LncRNA PWAR6 regulates proliferation and migration by epigenetically silencing YAP1 in tumorigenesis of pancreatic ductal adenocarcinoma

Abstract Long non‐coding RNAs (lncRNAs) are a novel class of regulators in multiple cancer biological processes. However, the functions of lncRNAs in pancreatic ductal adenocarcinoma (PDAC) remain largely unknown. In this study, we identified PWAR6 as a frequently down‐regulated lncRNA in PDAC samples as well as a panel of pancreatic cancer cell lines. Down‐regulated PWAR6 was associated with multiple clinical outcomes, including advanced tumour stage, distant metastasis, and overall survival of PDAC patients. In our cell‐based assays, ectopic expression of PWAR6 dramatically repressed PDAC cells proliferation, invasion and migration, accelerated apoptosis, and induced cell cycle arrest at G0/G1 phase. In contrast, depletion of PWAR6 mediated by siRNA exhibited opposite effects on PDAC cell behaviours. In vivo study further validated the anti‐tumour role of PWAR6 in PDAC. By taking advantage of available online sources, we also identified YAP1 as a potential PWAR6 target gene. Negative correlation between YAP1 and PWAR6 expressions were observed in both online database and our PDAC samples. Notably, rescue experiments further indicated that YAP1 is an important downstream effector involved in PWAR6‐mediated functions. Mechanistically, PWAR6 could bind to methyltransferase EZH2, a core component of Polycomb Repressive Complex 2 (PRC2) in regulating gene expression, and scaffold EZH2 to the promoter region of YAP1, resulting in epigenetic repression of YAP1. In conclusion, our data manifest the vital roles of PWAR6 in PDAC tumorigenesis and underscore the potential of PWAR6 as a promising target for PDAC diagnosis and therapy.


| INTRODUC TI ON
Pancreatic ductal adenocarcinoma (PDAC), a highly aggressive malignancy with limited efficacy of available therapies, accounts for the fourth leading cause of cancer-related death worldwide. 1,2 Due to the lack of early diagnosis and its rapid progression, the 5-year survival rate in PDAC is about 6%. 1 Given the fact that PDAC is resistant to most therapies currently used in clinic, there is a strong demand to identify novel targets for development of new treatment options that could potentially improve clinical outcomes for PDAC patients. 3 However, the complexity of its pathogenic lesions makes the study of the molecular mechanisms underlying PDAC very challenging.
Long non-coding RNAs (lncRNAs) are a subclass of non-coding transcripts with a length >200 nucleotides. 4 While previously considered as noises of genetic materials, lncRNAs have now been greatly appreciated for its involvement in various biological processes, including transcriptional regulation, RNA processing, translational control, epigenetic modification, and posttranslational modification. [5][6][7] In human cancers, especially in pancreatic cancer, aberrant lncRNA expression in PDAC indicates its vital roles in tumorigenesis. 8 Indeed, various lncRNAs have been reported to participate in the development, progression, metastasis, and chemotherapy-resistance of pancreatic cancer. [9][10][11] For example, elevated expression of lncRNA-BX111 in pancreatic cancer tissues is associated with shorter overall survival time of patients. By activating the transcription of ZEB1 through recruiting transcriptional factor Y-box protein (YB1) to its promoter region, lncRNA-BX111 promotes the epithelia-mesenchymal transition (EMT) in pancreatic cancer cells. 9 Research from Li et al 10 suggested that lncRNA NORAD acts as a ceRNA to regulate the expression of the small GTP binding protein RhoA through competition for hsa-miR-125a-3p, and thus promotes progression of pancreatic tumour. In addition, some tumour-specific expressing lncRNAs are ideal and excellent targets for designing the novel therapeutic strategies against human malignancies 12 In the present study, we investigated the clinical significance and biological functions of a poorly understood lncRNA in PDAC.
Prader Willi/Angelman Region RNA 6 (PWAR6) is a 4618-bp nonconserved lncRNA located on chromosome 15 in humans and contains one exon. The non-coding nature of lncRNA PWAR6 was confirmed by coding-potential analysis ( Figure S1). PWAR6 was identified as a protective lncRNA in glioma as it exhibits grade-specific dynamic expression and regulates hallmark-related genes, 13 but its characteristic in PDAC remains largely elusive.
Here, we assessed the expression patterns of PWAR6 in PDAC tissues and cell lines as well as its correlations with clinical outcomes in PDAC. In addition, we determined the biological functions of PWAR6 in PDAC via conducting a series of cell-based experiments. Finally, we explored the molecular mechanism of PWAR6 in PDAC cell proliferation and metastasis, providing novel insight in determining PWAR6 as a potential therapeutic and prognostic target in PDAC.

| Cell transfection
Scramble siRNA, siRNAs for PWAR6 and expression vectors for YAP1 were synthesized and obtained from Genepharma.
The siRNAs and plasmid were transfected into PDAC cells using   were performed in triplicate and repeated at least three times.

| Cell invasion and migration assay
Transfected cells were plated onto the upper insertion chamber (Millipore), that was either coated (to assess invasion) or non-coated (to assess migration) with 100 µL matrigel (BD Biosciences), in serum-free media. While the lower chamber was supplemented with culture medium containing 10% FBS. After 24 hours incubation, the cells remaining on the upper membrane were removed with cotton wool. Cells on the bottom of the filter were fixed with methanol, stained with 0.1% crystal violet, imaged and counted using an IX71 inverted microscope (Olympus). The experiments were performed in triplicate and repeated at least three times.

| Mouse xenograft experiments
The animal protocol of this study was approved by the Institutional Animal Care and Use Committee (IACUC) of The First Affiliated Hospital to Nanchang University (Nanchang, China). Seven-week-old female NOD/SCID mice were randomized into two groups (five mice in each). To assess the effect of sh-PWAR6 on PDAC tumorigenesis, 5 × 10 6 BxPC-3 cells infected with sh-LINC01133 or sh-scramble were injected into the right flanks of NOD/SCID mice. Four weeks later, the mice were sacrificed and the tumours were harvested and weighed.

| Western blot
Briefly, cell lysates were prepared in RIPA buffer, separated by 8% SDS-PAGE and then transferred to PVDF membranes (Millipore).
Membrane was blocked with 5% non-fat milk in TBS at 4°C overnight and then incubated with specific antibodies at room temperature for 1 hour, followed by incubation with HRP-conjugated secondary antibody at room temperature for another 1 hour. The bands were visualized using electrochemiluminescent (ECL) detection system (Thermo Fisher Scientific). Anti-EZH2 and anti-GAPDH antibodies were purchased from Abcam, anti-YAP1, anti-caspase-3, anti-BAX and anti-Bcl-2 antibodies were obtained from Cell Signaling Technology (Massachusetts).

| Quantitative reverse transcription PCR
Total RNA from tissues or cell lines were extracted by using Trizol reagent (Invitrogen) according to the manufacturer's instructions. RNA was then reverse-transcribed to cDNA using an miScript SYBR Green PCR kit (Invitrogen). SYBR Green PCR Master Mix (Applied Biosystems) was used to quantify the mRNA levels of target genes. qRT-PCR was performed on an ABI7500 system (Applied Biosystems) and the conditions were as follows: an ini- GAPDH: 5′-CAG GGCTGCTTTTAACTCTGGT-3′ (forward) and 5′-GATTTTGGAGGGATCTCGCT-3′ (reverse). The relative mRNA expression levels were calculated using the 2 − ΔΔC T method.

| Subcellular fractionation
The Cytoplasmic and Nuclear RNA Purification Kit (Norgen) was used to separate the nuclear and cytoplasmic fractions according to the manufacturer's instructions.

| RNA immunoprecipitation
RNA immunoprecipitation (RIP) experiments were performed using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore), according to the manufacturer's instructions. Antibodies for RIP assays of EZH2 and SUZ12 were from Abcam. IgG was used as a negative control and obtained from Santa Cruz Biotechnology.

| RNA pull-down assay
Biotin-labelled PWAR6 was obtained by T7 RNA polymerase (Promega) and biotin RNA tagged mixtures (Roche). Cell lysates were prepared in RIP buffer and then mixed with biotin-labelled PWAR6 RNAs. After incubation at 4°C for 1 hour, the streptavidin-coated beads (Thermo Fisher Scientific) were added in the reaction. The precipitated proteins were separated by SDS-PAGE and detected by western blot analysis.

| Chromatin immunoprecipitation (ChIP) assay
The ChIP assay was carried out using the EZ ChIP™ Chromatin Standard qRT-PCR was performed to quantify the immunoprecipitated DNA.

| Statistical analysis
Statistical analysis was performed using SPSS 22.0. The relationship between the expression level of PWAR6 and clinicopathological characteristics of PDAC patients was analysed by chi-square test. OS rates were calculated by Kaplan-Meier method and compared using the log-rank test. Pearson correlation analysis was applied to assess the correlation between PWAR6 and YAP1.
Student's t test or one-way ANOVA analysis was used for calculating intergroup differences. A P value <0.05 was considered statistically significant.

| LncRNA PWAR6 is down-regulated in human PDAC and correlates with poor prognosis
The mRNA expression level of PWAR6 was evaluated in 63 PDAC samples and their paired benign pancreatic tissues by qRT-PCR. As shown in Figure 1A Figure 1E). In addition, the data retrieved from KM-Plotter database (http://kmplot.com/analy sis/) further confirmed the prognostic role of PWAR6 in PDAC patients ( Figure 1F). Taken together, these results suggest that PWAR6 is frequently downregulated in PDAC and associated with advanced tumour stage, distant metastasis status and poor prognosis, indicating its potential tumour suppressor role in PDAC.

| LncRNA PWAR6 regulates PDAC cell progression both in vitro and in vivo
To investigate whether PWAR6 is involved in the tumorigenesis of  were caused non-specifically, we re-introduced PWAR6 into cells that were already transfected with PWAR6 siRNAs, and the results shown in Figure 2K,L clearly demonstrated that in addition to reproduced effects of PWAR6 knockdown on BxPC-3 cell viability and migration, these effects were successfully rescued by PWAR6 overexpression. Collectively, these findings confirmed the tumour suppressor role of PWAR6 in PDAC cells.

| Overexpression of LncRNA PWAR6 inhibits cell cycle and promotes cell apoptosis
The above data indicate that PWAR6 regulates cell proliferation and migration in PDAC, and these results promoted us to further investigate whether PWAR6 could influence cell cycle and cell apoptosis. As expected, flow cytometry analysis revealed that PWAR6 overexpression led to a significant accumulation of population in G1/G0 phase and a decrease in the percentage of cells in S phase ( Figure 3A). On the contrary, in response to PWAR6 knockdown, the cell population was skewed towards S phase and the percentage of cells in G1/G0 phase decreased ( Figure 3B).
The cell apoptosis was analysed by flow cytometry using Annexin V FITC/PI staining in AsPC-1 and BxPC-3 cells as shown in Figure 3C. Cells transfected with PWAR6 plasmid showed significantly increased percentage of apoptotic cells, which is 23.02% as compared to 8.97% in control cells for AsPC-1 and 20.93% as compared to 6.35% in control cells for BxPC-3. In addition, the expression levels of apoptosis-related proteins, such as caspase-3, BAX and Bcl-2 were also tested in PWAR6 overexpressed AsPC-1 cells. As indicated in Figure 3D, PWAR6 up-regulation increased the expression of caspase-3 and BAX, while decreased the expression of Bcl-2. Besides, the regulation of PWAR6 can also affect the level of a well-documented tumour suppressor P53. We found that overexpression of PWAR6 elevates p53 protein level, while knockdown of PWAR6 inhibits p53 protein level ( Figure 3E). Together, all these results demonstrated that PWAR6 overexpression inhibits cell cycle and promotes cell apoptosis.

| LncRNA PWAR6 is responsible for the epigenetic repression of YAP1 by interacting with PRC2
We next sought to further explore the molecular mechanisms underlying PWAR6 functions. We first searched for potential PWAR6 target genes by employing starBase v3.0 Pan-Cancer Analysis and Networks Platform (http://starb ase.sysu.edu.cn/). Interestingly, the result in Figure 4A showed that PWAR6 exhibited a weak negative correlation trend with YAP1, a main downstream effector of Hippo pathway. Dysregulation of YAP1 affects transcription of various genes related to cell proliferation and migration, subsequently contributing to cancer development. 14  According to previous reports, numerous lncRNAs could bind to chromatin-modifying enzymes to promote epigenetic activation or silencing of its target gene expression. 15 In order to better understand detailed mechanism of PWAR6 functions, we then examined the location of PWAR6 inside a cell. The results shown in Figure 5A demonstrated that PWAR6 was primarily located in the nucleus, where F I G U R E 5 LncRNA is responsible for the epigenetic repression of YAP1 by interacting with PRC2. A, The PWAR6 subcellular location was determined in AsPC-1 and BxPC-3 cells. U6 was used as a nuclear marker and GADPH was used as a cytoplasmic marker. B, The binding probability between PWAR6 and PRC2 complex (SUZ12, EZH2, EED) was predicted in RNA-protein interaction prediction website (RF and SVM scores >0.5). C, RIP assay validated the interaction between PWAR6 and EZH2/SUZ12 in PDAC cells. D, RNA pull-down assay demonstrated that PWAR6 could specially retrieved EZH2 in PDAC cells. E, The results of ChIP assay shown that PWAR6 overexpression could dramatically enhancing the binding ability of EZH2 to YAP1 promoter region and promoting the methylation of H3K27me3 in PDAC cells, while PWAR6 down-regulation contributed to the contrary results potential regulation on transcription mediated by PWAR6 could occur.
Polycomb Repressive Complex 2 (PRC2), a multimeric enzymatic complex composed of EZH2, SUZ12 and EED, has histone methyltransferase activity and primarily trimethylates histone H3 on lysine 27. 16 About 20% lncRNAs have been suggested to associate with PRC2 physically. 17 Therefore, we hypothesized that PWAR6 could regulate its downstream target gene through binding to PRC2. To test this hypothesis, we performed RNA-protein interaction prediction (http:// pridb.gdcb.iasta te.edu/RPISe q/refer ences.php) and found a high possibility for PWAR6 to interact with EZH2, SUZ12 and EED ( Figure 5B).
Next, RNA-RIP assay was carried out to verify the interaction between PWAR6 and PRC2. As shown in Figure 5C, PWAR6 could be specifically pulled down by EZH2 and SUZ12 antibodies, but not IgG negative control. In addition, RNA pull-down assay demonstrated that PWAR6, rather than the vector control and PWAR6 antisense, could specifically retrieve EZH2 in PDAC cells ( Figure 5D). We speculated that PWAR6 might epigenetically inhibit the expression of YAP1 by scaffolding EZH2. Our ChIP assay followed by qPCR analysis indicated that PWAR6 overexpression significantly enhanced EZH2 binding to and H3K27me3 levels on the YAP1 promoter region ( Figure 5E, left panel). In contrast, the binding of EZH2 and the H3K27me3 levels were decreased in the promoter region of YAP1 when PWAR6 was knocked down ( Figure 5E, right panel). Taken together, our data clearly indicate that PWAR6 binds to EZH2 and facilitates EZH2/PRC2-mediated epigenetic modification of YAP1 promoter for suppression.

| LncRNA PWAR6 promotes cancer progression via negatively regulating YAP1
The above finding that PWAR6 could negatively regulate YAP1 expression promoted us to assume that PWAR6 could also affect the target genes of Hippo-YAP1 pathway, then contributed to the PDAC tumorigenesis. As expected, we analysed the data of the PDAC patient cohort collected in the GEPIA database; as shown in Figure S2A, PWAR6 expression negatively correlated with the levels of some Hippo-YAP1 downstream genes (BIRC5, CCNB1, CCNE1, CDC20 and FOXM1) in PDAC specimens. qRT-PCR assay further confirmed that the regulation of PWAR6 could significantly influenced the mRNA expression of these genes ( Figure S2B). Next, to ascertain YAP1 was involved in PWAR6- The transwell invasion and migration assays revealed that overexpression of YAP1 recovered the inhibition of cell migration ability induced by PWAR6 overexpression (Figure 6D,E). Flow cytometry analysis revealed that YAP1 overexpression counteracted G0/ G1 phase arrest induced by PWAR6 ( Figure 6F, left panel). On the contrary, YAP1 knockdown restored cell cycle promoted by PWAR6 siRNA (Figure 6F, right panel). Furthermore, the apoptosis analysis showed that cell apoptosis enhanced by PWAR6 was significantly opposed by YAP1 co-transfection ( Figure 6G). The expression level of YAP1 was confirmed in Figure 6H as transfected with above indicated plasmids. These data suggest that YAP1 is a critical target gene of PWAR6 involved in PDAC tumorigenesis. serves as a promising biomarker for PDAC patients.

| D ISCUSS I ON
The above results suggested the tissue specificity and the tumour suppressor role of PWAR6 in PDAC. However, the genes that were affected by PWAR6 remains undocumented in PDAC. We thus screened a bunch of PWAR6-related genes, among which YAP1 caught our attention. YAP1 is a transcriptional coactivator in Hippo pathway and considered to be encoded by a proto-oncogene. 28 By mediating its target gene transcription in most cancer cells, YAP1 plays multiple regulatory roles in cancer progression. 29 In PDAC, overexpression of YAP1 correlated with liver metastasis and poor prognosis. 30 The bioinformatic analysis using GEPIA server indicated a negative correlation trend between PWAR6 and YAP1. We further tested the expression pattern of YAP1 in PDAC tissues and adjacent pancreatic tissues by performing qRT-PCR assay. It was evident that YAP1 was up-regulated in PDAC tissues, and the Pearson analysis further reinforced the inverse correlation between PWAR6 and YAP1, which was also validated in our western blot and qRT-PCR assays as PWAR6 suppressed the expression of YAP1. As to the mechanism of the regulation of YAP1 by PWAR6 remains unclear, which arouse our interest to explore.
Given that the subcellular location of lncRNAs may decide its functionality, we first analysed the distribution and subcellular location of PWAR6 in PDAC cells. The subcellular localization patterns of lncRNAs and their biological functions are closely related. In the cytoplasmic, lncRNAs target mRNA transcripts and modulate its stability and translation, whereas the nuclear lncRNAs are important epigenetic modulators of nuclear functions. 31 The subcellular fractionation assay demonstrated that PWAR6 primarily localizes within the nucleus of PDAC cells. In the nucleus, many lncRNAs act by influencing the epigenetic status of protein-coding genes through direct interactions with chromatin-modifying factors. 32 PRC2, one of the most studied chromatin-modifying factors, is a histone methyltransferase required for epigenetic silencing during cancer development. 33 The binding probability between PWAR6 and PRC2 was analysed by RNA-protein interaction prediction (http://pridb.gdcb. iasta te.edu/RPISe q/). Both the RF and SVM scores were higher than 0.5, indicating a high possibility for PWAR6 and PRC2 physical interaction. The methyltransferase EZH2 is the core subunit of the PRC2 complex, and previous studies have shown that numerous lncRNAs can bind to EZH2. 34 For instance, lncRNA UCA1 mediates the expression of p21 and SPRY1 through interacting with EZH2, thus facilitating tumour progression of gastric cancer. 35 LINC01133 inhibits breast cancer invasion and metastasis through repressing SOX4 expression by recruiting EZH2 to SOX4 promoter. 36 In our present study, through RIP and RNA pull-down assays, we identified direct interaction between PWAR6 and EZH2. ChIP assay using EZH2 and H3K27me3 antibodies demonstrated that EZH2 can directly bind to YAP1 promoter region and induce trimethylation of H3K27 in PDAC cells. Therefore, we speculated that PWAR6 exerts its tumour suppressive role by negatively regulating YAP1. As expected, PWAR6-induced cell proliferation inhibition, cell cycle arrest and cell apoptosis could be partially reversed by YAP1 co-transfection.
In conclusion, our study provided evidence for the first time that PWAR6 exerts tumour suppressor activity in human PDAC cells by interacting with EZH2 and facilitating its repression of YAP1, thus mediating proliferation, apoptosis and metastasis of PDAC. These findings indicate that PWAR6 is a critical molecule for PDAC tumorigenesis and could be a promising target for novel intervention and/ or therapeutics in PDAC.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest in this study.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.