAT38), which is specifically overexpressed in PCa. We further demonstrated that knockdown of PCSEAT results in the reduction of PCa cell growth and motility, and overexpression of PCSEAT reverses these phenotypes. Furthermore, bioactive PCSEAT is incorporated into exosomes and transmitted to adjacent cells, thus promoting cell proliferation and motility. Mechanistically, we found that PCSEAT promotes cell proliferation, at least in part by affecting miR-143-3p- and miR-24-2-5p-mediated regulation of EZH2, suggesting that PCSEAT and EZH2 competitively ‘sponge’ miR-143-3p and miR-24-2-5p. Overall, our results reveal that PCSEAT is specifically overexpressed in PCa patients and a potential oncogene in PCa cells via mediating EZH2 activity, indicating that PCSEAT may be a potential therapeutic target in PCa. © 2018 Elsevier Inc. All rights reserved.
Keywords: Prostate cancer PCSEAT EZH2 Competing endogenous RNA Exosome
1. Introduction As the most common malignant tumor in men, prostate cancer (PCa) is a major cause of cancer deaths [1]. To understand the underlying biology, numerous studies have been made to identify recurrent somatic mutations (SPOP, FOXA1, IDH1, and TP53), changes in copy number (MYC, RB1, PTEN, and CHD1), and the oncogenic structural DNA rearrangements (E26 transformationspecific (ETS) fusions) in primary PCa [2] and genomic alterations (AR, PIK3CA/B, R-spondin, BRAF/RAF1, APC, b-catenin, and ZBTB16/ PLZF) in advanced PCa [3]. It has been revealed that a large portion of the human genome is
* Corresponding author. NO.88, Keling Road, High-tech District, Suzhou, China. E-mail address: [email protected] (S. Gao).
transcribed into noncoding transcripts [4], including long noncoding RNAs (lncRNAs) with >200 bp in length [5]. Dysregulated expression of lncRNAs signal the spectrum of disease progression and can be used as the independent biomarkers and/or the therapeutic targets in cancer [6,7]. Recently, many lncRNAs have been shown to act as tumor suppressor genes or oncogenes via modulation of various signaling pathways in PCa, such as H19, MALAT1, PCA3, PCAT-1, and SChLAP1 [8]. The histone methyltransferase, polycomb group protein enhancer of zeste homolog 2 (EZH2) maintains the epigenomic integrity of a cell, which is capable of preserving proper chromatin structure, ensuring appropriate gene expression, and regulating a cohort of tumor and metastasis suppressor genes [9]. Moreover, EZH2 is particularly culpable in PCa's development and progression to advanced disease stages [10]. Indeed, it has been shown that some lncRNAs can directly or indirectly interact with EZH2 in PCa,
https://doi.org/10.1016/j.bbrc.2018.05.157 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: X. Yang, et al., The long non-coding RNA PCSEAT exhibits an oncogenic property in prostate cancer and functions as a competing endogenous RNA that associates with EZH2, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.157
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Chinese Academy of Sciences Cell Bank. 293T cells were cultured in DMEM medium supplemented with 10% fetal calf serum. RWPE1 cells were cultured in Keratinocyte-SFM medium supplemented with Gentamicin Solution. The other cells lines were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum. All cell lines were verified using short tandem repeat assays by Genetic Testing Biotechnology, Suzhou, China. GSK343 was purchased from Selleck Chemicals.
such as PCAT-1 [11] and ANRI [12]. Exosomes, ranging from 50 to 150 nm in diameter, are an extracellular vesicle that are formed within multivesicular bodies [13] and can be secreted from a variety of cell types and mediate a broad spectrum of effects on recipient cells [14]. For example, exosomes released from melanoma cells create the optimal environment for metastatic cancer cells, such as promoting angiogenesis, stromal remodeling, and lymph node incompetence [15]. Furthermore, colorectal cancer cells can shed Fas ligand loaded vesicles, which reduces the ability of T cell to induce cancer cells apoptosis [16]. Here, we reported the clinical implication and function of the lncRNA PCa specific expression and EZH2-associated transcript (PCSEAT, annotated as PRCAT38). PCSEAT promotes prostate tumor growth and migration by acting in part through ‘sponging’ miR143-3p and miR-24-2-5p to regulate EZH2 expression. Moreover, we find that PCSEAT can be incorporated into exosomes and transmitted to neighbor cells, thus promoting cell proliferation.
Cells were plated in 96-well plates with a density of 1 103 or 2 103 cells per well. Cell proliferation was determined with a Celltiter glow (CTG) kit measured with a multi-mode microplate reader (Synergy HTX, BioTek, Winooski, United States). PC3 cells were exposed to vehicle (0.5% DMSO) or GSK343 at the IC50 (half maximal inhibitory concentration) for 72 h, and proliferation was assessed. All experiments were performed in triplicate.
2. Materials and methods
2.3. Clone formation assay
2.1. Cell lines, cell culture and reagent
Cells were plated at 500 or 1000 cells per well in 6-well plates and incubated for 2 weeks at 37 C in a 5% CO2 humidified environment. Colonies were stained with Coomassie Blue Staining Solution (0.1% (w/v) Coomassie Blue R-250, 25% (v/v) dimethylcarbinol, and
The 293T, PC3, DU145, LNCaP, 22Rv1, RWPE-1, 769-P, 786-O, ACHN, Calu-1 and NCI-H1299 cells lines were obtained from the
2.2. Cell proliferation assay
Fig. 1. PCSEAT is expressed in PCa. (A) PCSEAT was selectively expressed in PRAD across the 21 cancer types (Mitranscriptome) (means ± SEM; P < 0.0001). (B) ROC curve for prediction of PRAD using PCSEAT expression levels from TCGA. (95% CI, P < 0.0001). (C, D) In situ hybridization of PCSEAT in 90 pairs of Chinese PCa tissues was performed. Representative images of PCSEAT staining in PCa tissues (cancer) and adjacent normal tissues (paratumor) (Scale bars ¼ 200 mm) (top). Histogram representation of PCSEAT expression levels for PCa tissues and adjacent normal tissues (bottom). (n ¼ 90; means ± SEM; P < 0.0001). (E) Genomic landscape of PCSEAT gene: ChIP-seq data for H3K4me3, H3K27ac, and H3K36me3 demonstrated enrichment at the PSCEAT gene. (F) The protein-coding potential of PCSEAT transcript was scored using PhyloCSF. HOTAIR and SChLAP1 were non-coding transcript controls (PhyloCSF score < 0). GAPDH and ACTB were coding transcript controls (PhyloCSF score > 0).
Please cite this article in press as: X. Yang, et al., The long non-coding RNA PCSEAT exhibits an oncogenic property in prostate cancer and functions as a competing endogenous RNA that associates with EZH2, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.157
X. Yang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
10% (v/v) glacial acetic acid) after fixed with methanol, and then counted. All experiments were performed in triplicate.
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Data was visualized using the UCSC Genome Browser [20]. 2.7. Western blotting
2.4. Exosome isolation DU145 cells were grown to confluent monolayers, washed three times with PBS, and grown in serum-free media for 3 days. Exosomes were collected by differential centrifugation from conditioned media, resuspended in PBS [17]. 2.5. LncRNAs, miRNA, and mRNA expression data Differentially expressed lncRNAs were analyzed based on the clinical information of 42 normal tissues and 173 tumor samples from The Cancer Genome Atlas (TCGA) and the matched expression profiles of 12382 lncRNAs from Mitranscriptome (http:// mitranscriptome.com/). TCGA prostate adenocarcinoma patients' microRsNA expressions (Level 3 data, illuminahiseq mirnaseq) and mRNA expressions (level 3 data, RNA-seq Version 2) were downloaded from FireBrowse (http://firebrowse.org/). FireBrowse portals serve TCGA data. 2.6. PC3 ChIP-Seq data Sequencing data from GSE96019, GSE96399 and GSE96418 were downloaded from GEO. Reads from the PC3 cell H3K4me3, H3K27ac and H3K36me3 ChIP-Seq samples were mapped to human genome version hg19 using HISAT2. Peak calling was performed using MACS [18] according to the published protocols [19].
Total cell lysates were denatured in SDS (Sigma, St Louis, MO, USA) loading buffer and boiled for 10 min. Samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (General Electric, Fairfield, CT, USA). Membranes were blocked with 5% milk in Tris-buffered salineeTween 20 (TBST) and incubated overnight at 4 C with primary antibodies. Membranes were then washed and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies for 1.5 h at room temperature. Membranes were washed again, and bands were visualized with Immobilon Western Chemilum HRP Substrate from Millipore (Merck KGaA, Darmstadt, Germany). 2.8. Statistical analyses Statistical analyses were performed using the Student's t-test. A P-value less than 0.05 was considered to be statistically significant in Student's t-test. No criteria were applied for inclusion and exclusion of data. No outliers were taken into account, and all collected data were subjected to statistical analyses. 3. Results 3.1. PCSEAT is specifically upregulated in human PCa tissues To explore the potential lncRNAs involved in PCa oncogenesis, we
Fig. 2. PCSAET promotes PCa cell proliferation and migration in vitro. (A, B) Proliferation of PCa cells was assessed by CTG assays. (C, D) Clone formation assays were performed in PCa cells. Representative graphs are shown. (E, F) Wound healing assays were made in PC3 and DU145 cells for 12 or 72 h of recovery, respectively. All of the cells were grown in serum-free medium during the recovery. The graphs represent relative wound healing rates. The data depict the count number from three independent experiments (means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001).
Please cite this article in press as: X. Yang, et al., The long non-coding RNA PCSEAT exhibits an oncogenic property in prostate cancer and functions as a competing endogenous RNA that associates with EZH2, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.157
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examined clinical data of patients in TCGA and matched expression profiles of lncRNAs from Mitranscriptome (Supplementary Table 1). We noted an lncRNA (annotation as PRCAT38), named PCSEAT, which was selective expression in prostate adenocarcinoma (PRAD) tissues across 21 types of cancer and corresponding adjacent normal tissues (TCGA) (P < 0.0001; Fig. 1A). Receiver operating characteristic (ROC) curve [21] was determined to evaluate the sensitivity and specificity of PCSEAT expression in predicting PRAD tissues from normal tissues. Notably, PCSEAT displayed considerably predictive significance, with an area under the curve (AUC) of 0.950 (95% confidence interval: 0.904e0.996; P < 0.0001; Fig. 1B). Furthermore, we used an in-situ hybridization assay to examine the subcellular localization and the expression levels of PCSEAT in 90 pairs of formalin-fixed, paraffinembedded Chinese human PCa samples. We confirmed the relative nuclear enrichment of PCSEAT and that the expression levels of PCSEAT were significantly increased in the PCa samples compared to the adjacent normal tissues (Fig. 1C and D and Supplementary Table 2). To more precisely explore the function of PCSEAT, we defined a 2413-bp polyadenylated gene composed of only one exon on chromosome 21q22.3 using 50 and 30 rapid amplification of cDNA ends and reverse transcription PCR (RT-PCR) (Figs. 1E and S1 and Supplementary Table 3). Published PCa chromatin immunoprecipitation and sequencing (ChIP-seq) data [22] confirm that the transcriptional start site of PCSEAT is marked by H3K4me3, a marker of an active promoter [23], and H3K27ac, a marker of active enhancers and
promoters [23]. Its genetic locus also harbored H3K36me3 (Fig. 1E), an epigenetic signature consistent with lncRNAs [23]. We did not find protein-coding potential in PCSEAT by PhyloCSF [24] (Fig. 1F). These data suggest that PCSEAT is specifically upregulated in PCa. 3.2. The lncRNA PCSEAT promotes PCa cell proliferation and motility To evaluate the biological effects of PCSEAT on PCa cells, we performed gain- and loss-of-function studies. We established cell lines with stably overexpressed PCSEAT and PCSEAT-specific knockdown, respectively (Figs. S2A and S2B). CTG assays demonstrated that knockdown of PCSEAT significantly inhibited PC3 cells proliferation (Fig. 2A). Additionally, we found that both shRNAs significantly reduced the clonogenicity and motility of PC3 cells (Fig. 2C and E). Conversely, our results demonstrated that overexpression of PCSEAT led to a significant increase in the proliferative capacity, clonogenicity and motility of DU145 cells (Fig. 2B, D and 2F). All together, these results indicate that PCSEAT promotes the growth and motility of PCa cells. 3.3. PCSEAT regulates EZH2 by competitively ‘sponge’ miR-143-3p and miR-24-2-5p It has been proposed that lncRNAs can regulate the expression
Fig. 3. PCSEAT regulates EZH2 through miR-143-3p and 31 miR-24-2-5p. (A) The protein levels of EZH2 and H3K27me3 in stable PCSEAT knockdown PC3 cells and stable PCSEAT-overexpressing DU145 cells are shown. (B) The dual-luciferase reporter assay was performed in 293T cells containing reporter vectors by co-transfecting the cells with miR-143-3p or miR-24-2-5p mimic and PCSEAT transcript. (C)The proliferation was measured by CTG assays and protein levels of EZH2 were analyzed by western blot in DU145 cells transfected with the indicated vectors and miRNA mimics. (D, E) Proliferation of PCa cells was assessed by CTG assays after incubation for 120 h. DU145 (E) and PC3 (F) cells were transfected with the indicated vectors. The protein levels of EZH2 were analyzed by western blot in the above conditions. (F) PCSEAT-knockdown or the control PC3 cells were treated with GSK343 for 72 h, and cell proliferation was assessed. The data graphs depict the count number from three independent experiments (means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001).
Please cite this article in press as: X. Yang, et al., The long non-coding RNA PCSEAT exhibits an oncogenic property in prostate cancer and functions as a competing endogenous RNA that associates with EZH2, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.157
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of target genes by competitively interacting with microRNA (miRNA), termed competitive endogenous RNA (ceRNA) [25]. To explore if PCSEAT mediated modulation of PCa cells proliferation and motility in such a way, we designed a strategy. First, we performed a search for miRNAs that have complementary base pairing with PCSEAT, using PITA algorithm (Supplementary Table 4). We then analyzed the correlation of the expression levels between PCSEAT and the mature miRNAs from TCGA, and retained 23 miRNAs (r < 0.25, P < 0.0001) (Supplementary Table 5). Second, we analyzed the correlation between PCSEAT and genes that were expressed in prostate cancer samples and identified 323 genes (r > 0.5, P < 0.0001) (Supplementary Table 5). Third, we searched if those miRNAs may target these coding genes using PITA algorithm. Based on these, we constructed a detailed ceRNA map (Fig. S3A). miR-143-3p and miR-24-2-5p, as the common targets of EZH2 and PCSEAT, were identified from the above defined threshold (Fig. S3B). First, we observed a significant downregulation of the EZH2 and its effector H3K27me3 protein levels upon PCSEAT knockdown in PC3 cells and an upregulation of the EZH2 and H3K27me3 protein levels in PCSEAT-overexpressing DU145 cells (Fig. 3A), but we did not observe a significant change in EZH2 mRNA levels (Fig. S4A), suggesting that PCSEAT regulates the expression of EZH2 at the
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post-transcriptional level and enzymatic activity of EZH2. Next, we confirmed the presence of miR-143-3p and miR-24-25p binding sites in PCSEAT and EZH2 gene using dual-luciferase reporter assays (Figs. 3B, S3C, S3D and S3E). Consequently, we observed that overexpression of miR-143-3p and miR-24-2-5p significantly abrogated the increase of the cell proliferation and the protein level of EZH2 in PCSEAT overexpression cells (Fig. 3C). Collectively, these data suggest that PCSEAT and EZH2 competitively ‘sponge’ miR-143-3p and miR-24-2-5p, which negatively regulate PCSEAT-mediated tumor activity. Then, we observed that knockdown of EZH2 abrogated the effect of PCSEAT overexpression on promoting DU145 cell proliferation and clonogenicity (Figs. 3D and S4B). Overexpression of EZH2 partially reversed the effect of PCSEAT knockdown on suppressing PC3 cell proliferation and clonogenicity (Figs. 3E and S4C). Additionally, the protein level of EZH2 also showed the similar trends (Fig. 3D and E). Collectively, these data suggest that PCSEAT partially regulates PCa cells proliferation and clonogenicity by positively regulating EZH2. Besides, we observed that PCSEAT knockdown PC3 cells were resistant to EZH2 inhibitor GSK343 compared to the control cells (Fig. 3F), suggesting that expression of PCSEAT may serve as a biomarker for EZH2 inhibitors.
Fig. 4. PCSEAT-rich exosomes function in recipient cells. (A) Electron microscopy image and NanoSight particle-tracking analysis of the size distributions and number of purified exosomes collected from DU145 cells was shown (Scale bar ¼ 50 nm). (B, C) The amounts of PCSEAT transcripts (B) and the protein levels of EZH2 (C) in DU145-derived exosomes with empty vector or PCSEAT overexpression were determined. (D) The amounts of PCSEAT transcripts in DU145 cells fed with the exosomes derived from PCSEAT-overexpressing or the control DU145 cells were quantified by qRTPCR. (E) Proliferation of DU145 cells treated with exosomes from PCSEAT-overexpressing or the control DU145 cells were assessed by CTG assays after incubation for 72 h (F, G) Clone formation assays and transwell assays were performed in DU145 cells that incubated with exosomes derived from PCSEAT-overexpressing or the control DU145 cells, respectively. Representative graphs are shown. (H) Proliferation of the exosomes fed RWPE-1 cells were assessed by CTG assays after incubation for 72 h. The data depict the count number from three independent experiments (means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001).
Please cite this article in press as: X. Yang, et al., The long non-coding RNA PCSEAT exhibits an oncogenic property in prostate cancer and functions as a competing endogenous RNA that associates with EZH2, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.157
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3.4. Intercellular transfer of PCSEAT by exosomes functions in PCa cells Indeed, recent studies demonstrate that lncRNAs-contained exosomes can function to disseminate cell signals that alter and/ or control neighboring cell fate [15,16]. Thus, we asked whether PCSEAT may also function through exosomes. Electron microscopy, Nanosight particle tracking analysis and western blot assays confirmed the quality, number, size and identity of the isolated exosomes (Figs. 4A and S5). Especially, we detected a significant increase of PCSEAT and EZH2 in exosomes derived from PCSEAT overexpression cells, which was consistent with the cellular expression (Fig. 4B and C). To determine if the enrichment of PCSEAT in the exosomes has physiological consequences, we performed exosome feeding assays and found that DU145 cells that were incubated with exosomes derived from PCSEAT-overexpressing DU145 cells had a higher PCSEAT level and exhibited significantly increased proliferation, clonogenicity and migration ability compared to those incubated with control exosomes (Fig. 4D, E, 4F and 4G). Strikingly, exosomes derived from PCSEAToverexpressing DU145 cells promoted the proliferative ability of normal prostate cells (RWPE-1) and other different types of cancer cells (769-P, 786-O, ACHN, Calu-1, NCI-H1299) after fed (Figs. 4H and S6). Together, these data suggest that PCSEAT can be delivered by PCa cell-derived exosomes and received by other cells, thus promoting recipient cell proliferation. 4. Discussion In this study, we found that a novel lncRNA, PCSEAT, is specifically upregulated in PCa cells. As benign prostatic hyperplasia (BPH) and PCa are among the most common diseases of the prostate gland and share many similar traits [26], it is worth to evaluate the expression of PCSEAT in normal, BPH and PCa tissues in future study. EZH2 has been emerging as a robust biomarker and potential therapeutic target in metastatic PCa [9]. Additionally, extensive genetic (e.g., the fusion between the TMPRESS2 and ERG genes) and epigenetic aberrations have been found to be associated with different stages of PCa development, many of which are associated with disorders of EZH2 [9]. To date, few data are available on the association between lncRNAs and EZH2. The imprinted lncRNA Meg3 directly and specifically binds to PRC2 and most likely recruits EZH2 subunits to Dlk1, affecting the gene expression in cis [27]. The lncRNA Xist interacts with EZH2 through YY1, which captures the Xist-EZH2 complex, resulting in inactivation of the X chromosome [28]. The lncRNA HOTAIR is able to induce H3K27me3 and inhibit the HOXD locus and other target genes as direct targets of the EZH2 subunit. Importantly, HOTAIR overexpression enhances the occupancy of EZH2 in colorectal cancer cells [29]. In addition, there are several lncRNAs in PCa that can interact with EZH2 to function. The lncRNA PCAT-1 and EZH2 expression are almost mutually exclusive, but EZH2 inhibition result in increased expression of PCAT-1 in cell lines, indicating that it is an EZH2 target [11]. The antisense lncRNA ANRIL recruits PRC2 and PRC1 subunits (EZH2 and CBX7, respectively) to achieve INK4b/ARF/INK4a silencing, and is directly involved in epigenetic transcriptional repression observed in cancer [30]. Our findings describe a new way in which lncRNAs can be involved in epigenetic regulation via ceRNA network. This makes it possible for PCSEAT to become a target of diagnosis or treatment. Recent studies indicate that exosomes can serve as trafficking vesicles for functional lncRNAs, which can lead to phenotypic effects within recipient cells [31]. Several of the previously described lncRNAs, such as MALAT1, HOTAIR, and GAS5, which play an
important role in various cancers, are also highly expressed in exosomes from HeLa and MCF-7 cells, indicating that these exosomes are released by cancer cells to induce cancer-like phenotypes in recipient cells [32]. However, data on the role and expression of lncRNAs in PCa exosomes are limited [33e35]. Our data indicate that PCSEAT, EZH2 and exosomes may function together to transmit cellular signals. This effect is effective not only in PCa cells, but also in normal prostate cells, lung cancer cells and clear cell renal cell carcinoma cells. In fact, exosomes are currently being evaluated as possible ways to target drug delivery, and several clinical trials are under way [36]. Their functional relevance in cancer also makes it possible to use PCSEAT as a diagnostic and a cancer therapy target. More importantly, we partially elucidated a lncRNA that can function in exosomes to influence the progression of PCa. In summary, we demonstrated the detailed mechanistic insight of the PCSEAT-miR-143-3p/miR-24-2-5p-EZH2 axis and some PCSEAT secretion-uptake from cells in PCa (Fig. S7). These finding suggest that PCSEAT may be an important biomarker and target for PCa therapy. Conflicts of interest The authors declare no conflicts of interest related to the publication of this work. Acknowledgments This work was supported by National Natural Science Foundation of China (81773023 and 81472827); the National Key R&D Program of China (2016YFC1302100); Hundred-Talent Program (Y521031102) and Frontier Research Program (QYZDB-SSWSMC038) of Chinese Academy of Sciences, to Shan Gao. We also thank the ENCODE Consortium and the ENCODE production laboratory(s) generating the particular dataset(s). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.05.157. References [1] G. Attard, C. Parker, R.A. Eeles, et al., Prostate cancer, Lancet 387 (2015) 70e82. [2] R. Dan, E. Vanallen, Y.M. Wu, et al., Integrative clinical genomics of advanced prostate cancer, Cell 161 (2015) 1215e1228. [3] D. Robinson, E.M. Van Allen, Y.M. Wu, et al., Integrative clinical genomics of advanced prostate cancer, Cell 161 (2015) 1215e1228. € der, P.v. Leeuwen, et al., Performance of the prostate [4] M.J. Roobol, F.H. Schro cancer antigen 3 (PCA3) gene and prostate-specific antigen in prescreened men: exploring the value of for a first-line diagnostic test, Eur. Urol. 59 (2011) 10e11. [5] Z. Chang, W. Xinwen, W. Liang, et al., The mystery of "rubbish" DNA, Chin. Sci. Bull. 61 (2016) 3079e3084. [6] J.R. Prensner, M.K. Iyer, O.A. Balbin, et al., Transcriptome sequencing across a prostate cancer cohort identifies PCAT-1, an unannotated lincRNA implicated in disease progression, Nat. Biotechnol. 29 (2011) 742. [7] G. RA, S. N, W. KC, et al., Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis, Nature 464 (2010) 1071. [8] A.L. Walsh, A.V. Tuzova, E.M. Bolton, et al., Long noncoding RNAs and prostate carcinogenesis: the missing ‘linc’? Trends Mol. Med. 20 (2014) 428. [9] Y.A. Yang, J. Yu, EZH2, an epigenetic driver of prostate cancer, Protein & Cell 4 (2013) 331e341. [10] S. Varambally, S.M. Dhanasekaran, M. Zhou, et al., The polycomb group protein EZH2 is involved in progression of prostate cancer, Nature 419 (2002) 624e629. [11] J.R. Prensner, M.K. Iyer, O.A. Balbin, et al., Transcriptome sequencing across a prostate cancer cohort identifies PCAT-1, an unannotated lincRNA implicated in disease progression, Nat. Biotechnol. 29 (2011) 742e749. ~ ozcabello, et al., Molecular interplay of the noncoding [12] K.L. Yap, S. Li, A.M. Mun RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a, Mol. Cell 38 (2010) 662e674. [13] S.J. Gould, G. Raposo, As we wait: coping with an imperfect nomenclature for
Please cite this article in press as: X. Yang, et al., The long non-coding RNA PCSEAT exhibits an oncogenic property in prostate cancer and functions as a competing endogenous RNA that associates with EZH2, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.157
X. Yang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7 extracellular vesicles, J. Extracell. Vesicles 2 (2013). [14] W. Eduard, H.J. Johansson, M. Imre, et al., Cells release subpopulations of exosomes with distinct molecular and biological properties, Sci. Rep. 6 (2016) 22519. [15] J.L. Hood, R.S. San, S.A. Wickline, Exosomes released by melanoma cells prepare sentinel lymph nodes for tumor metastasis, Canc. Res. 71 (2011) 3792. [16] V. Huber, S. Fais, M. Iero, et al., Human colorectal cancer cells induce T-cell death through release of proapoptotic microvesicles: role in immune escape, Gastroenterology 128 (2005) 1796e1804. [17] S. Staubach, H. Razawi, F.G. Hanisch, Proteomics of MUC1-containing lipid rafts from plasma membranes and exosomes of human breast carcinoma cells MCF-7, Proteomics 9 (2009) 2820e2835. [18] Y. Zhang, T. Liu, C.A. Meyer, et al., Model-based analysis of ChIP-seq (MACS), Genome Biol. 9 (2008). R137. [19] J. Feng, T. Liu, Y. Zhang, Using MACS to identify peaks from ChIP-Seq data, Chapter 2, Current Protocols in Bioinformatics (2011). Unit 2.14. [20] W.J. Kent, C.W. Sugnet, T.S. Furey, et al., The human genome browser at UCSC, Genome Res. 12 (2002) 996e1006. [21] A.K. Akobeng, Understanding diagnostic tests 3: receiver operating characteristic curves, Acta Paediatr. 96 (2007) 644e647. [22] E.P. Consortium, An integrated encyclopedia of DNA elements in the human genome, Nature 489 (2012) 57e74. [23] G. Liang, J.C.Y. Lin, V. Wei, et al., Distinct localization of histone H3 acetylation and H3-K4 methylation to the transcription start sites in the human genome, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 7357e7362. [24] M.F. Lin, I. Jungreis, M. Kellis, PhyloCSF: a comparative genomics method to distinguish protein coding and non-coding regions, Bioinformatics 27 (2011) i275ei282. [25] L. Salmena, L. Poliseno, Y. Tay, et al., A ceRNA hypothesis: the Rosetta stone of a hidden RNA language? Cell 146 (2011) 353e358.
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[26] Ø. DD, S.E. Bojesen, The link between benign prostatic hyperplasia and prostate cancer, Nat. Rev. Urol. 10 (2013) 49e54. [27] J. Zhao, T.K. Ohsumi, J.T. Kung, et al., Genome-wide identification of polycomb-associated RNAs by RIP-seq, Mol. Cell 40 (2010) 939. [28] Y. Jeon, J.T. Lee, YY1 tethers Xist RNA to the inactive X nucleation center, Cell 146 (2011) 119. [29] R.A. Gupta, N. Shah, K.C. Wang, et al., Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis, Nature 464 (2010) 1071e1076. ~ ozcabello, et al., Molecular interplay of the non-coding [30] K.L. Yap, S. Li, A.M. Mun RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a, Mol. Cell 38 (2010) 662. [31] T. Kogure, I.K. Yan, W.L. Lin, et al., Extracellular vesicleemediated transfer of a novel long noncoding RNA TUC339 a mechanism of intercellular signaling in human hepatocellular cancer, Genes & Cancer 4 (2013) 261. € [32] U. Gezer, E. Ozgür, M. Cetinkaya, et al., Long non-coding RNAs with low expression levels in cells are enriched in secreted exosomes, Cell Biol. Int. 38 (2014) 1076. [33] A. Alireza, B. Samuel, P.J. Kennedy, et al., Long non-coding RNAs harboring miRNA seed regions are enriched in prostate cancer exosomes, Sci. Rep. 6 (2016) 24922. € [34] M. Is¸ın, E. Uysaler, E. Ozgür, et al., Exosomal lncRNA-p21 levels may help to distinguish prostate cancer from benign disease, Journal of Urological Surgery 6 (2015) 168. [35] A. Ahadi, S. Khoury, M. Losseva, et al., A comparative analysis of lncRNAs in prostate cancer exosomes and their parental cell lines, Genomics Data 9 (2016) 7e9. [36] O. Shin-Ichiro, G.P.C. Drummen, K. Masahiko, Focus on extracellular vesicles: development of extracellular vesicle-based therapeutic systems, Int. J. Mol. Sci. 17 (2016) 172.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
The long non-coding RNA PCSEAT exhibits an oncogenic property in prostate cancer and functions as a competing endogenous RNA that associates with EZH2 Xiaohui Yang a, b, Liang Wang c, Rong Li b, Yuhui Zhao c, Yinmin Gu b, Siying Liu b, Tianyou Cheng d, Kuohsiang Huang b, Yi Yuan e, Dalong Song e, Shan Gao b, d, e, * a
Laboratory for Noncoding RNA & Cancer, School of Life Sciences, Shanghai University, Shanghai, 200444, China CAS Key Laboratory of Bio-medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163, China c CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China d Shanxi Academy of Advanced Research and Innovation, Taiyuan, 030032, China e Medical College, Guizhou University, Guiyang, 550025, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 May 2018 Accepted 22 May 2018 Available online xxx
Prostate cancer (PCa) is the most common malignancy and the leading cause of cancer deaths in males. Recent studies demonstrate that long non-coding RNAs (lncRNAs) are involved in many aspects of PCa. However, their biological roles in PCa remain imperfectly understood. Here, we characterized an lncRNA, PCa specific expression and EZH2-associated transcript (PCSEAT, annotated as PRCAT38), which is specifically overexpressed in PCa. We further demonstrated that knockdown of PCSEAT results in the reduction of PCa cell growth and motility, and overexpression of PCSEAT reverses these phenotypes. Furthermore, bioactive PCSEAT is incorporated into exosomes and transmitted to adjacent cells, thus promoting cell proliferation and motility. Mechanistically, we found that PCSEAT promotes cell proliferation, at least in part by affecting miR-143-3p- and miR-24-2-5p-mediated regulation of EZH2, suggesting that PCSEAT and EZH2 competitively ‘sponge’ miR-143-3p and miR-24-2-5p. Overall, our results reveal that PCSEAT is specifically overexpressed in PCa patients and a potential oncogene in PCa cells via mediating EZH2 activity, indicating that PCSEAT may be a potential therapeutic target in PCa. © 2018 Elsevier Inc. All rights reserved.
Keywords: Prostate cancer PCSEAT EZH2 Competing endogenous RNA Exosome
1. Introduction As the most common malignant tumor in men, prostate cancer (PCa) is a major cause of cancer deaths [1]. To understand the underlying biology, numerous studies have been made to identify recurrent somatic mutations (SPOP, FOXA1, IDH1, and TP53), changes in copy number (MYC, RB1, PTEN, and CHD1), and the oncogenic structural DNA rearrangements (E26 transformationspecific (ETS) fusions) in primary PCa [2] and genomic alterations (AR, PIK3CA/B, R-spondin, BRAF/RAF1, APC, b-catenin, and ZBTB16/ PLZF) in advanced PCa [3]. It has been revealed that a large portion of the human genome is
* Corresponding author. NO.88, Keling Road, High-tech District, Suzhou, China. E-mail address: [email protected] (S. Gao).
transcribed into noncoding transcripts [4], including long noncoding RNAs (lncRNAs) with >200 bp in length [5]. Dysregulated expression of lncRNAs signal the spectrum of disease progression and can be used as the independent biomarkers and/or the therapeutic targets in cancer [6,7]. Recently, many lncRNAs have been shown to act as tumor suppressor genes or oncogenes via modulation of various signaling pathways in PCa, such as H19, MALAT1, PCA3, PCAT-1, and SChLAP1 [8]. The histone methyltransferase, polycomb group protein enhancer of zeste homolog 2 (EZH2) maintains the epigenomic integrity of a cell, which is capable of preserving proper chromatin structure, ensuring appropriate gene expression, and regulating a cohort of tumor and metastasis suppressor genes [9]. Moreover, EZH2 is particularly culpable in PCa's development and progression to advanced disease stages [10]. Indeed, it has been shown that some lncRNAs can directly or indirectly interact with EZH2 in PCa,
https://doi.org/10.1016/j.bbrc.2018.05.157 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: X. Yang, et al., The long non-coding RNA PCSEAT exhibits an oncogenic property in prostate cancer and functions as a competing endogenous RNA that associates with EZH2, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.157
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Chinese Academy of Sciences Cell Bank. 293T cells were cultured in DMEM medium supplemented with 10% fetal calf serum. RWPE1 cells were cultured in Keratinocyte-SFM medium supplemented with Gentamicin Solution. The other cells lines were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum. All cell lines were verified using short tandem repeat assays by Genetic Testing Biotechnology, Suzhou, China. GSK343 was purchased from Selleck Chemicals.
such as PCAT-1 [11] and ANRI [12]. Exosomes, ranging from 50 to 150 nm in diameter, are an extracellular vesicle that are formed within multivesicular bodies [13] and can be secreted from a variety of cell types and mediate a broad spectrum of effects on recipient cells [14]. For example, exosomes released from melanoma cells create the optimal environment for metastatic cancer cells, such as promoting angiogenesis, stromal remodeling, and lymph node incompetence [15]. Furthermore, colorectal cancer cells can shed Fas ligand loaded vesicles, which reduces the ability of T cell to induce cancer cells apoptosis [16]. Here, we reported the clinical implication and function of the lncRNA PCa specific expression and EZH2-associated transcript (PCSEAT, annotated as PRCAT38). PCSEAT promotes prostate tumor growth and migration by acting in part through ‘sponging’ miR143-3p and miR-24-2-5p to regulate EZH2 expression. Moreover, we find that PCSEAT can be incorporated into exosomes and transmitted to neighbor cells, thus promoting cell proliferation.
Cells were plated in 96-well plates with a density of 1 103 or 2 103 cells per well. Cell proliferation was determined with a Celltiter glow (CTG) kit measured with a multi-mode microplate reader (Synergy HTX, BioTek, Winooski, United States). PC3 cells were exposed to vehicle (0.5% DMSO) or GSK343 at the IC50 (half maximal inhibitory concentration) for 72 h, and proliferation was assessed. All experiments were performed in triplicate.
2. Materials and methods
2.3. Clone formation assay
2.1. Cell lines, cell culture and reagent
Cells were plated at 500 or 1000 cells per well in 6-well plates and incubated for 2 weeks at 37 C in a 5% CO2 humidified environment. Colonies were stained with Coomassie Blue Staining Solution (0.1% (w/v) Coomassie Blue R-250, 25% (v/v) dimethylcarbinol, and
The 293T, PC3, DU145, LNCaP, 22Rv1, RWPE-1, 769-P, 786-O, ACHN, Calu-1 and NCI-H1299 cells lines were obtained from the
2.2. Cell proliferation assay
Fig. 1. PCSEAT is expressed in PCa. (A) PCSEAT was selectively expressed in PRAD across the 21 cancer types (Mitranscriptome) (means ± SEM; P < 0.0001). (B) ROC curve for prediction of PRAD using PCSEAT expression levels from TCGA. (95% CI, P < 0.0001). (C, D) In situ hybridization of PCSEAT in 90 pairs of Chinese PCa tissues was performed. Representative images of PCSEAT staining in PCa tissues (cancer) and adjacent normal tissues (paratumor) (Scale bars ¼ 200 mm) (top). Histogram representation of PCSEAT expression levels for PCa tissues and adjacent normal tissues (bottom). (n ¼ 90; means ± SEM; P < 0.0001). (E) Genomic landscape of PCSEAT gene: ChIP-seq data for H3K4me3, H3K27ac, and H3K36me3 demonstrated enrichment at the PSCEAT gene. (F) The protein-coding potential of PCSEAT transcript was scored using PhyloCSF. HOTAIR and SChLAP1 were non-coding transcript controls (PhyloCSF score < 0). GAPDH and ACTB were coding transcript controls (PhyloCSF score > 0).
Please cite this article in press as: X. Yang, et al., The long non-coding RNA PCSEAT exhibits an oncogenic property in prostate cancer and functions as a competing endogenous RNA that associates with EZH2, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.157
X. Yang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
10% (v/v) glacial acetic acid) after fixed with methanol, and then counted. All experiments were performed in triplicate.
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Data was visualized using the UCSC Genome Browser [20]. 2.7. Western blotting
2.4. Exosome isolation DU145 cells were grown to confluent monolayers, washed three times with PBS, and grown in serum-free media for 3 days. Exosomes were collected by differential centrifugation from conditioned media, resuspended in PBS [17]. 2.5. LncRNAs, miRNA, and mRNA expression data Differentially expressed lncRNAs were analyzed based on the clinical information of 42 normal tissues and 173 tumor samples from The Cancer Genome Atlas (TCGA) and the matched expression profiles of 12382 lncRNAs from Mitranscriptome (http:// mitranscriptome.com/). TCGA prostate adenocarcinoma patients' microRsNA expressions (Level 3 data, illuminahiseq mirnaseq) and mRNA expressions (level 3 data, RNA-seq Version 2) were downloaded from FireBrowse (http://firebrowse.org/). FireBrowse portals serve TCGA data. 2.6. PC3 ChIP-Seq data Sequencing data from GSE96019, GSE96399 and GSE96418 were downloaded from GEO. Reads from the PC3 cell H3K4me3, H3K27ac and H3K36me3 ChIP-Seq samples were mapped to human genome version hg19 using HISAT2. Peak calling was performed using MACS [18] according to the published protocols [19].
Total cell lysates were denatured in SDS (Sigma, St Louis, MO, USA) loading buffer and boiled for 10 min. Samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (General Electric, Fairfield, CT, USA). Membranes were blocked with 5% milk in Tris-buffered salineeTween 20 (TBST) and incubated overnight at 4 C with primary antibodies. Membranes were then washed and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies for 1.5 h at room temperature. Membranes were washed again, and bands were visualized with Immobilon Western Chemilum HRP Substrate from Millipore (Merck KGaA, Darmstadt, Germany). 2.8. Statistical analyses Statistical analyses were performed using the Student's t-test. A P-value less than 0.05 was considered to be statistically significant in Student's t-test. No criteria were applied for inclusion and exclusion of data. No outliers were taken into account, and all collected data were subjected to statistical analyses. 3. Results 3.1. PCSEAT is specifically upregulated in human PCa tissues To explore the potential lncRNAs involved in PCa oncogenesis, we
Fig. 2. PCSAET promotes PCa cell proliferation and migration in vitro. (A, B) Proliferation of PCa cells was assessed by CTG assays. (C, D) Clone formation assays were performed in PCa cells. Representative graphs are shown. (E, F) Wound healing assays were made in PC3 and DU145 cells for 12 or 72 h of recovery, respectively. All of the cells were grown in serum-free medium during the recovery. The graphs represent relative wound healing rates. The data depict the count number from three independent experiments (means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001).
Please cite this article in press as: X. Yang, et al., The long non-coding RNA PCSEAT exhibits an oncogenic property in prostate cancer and functions as a competing endogenous RNA that associates with EZH2, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.157
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examined clinical data of patients in TCGA and matched expression profiles of lncRNAs from Mitranscriptome (Supplementary Table 1). We noted an lncRNA (annotation as PRCAT38), named PCSEAT, which was selective expression in prostate adenocarcinoma (PRAD) tissues across 21 types of cancer and corresponding adjacent normal tissues (TCGA) (P < 0.0001; Fig. 1A). Receiver operating characteristic (ROC) curve [21] was determined to evaluate the sensitivity and specificity of PCSEAT expression in predicting PRAD tissues from normal tissues. Notably, PCSEAT displayed considerably predictive significance, with an area under the curve (AUC) of 0.950 (95% confidence interval: 0.904e0.996; P < 0.0001; Fig. 1B). Furthermore, we used an in-situ hybridization assay to examine the subcellular localization and the expression levels of PCSEAT in 90 pairs of formalin-fixed, paraffinembedded Chinese human PCa samples. We confirmed the relative nuclear enrichment of PCSEAT and that the expression levels of PCSEAT were significantly increased in the PCa samples compared to the adjacent normal tissues (Fig. 1C and D and Supplementary Table 2). To more precisely explore the function of PCSEAT, we defined a 2413-bp polyadenylated gene composed of only one exon on chromosome 21q22.3 using 50 and 30 rapid amplification of cDNA ends and reverse transcription PCR (RT-PCR) (Figs. 1E and S1 and Supplementary Table 3). Published PCa chromatin immunoprecipitation and sequencing (ChIP-seq) data [22] confirm that the transcriptional start site of PCSEAT is marked by H3K4me3, a marker of an active promoter [23], and H3K27ac, a marker of active enhancers and
promoters [23]. Its genetic locus also harbored H3K36me3 (Fig. 1E), an epigenetic signature consistent with lncRNAs [23]. We did not find protein-coding potential in PCSEAT by PhyloCSF [24] (Fig. 1F). These data suggest that PCSEAT is specifically upregulated in PCa. 3.2. The lncRNA PCSEAT promotes PCa cell proliferation and motility To evaluate the biological effects of PCSEAT on PCa cells, we performed gain- and loss-of-function studies. We established cell lines with stably overexpressed PCSEAT and PCSEAT-specific knockdown, respectively (Figs. S2A and S2B). CTG assays demonstrated that knockdown of PCSEAT significantly inhibited PC3 cells proliferation (Fig. 2A). Additionally, we found that both shRNAs significantly reduced the clonogenicity and motility of PC3 cells (Fig. 2C and E). Conversely, our results demonstrated that overexpression of PCSEAT led to a significant increase in the proliferative capacity, clonogenicity and motility of DU145 cells (Fig. 2B, D and 2F). All together, these results indicate that PCSEAT promotes the growth and motility of PCa cells. 3.3. PCSEAT regulates EZH2 by competitively ‘sponge’ miR-143-3p and miR-24-2-5p It has been proposed that lncRNAs can regulate the expression
Fig. 3. PCSEAT regulates EZH2 through miR-143-3p and 31 miR-24-2-5p. (A) The protein levels of EZH2 and H3K27me3 in stable PCSEAT knockdown PC3 cells and stable PCSEAT-overexpressing DU145 cells are shown. (B) The dual-luciferase reporter assay was performed in 293T cells containing reporter vectors by co-transfecting the cells with miR-143-3p or miR-24-2-5p mimic and PCSEAT transcript. (C)The proliferation was measured by CTG assays and protein levels of EZH2 were analyzed by western blot in DU145 cells transfected with the indicated vectors and miRNA mimics. (D, E) Proliferation of PCa cells was assessed by CTG assays after incubation for 120 h. DU145 (E) and PC3 (F) cells were transfected with the indicated vectors. The protein levels of EZH2 were analyzed by western blot in the above conditions. (F) PCSEAT-knockdown or the control PC3 cells were treated with GSK343 for 72 h, and cell proliferation was assessed. The data graphs depict the count number from three independent experiments (means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001).
Please cite this article in press as: X. Yang, et al., The long non-coding RNA PCSEAT exhibits an oncogenic property in prostate cancer and functions as a competing endogenous RNA that associates with EZH2, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.157
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of target genes by competitively interacting with microRNA (miRNA), termed competitive endogenous RNA (ceRNA) [25]. To explore if PCSEAT mediated modulation of PCa cells proliferation and motility in such a way, we designed a strategy. First, we performed a search for miRNAs that have complementary base pairing with PCSEAT, using PITA algorithm (Supplementary Table 4). We then analyzed the correlation of the expression levels between PCSEAT and the mature miRNAs from TCGA, and retained 23 miRNAs (r < 0.25, P < 0.0001) (Supplementary Table 5). Second, we analyzed the correlation between PCSEAT and genes that were expressed in prostate cancer samples and identified 323 genes (r > 0.5, P < 0.0001) (Supplementary Table 5). Third, we searched if those miRNAs may target these coding genes using PITA algorithm. Based on these, we constructed a detailed ceRNA map (Fig. S3A). miR-143-3p and miR-24-2-5p, as the common targets of EZH2 and PCSEAT, were identified from the above defined threshold (Fig. S3B). First, we observed a significant downregulation of the EZH2 and its effector H3K27me3 protein levels upon PCSEAT knockdown in PC3 cells and an upregulation of the EZH2 and H3K27me3 protein levels in PCSEAT-overexpressing DU145 cells (Fig. 3A), but we did not observe a significant change in EZH2 mRNA levels (Fig. S4A), suggesting that PCSEAT regulates the expression of EZH2 at the
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post-transcriptional level and enzymatic activity of EZH2. Next, we confirmed the presence of miR-143-3p and miR-24-25p binding sites in PCSEAT and EZH2 gene using dual-luciferase reporter assays (Figs. 3B, S3C, S3D and S3E). Consequently, we observed that overexpression of miR-143-3p and miR-24-2-5p significantly abrogated the increase of the cell proliferation and the protein level of EZH2 in PCSEAT overexpression cells (Fig. 3C). Collectively, these data suggest that PCSEAT and EZH2 competitively ‘sponge’ miR-143-3p and miR-24-2-5p, which negatively regulate PCSEAT-mediated tumor activity. Then, we observed that knockdown of EZH2 abrogated the effect of PCSEAT overexpression on promoting DU145 cell proliferation and clonogenicity (Figs. 3D and S4B). Overexpression of EZH2 partially reversed the effect of PCSEAT knockdown on suppressing PC3 cell proliferation and clonogenicity (Figs. 3E and S4C). Additionally, the protein level of EZH2 also showed the similar trends (Fig. 3D and E). Collectively, these data suggest that PCSEAT partially regulates PCa cells proliferation and clonogenicity by positively regulating EZH2. Besides, we observed that PCSEAT knockdown PC3 cells were resistant to EZH2 inhibitor GSK343 compared to the control cells (Fig. 3F), suggesting that expression of PCSEAT may serve as a biomarker for EZH2 inhibitors.
Fig. 4. PCSEAT-rich exosomes function in recipient cells. (A) Electron microscopy image and NanoSight particle-tracking analysis of the size distributions and number of purified exosomes collected from DU145 cells was shown (Scale bar ¼ 50 nm). (B, C) The amounts of PCSEAT transcripts (B) and the protein levels of EZH2 (C) in DU145-derived exosomes with empty vector or PCSEAT overexpression were determined. (D) The amounts of PCSEAT transcripts in DU145 cells fed with the exosomes derived from PCSEAT-overexpressing or the control DU145 cells were quantified by qRTPCR. (E) Proliferation of DU145 cells treated with exosomes from PCSEAT-overexpressing or the control DU145 cells were assessed by CTG assays after incubation for 72 h (F, G) Clone formation assays and transwell assays were performed in DU145 cells that incubated with exosomes derived from PCSEAT-overexpressing or the control DU145 cells, respectively. Representative graphs are shown. (H) Proliferation of the exosomes fed RWPE-1 cells were assessed by CTG assays after incubation for 72 h. The data depict the count number from three independent experiments (means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001).
Please cite this article in press as: X. Yang, et al., The long non-coding RNA PCSEAT exhibits an oncogenic property in prostate cancer and functions as a competing endogenous RNA that associates with EZH2, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.157
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3.4. Intercellular transfer of PCSEAT by exosomes functions in PCa cells Indeed, recent studies demonstrate that lncRNAs-contained exosomes can function to disseminate cell signals that alter and/ or control neighboring cell fate [15,16]. Thus, we asked whether PCSEAT may also function through exosomes. Electron microscopy, Nanosight particle tracking analysis and western blot assays confirmed the quality, number, size and identity of the isolated exosomes (Figs. 4A and S5). Especially, we detected a significant increase of PCSEAT and EZH2 in exosomes derived from PCSEAT overexpression cells, which was consistent with the cellular expression (Fig. 4B and C). To determine if the enrichment of PCSEAT in the exosomes has physiological consequences, we performed exosome feeding assays and found that DU145 cells that were incubated with exosomes derived from PCSEAT-overexpressing DU145 cells had a higher PCSEAT level and exhibited significantly increased proliferation, clonogenicity and migration ability compared to those incubated with control exosomes (Fig. 4D, E, 4F and 4G). Strikingly, exosomes derived from PCSEAToverexpressing DU145 cells promoted the proliferative ability of normal prostate cells (RWPE-1) and other different types of cancer cells (769-P, 786-O, ACHN, Calu-1, NCI-H1299) after fed (Figs. 4H and S6). Together, these data suggest that PCSEAT can be delivered by PCa cell-derived exosomes and received by other cells, thus promoting recipient cell proliferation. 4. Discussion In this study, we found that a novel lncRNA, PCSEAT, is specifically upregulated in PCa cells. As benign prostatic hyperplasia (BPH) and PCa are among the most common diseases of the prostate gland and share many similar traits [26], it is worth to evaluate the expression of PCSEAT in normal, BPH and PCa tissues in future study. EZH2 has been emerging as a robust biomarker and potential therapeutic target in metastatic PCa [9]. Additionally, extensive genetic (e.g., the fusion between the TMPRESS2 and ERG genes) and epigenetic aberrations have been found to be associated with different stages of PCa development, many of which are associated with disorders of EZH2 [9]. To date, few data are available on the association between lncRNAs and EZH2. The imprinted lncRNA Meg3 directly and specifically binds to PRC2 and most likely recruits EZH2 subunits to Dlk1, affecting the gene expression in cis [27]. The lncRNA Xist interacts with EZH2 through YY1, which captures the Xist-EZH2 complex, resulting in inactivation of the X chromosome [28]. The lncRNA HOTAIR is able to induce H3K27me3 and inhibit the HOXD locus and other target genes as direct targets of the EZH2 subunit. Importantly, HOTAIR overexpression enhances the occupancy of EZH2 in colorectal cancer cells [29]. In addition, there are several lncRNAs in PCa that can interact with EZH2 to function. The lncRNA PCAT-1 and EZH2 expression are almost mutually exclusive, but EZH2 inhibition result in increased expression of PCAT-1 in cell lines, indicating that it is an EZH2 target [11]. The antisense lncRNA ANRIL recruits PRC2 and PRC1 subunits (EZH2 and CBX7, respectively) to achieve INK4b/ARF/INK4a silencing, and is directly involved in epigenetic transcriptional repression observed in cancer [30]. Our findings describe a new way in which lncRNAs can be involved in epigenetic regulation via ceRNA network. This makes it possible for PCSEAT to become a target of diagnosis or treatment. Recent studies indicate that exosomes can serve as trafficking vesicles for functional lncRNAs, which can lead to phenotypic effects within recipient cells [31]. Several of the previously described lncRNAs, such as MALAT1, HOTAIR, and GAS5, which play an
important role in various cancers, are also highly expressed in exosomes from HeLa and MCF-7 cells, indicating that these exosomes are released by cancer cells to induce cancer-like phenotypes in recipient cells [32]. However, data on the role and expression of lncRNAs in PCa exosomes are limited [33e35]. Our data indicate that PCSEAT, EZH2 and exosomes may function together to transmit cellular signals. This effect is effective not only in PCa cells, but also in normal prostate cells, lung cancer cells and clear cell renal cell carcinoma cells. In fact, exosomes are currently being evaluated as possible ways to target drug delivery, and several clinical trials are under way [36]. Their functional relevance in cancer also makes it possible to use PCSEAT as a diagnostic and a cancer therapy target. More importantly, we partially elucidated a lncRNA that can function in exosomes to influence the progression of PCa. In summary, we demonstrated the detailed mechanistic insight of the PCSEAT-miR-143-3p/miR-24-2-5p-EZH2 axis and some PCSEAT secretion-uptake from cells in PCa (Fig. S7). These finding suggest that PCSEAT may be an important biomarker and target for PCa therapy. Conflicts of interest The authors declare no conflicts of interest related to the publication of this work. Acknowledgments This work was supported by National Natural Science Foundation of China (81773023 and 81472827); the National Key R&D Program of China (2016YFC1302100); Hundred-Talent Program (Y521031102) and Frontier Research Program (QYZDB-SSWSMC038) of Chinese Academy of Sciences, to Shan Gao. We also thank the ENCODE Consortium and the ENCODE production laboratory(s) generating the particular dataset(s). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.05.157. References [1] G. Attard, C. Parker, R.A. Eeles, et al., Prostate cancer, Lancet 387 (2015) 70e82. [2] R. Dan, E. Vanallen, Y.M. Wu, et al., Integrative clinical genomics of advanced prostate cancer, Cell 161 (2015) 1215e1228. [3] D. Robinson, E.M. Van Allen, Y.M. Wu, et al., Integrative clinical genomics of advanced prostate cancer, Cell 161 (2015) 1215e1228. € der, P.v. Leeuwen, et al., Performance of the prostate [4] M.J. Roobol, F.H. Schro cancer antigen 3 (PCA3) gene and prostate-specific antigen in prescreened men: exploring the value of for a first-line diagnostic test, Eur. Urol. 59 (2011) 10e11. [5] Z. Chang, W. Xinwen, W. Liang, et al., The mystery of "rubbish" DNA, Chin. Sci. Bull. 61 (2016) 3079e3084. [6] J.R. Prensner, M.K. Iyer, O.A. Balbin, et al., Transcriptome sequencing across a prostate cancer cohort identifies PCAT-1, an unannotated lincRNA implicated in disease progression, Nat. Biotechnol. 29 (2011) 742. [7] G. RA, S. N, W. KC, et al., Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis, Nature 464 (2010) 1071. [8] A.L. Walsh, A.V. Tuzova, E.M. Bolton, et al., Long noncoding RNAs and prostate carcinogenesis: the missing ‘linc’? Trends Mol. Med. 20 (2014) 428. [9] Y.A. Yang, J. Yu, EZH2, an epigenetic driver of prostate cancer, Protein & Cell 4 (2013) 331e341. [10] S. Varambally, S.M. Dhanasekaran, M. Zhou, et al., The polycomb group protein EZH2 is involved in progression of prostate cancer, Nature 419 (2002) 624e629. [11] J.R. Prensner, M.K. Iyer, O.A. Balbin, et al., Transcriptome sequencing across a prostate cancer cohort identifies PCAT-1, an unannotated lincRNA implicated in disease progression, Nat. Biotechnol. 29 (2011) 742e749. ~ ozcabello, et al., Molecular interplay of the noncoding [12] K.L. Yap, S. Li, A.M. Mun RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a, Mol. Cell 38 (2010) 662e674. [13] S.J. Gould, G. Raposo, As we wait: coping with an imperfect nomenclature for
Please cite this article in press as: X. Yang, et al., The long non-coding RNA PCSEAT exhibits an oncogenic property in prostate cancer and functions as a competing endogenous RNA that associates with EZH2, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.157
X. Yang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7 extracellular vesicles, J. Extracell. Vesicles 2 (2013). [14] W. Eduard, H.J. Johansson, M. Imre, et al., Cells release subpopulations of exosomes with distinct molecular and biological properties, Sci. Rep. 6 (2016) 22519. [15] J.L. Hood, R.S. San, S.A. Wickline, Exosomes released by melanoma cells prepare sentinel lymph nodes for tumor metastasis, Canc. Res. 71 (2011) 3792. [16] V. Huber, S. Fais, M. Iero, et al., Human colorectal cancer cells induce T-cell death through release of proapoptotic microvesicles: role in immune escape, Gastroenterology 128 (2005) 1796e1804. [17] S. Staubach, H. Razawi, F.G. Hanisch, Proteomics of MUC1-containing lipid rafts from plasma membranes and exosomes of human breast carcinoma cells MCF-7, Proteomics 9 (2009) 2820e2835. [18] Y. Zhang, T. Liu, C.A. Meyer, et al., Model-based analysis of ChIP-seq (MACS), Genome Biol. 9 (2008). R137. [19] J. Feng, T. Liu, Y. Zhang, Using MACS to identify peaks from ChIP-Seq data, Chapter 2, Current Protocols in Bioinformatics (2011). Unit 2.14. [20] W.J. Kent, C.W. Sugnet, T.S. Furey, et al., The human genome browser at UCSC, Genome Res. 12 (2002) 996e1006. [21] A.K. Akobeng, Understanding diagnostic tests 3: receiver operating characteristic curves, Acta Paediatr. 96 (2007) 644e647. [22] E.P. Consortium, An integrated encyclopedia of DNA elements in the human genome, Nature 489 (2012) 57e74. [23] G. Liang, J.C.Y. Lin, V. Wei, et al., Distinct localization of histone H3 acetylation and H3-K4 methylation to the transcription start sites in the human genome, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 7357e7362. [24] M.F. Lin, I. Jungreis, M. Kellis, PhyloCSF: a comparative genomics method to distinguish protein coding and non-coding regions, Bioinformatics 27 (2011) i275ei282. [25] L. Salmena, L. Poliseno, Y. Tay, et al., A ceRNA hypothesis: the Rosetta stone of a hidden RNA language? Cell 146 (2011) 353e358.
7
[26] Ø. DD, S.E. Bojesen, The link between benign prostatic hyperplasia and prostate cancer, Nat. Rev. Urol. 10 (2013) 49e54. [27] J. Zhao, T.K. Ohsumi, J.T. Kung, et al., Genome-wide identification of polycomb-associated RNAs by RIP-seq, Mol. Cell 40 (2010) 939. [28] Y. Jeon, J.T. Lee, YY1 tethers Xist RNA to the inactive X nucleation center, Cell 146 (2011) 119. [29] R.A. Gupta, N. Shah, K.C. Wang, et al., Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis, Nature 464 (2010) 1071e1076. ~ ozcabello, et al., Molecular interplay of the non-coding [30] K.L. Yap, S. Li, A.M. Mun RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a, Mol. Cell 38 (2010) 662. [31] T. Kogure, I.K. Yan, W.L. Lin, et al., Extracellular vesicleemediated transfer of a novel long noncoding RNA TUC339 a mechanism of intercellular signaling in human hepatocellular cancer, Genes & Cancer 4 (2013) 261. € [32] U. Gezer, E. Ozgür, M. Cetinkaya, et al., Long non-coding RNAs with low expression levels in cells are enriched in secreted exosomes, Cell Biol. Int. 38 (2014) 1076. [33] A. Alireza, B. Samuel, P.J. Kennedy, et al., Long non-coding RNAs harboring miRNA seed regions are enriched in prostate cancer exosomes, Sci. Rep. 6 (2016) 24922. € [34] M. Is¸ın, E. Uysaler, E. Ozgür, et al., Exosomal lncRNA-p21 levels may help to distinguish prostate cancer from benign disease, Journal of Urological Surgery 6 (2015) 168. [35] A. Ahadi, S. Khoury, M. Losseva, et al., A comparative analysis of lncRNAs in prostate cancer exosomes and their parental cell lines, Genomics Data 9 (2016) 7e9. [36] O. Shin-Ichiro, G.P.C. Drummen, K. Masahiko, Focus on extracellular vesicles: development of extracellular vesicle-based therapeutic systems, Int. J. Mol. Sci. 17 (2016) 172.
Please cite this article in press as: X. Yang, et al., The long non-coding RNA PCSEAT exhibits an oncogenic property in prostate cancer and functions as a competing endogenous RNA that associates with EZH2, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.157