WT1-AS Expression Research Articles

WT1-AS/IGF2BP2 Axis Is a Potential Diagnostic and Prognostic Biomarker for Lung Adenocarcinoma According to ceRNA Network Comprehensive Analysis Combined with Experiments.

Lung adenocarcinoma (LUAD) is one of the most common malignancies, and there is still a lack of effective biomarkers for early detection and prognostic prediction. Here, we comprehensively analyze the characteristics of. an RNA sequencing data set of LUAD samples. In total, 395 long non-coding RNAs (lncRNAs), 89 microRNAs (miRNAs), and 872 mRNAs associated with c-Myc were identified, which were differentially expressed between tumor and normal tissues. The most relevant pathway was found to be WT1-AS–miR-200a-3p–IGF2BP2 according to the rules of competitive endogenous RNA (ceRNA) regulation. WT1-AS and IGF2BP2 expression were positively correlated and increased in LUAD samples, while miR-200a-3p had relatively low expression. The high expression of WT1-AS and IGF2BP2 was associated with poor prognosis in LUAD patients, while low expression of miR-200a-3p predicted reduced survival (p < 0.05). The analysis of the multi-gene regulation model indicated that the WT1-AS (downregulation)–miR-200a-3p (upregulation)–IGF2BP2 (downregulation) pattern significantly improved the survival of LUAD patients. Finally, reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were detected in LUAD cells, and the results are consistent with the bioinformatics analysis. In summary, the WT1-AS/IGF2BP2 axis is a potential prognostic biomarker in LUAD and is expected to become an effective target for diagnosis and treatment.

Overexpression of long non-coding RNA WT1-AS or silencing of PIK3AP1 are inhibitory to cervical cancer progression

ABSTRACT Accumulating evidence demonstrate that long non-coding RNAs (lncRNAs) play an important role in regulating the biological function of cervical cancer cells. However, the regulatory role of lncRNA Wilms tumor 1 homolog antisense RNA (WT1-AS) in cervical cancer cells remains uncertain. In this study, we explored the participation of WT1-AS in cervical cancer by first using the reverse transcription quantitative polymerase-chain reaction (RT-qPCR) was to analyze the expression of WT1-AS and phosphoinositide-3-kinase adaptor protein 1 (PIK3AP1) in cervical cancer tissues and cells. Dual-luciferase reporter gene assay, RNA pull-down/RNA immunoprecipitation (RIP) assays and Chromatin Immunoprecipitation (ChIP) assay were conducted to explore the interactions among WT1-AS, PIK3AP1, and SPI1. Gain- and loss-of-function approaches were carried out to determine the effects of lncRNA WT1-AS, PIK3AP1 on cell biological characteristics, followed by assays of cell proliferation, autophagy, and apoptosis abilities using, respectively, EdU, monodansylcadaverine (MDC) staining, and flow cytometry. Finally, we measured growth of xenograft tumors in nude mice. We found decreased expression of lncRNA WT1-AS and increased expression of PIK3AP1 in cervical cancer samples. Moreover, PIK3AP1 was negatively regulated by WT1-AS, which promoted apoptosis, but inhibited cell proliferation and autophagy of cervical cancer cells. Furthermore, WT1-AS inhibited PIK3AP1 expression by recruiting SPI1, and inhibited the progression of cervical cancer through the SPI1/PIK3AP1 axis in vivo and in vitro. In summary, lncRNA WT1-AS repressed the development of cervical cancer by reducing PIK3AP1 expression through an interaction with SPI1, which may suggest new therapeutic approaches for treating cervical cancer. Abbreviations: HPV, human papillomavirus; lncRNAs, Long non-coding RNAs; WT1-AS, wilms tumor 1 antisense RNA; HCC, hepatocellular carcinoma; SFFV, Spleen focus forming virus; SPI1, Spleen focus forming virus proviral integration oncogene 1; TF, transcription factor; PIK3AP1, Phosphoinositide-3-kinase adaptor protein 1; NCBI, National Center for Biotechnology Information; oe, overexpressed; sh-PIK3AP1, short hairpin RNA against PIK3AP1; RIPA, radioimmunoprecipitation; PMSF, phenylmethylsulfonyl fluoride; HRP, horseradish peroxidase; IgG, immunoglobulin G; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction; EP, Eppendorf; RIP, RNA-binding protein immunoprecipitation; CHIP, Chromatin immunoprecipitation; EdU, 5-ethynyl-2ʹ-deoxyuridine; PI, propidium iodide; MDC, Monodansylcadaverine; PFA, paraformaldehyde; SPF, specific pathogen free; TV, tumor volume; DLG1-AS1, discs large MAGUK scaffold protein 1 antisense RNA 1; TOB1-AS1, transducer of epidermal growth factor receptor-2.1 antisense RNA 1; LC3II, light chain 3 type II; LC3I, light chain 3 type I; IRF4, interferon regulatory factor 4

Abnormal Expression Of WT1-As, MEG3 and ANRIL Long Non-Coding RNAs In Primary Myelofibrosis and Their Clinical Correlates

A growing body of evidence suggests that long non-coding RNAs (lncRNAs) are involved in the regulation of gene expression in normal and cancer cells by recruiting chromatin remodeling complexes to specific genomic loci [Rinn JL, et al. Cell. 2007; Mattick JS, et al. Bioessays 2009]. For this reason, lncRNAs deregulation can play key roles in malignant transformation and cancer cell behavior. [Kogo R, et al. Cancer Res 2011; Yang F, et al. Hepatology 2011].Despite the increasing number of studies on lncRNAs expression and their involvement in some solid tumors, lncRNAs are far from being extensively characterized in malignant hematopoiesis [Heuston EF, Front Genet 2011]. In particular, lncRNAs expression has never been investigated in cells from primary myelofibrosis (PMF) patients.PMF is a Philadelphia negative chronic Myeloproliferative Neoplasm (MPN) that originates from deregulated clonal proliferation of hematopoietic stem cell associated with overproduction of mature blood cells. Molecular mechanisms underlying MPN pathogenesis were partially unraveled in 2005-2006 with the identification of somatic gain-of-function mutations of JAK2 [James C, et al. Nature 2005; Kralovics R, et al. N Engl J Med 2005; Levine RL, et al. Cancer Cell 2005] and MPL [Pardanani AD, et al. Blood 2006] after which many other mutations were identified. Recently, several new molecular pathogenetic mechanisms were proposed, such as the aberrant expression of coding and non-coding RNAs.After extensive screening of the long non-coding RNA database (lncRNAdb), which is a database providing comprehensive annotations of eukaryotic lncRNAs (http://www.lncrnadb.org) [Amaral PP, Nucleic Acids Res 2011], we identified some previously described lncRNAs as potentially involved in hematological malignancies, such as CDKN2B-antisense (ANRIL), MEG3 and WT1-antisense lncRNAs.With the aim to describe possible abnormalities of lncRNAs in MPN, we investigated the expression of those lncRNAs in CD34+ cells from PMF patients. Since the perturbation of the antisense RNA could alter the expression of the sense gene, we also analyzed the expression of the ANRIL and WT1-as coding genes, called CDKN2B and WT1, to evaluate the correlation of each sense/antisense couple.This study included a total of 26 PMF patients and 16 healthy donors. The diagnosis of PMF was made according to World Health Organization (WHO) criteria.The results evidenced that the majority of PMF samples (74%) displayed a co-upregulation of WT1 and its antisense RNA compared to healthy controls. We found that the large part (61%) of patients harboring concordant upregulation of WT1 sense and antisense belongs to the intermediate-2 and high risk categories of DIPSS-plus scoring system (57.4% and 43.1% in the intermediate-1 and -2, respectively versus 0% in the high-risk category; P = .006) and presents low Hb level, higher percentage of circulating CD34+ cells and a blast count > 1% .Noteworthy, almost 90% of patients with increased expression of WT1 and its antisense showed concurrent upregulation of MEG3 and shared the same clinical variables associated with poor outcome. In fact, also patients harboring higher levels of MEG3 cluster in higher DIPSS-plus scoring system categories, suffer from anemia, have high levels of circulating CD34+ cells and a blast count >1%. These data suggest that not only high levels of WT1-as we previously described [Guglielmelli P, Stem Cells 2007], but also its antisense and MEG3 could identify PMF patients with more severe disease.Finally, we could observe a correlation between CDKN2B upregulation and the grade of BM fibrosis. In fact, the majority of samples having CDKN2B upregulation clustered in categories 2 and 3 (87.3%) as compared to only 4.5% in case of samples showing CDKN2B downregulation (P = .043). Noteworthy, 82% of patients overexpressing CDKN2B were JAK2V617F positive as compared to 22.1% among those with CDKN2B downregulation (P = .004)To our knowledge, this is the first study describing the expression profiles of human lncRNAs in CD34+ cells of PMF patients. Our data demonstrate that WT1-as, MEG3 and ANRIL lncRNAs are aberrantly expressed in PMF CD34+ cells compared to healthy controls. Moreover, different patterns of expression are correlated with some patient' clinical features. Therefore, our results suggest that a deregulated expression of these lncRNAs could play a role in PMF pathogenesis and progression. Disclosures:No relevant conflicts of interest to declare.

LncRNA WT1-AS is upregulated in osteoporosis and regulates the apoptosis of osteoblasts by interacting with p53.

In cervical cancer, cellular tumor antigen p53 (p53) interacts with long non-coding WT1 antisense RNA (WT1-AS) and this protein serves an important role in osteoporosis. The present study aimed to investigate the role of WT1-AS in osteoporosis. WT1-AS was upregulated in the plasma of patients with osteoporosis and was positively correlated with p53 expression. Altered expression of WT1-AS and p53 separated patients with osteoporosis from healthy controls. Expression levels of WT1-AS and p53 decreased with prolonged treatment. In osteoblasts, WT1-AS overexpression resulted in increased p53 expression, while WT1-AS small interfering RNA (siRNA) silencing resulted in decreased p53 expression. In addition, WT1-AS overexpression resulted in increased apoptosis rate, while WT1-AS siRNA silencing resulted in decreased apoptosis rate in osteoblasts. p53 overexpression attenuated the effects of WT1-AS siRNA silencing on cell apoptosis. Therefore, WT1-AS was upregulated during osteoporosis and regulated the apoptosis of osteoblasts by interacting with p53.

Unravelling the Role of LncRNA WT1-AS/miR-206/NAMPT Axis as Prognostic Biomarkers in Lung Adenocarcinoma.

Lung cancer is the world’s highest morbidity and mortality of malignant tumors, with lung adenocarcinoma (LUAD) as a major subtype. The competitive endogenous RNA (ceRNA) regulative network provides opportunities to understand the relationships among different molecules, as well as the regulative mechanisms among them in order to investigate the whole transcriptome landscape in cancer pathology. We designed this work to explore the role of a key oncogene, MYC, in the pathogenesis of LUAD, and this study aims to identify important long noncoding RNA (lncRNA)-microRNA (miRNA)- transcription factor (TF) interactions in non-small cell lung cancer (NSCLC) using a bioinformatics analysis. The Cancer Genome Atlas (TCGA) database, containing mRNA expression data of NSCLC, was used to determine the deferentially expressed genes (DEGs), and the ceRNA network was composed of WT1-AS, miR-206, and nicotinamide phosphoribosyltransferase (NAMPT) bashing on the MYC expression level. The Kaplan–Meier univariate survival analysis showed that these components may be closely related prognostic biomarkers and will become new ideas for NSCLC treatment. Moreover, the high expression of WT1-AS and NAMPT and low expression of miR-206 were associated with a shortened survival in NSCLC patients, which provided a survival advantage. In summary, the current study constructing a ceRNA-based WT1-AS/miR-206/NAMPT axis might be a novel important prognostic factor associated with the diagnosis and prognosis of LUAD.

LncRNA WT1-AS downregulates lncRNA UCA1 to suppress non-small cell lung cancer and predicts poor survival

BackgroundLncRNA WT1-AS inhibits gastric cancer, while its role in other cancers is unknown. We investigated the role of WT1-AS in non-small cell lung cancer (NSCLC).MethodsSixty-six NSCLC patients (40 males and 26 females; 36 to 68 years old; mean age 52.7 ± 6.4 years old) were selected from the 178 NSCLC patients operated on for lung cancer between 2010 and 2013. RT-qPCR was used to analyze the expression of lncRNA. Overexpression experiments were performed to assess interactions between lncRNAs. CCK-8 assay was carried to evaluate the roles of WT1-AS and UCA1 in regulating cell proliferation. Cell invasion and migration assays were performed to assess the roles of WT1-AS and UCA1 in regulating cell invasion and migration. Western-blot was performed to illustrate the effect of WT1-AS and UCA1 in EMT.ResultsWT1-AS was downregulated in NSCLC and was correlated with poor survival. The expression of WT1-AS in NSCLC was not correlated with clinical stages. LncRNA UCA1 was upregulated in cancer tissues and inversely correlated with WT1-AS. Overexpression of UCA1 did not affect WT1-AS, while overexpression of WT1-AS led to inhibited expression of UCA1. Overexpression of UCA1 resulted in increased proliferation, EMT, migration and invasion of NSCLC cells, while overexpression of WT1-AS showed opposite effects. In addition, overexpression of UCA1 inhibited the role of overexpression of WT1-AS.ConclusionsTherefore, overexpression of WT1-AS may inhibit the cell proliferation and EMT to decrease cell migration and invasion of NSCLC cells by downregulating UCA1.

LncRNA WT1-AS over-expression inhibits non-small cell lung cancer cell stemness by down-regulating TGF-\u03b21

BackgroundLncRNA WT1-AS is a recently identified potential tumor suppressor in gastric cancer. This study mainly explored the role of WT1-AS in non-small cell lung cancer (NSCLC).MethodsWT1-AS and TGF-β1 mRNA in two types of tissues of 74 NSCLC patients were detected by performing RT-qPCR experiments. WT1-AS and TGF-β1 expression vectors were established using the pcDNA3.1 vector. Protein concentration was measured by BCA assay. Mean values in this study were calculated using the data of three biological replicates of each experiment.ResultsWe found that WT1-AS was down-regulated, while TGF-β1 was upregulated in NSCLC tissues. Survival analysis showed that low levels of WT1-AS expression predicted poor survival of NSCLC patients. WT1-AS and TGF-β1 were inversely correlated in NSCLC tissues. Over-expression experiments revealed down-regulated TGF-β1 after WT1-AS over-expression, while TGF-β1 over-expression failed to affect WT1-AS. WT1-AS over-expression resulted in inhibited cancer cell stemness. TGF-β1 over-expression played an opposite role and attenuated the effects of TGF-β1 over-expression.ConclusionTherefore, WT1-AS over-expression may inhibit non-small cell lung cancer cell stemness by down-regulating TGF-β1.Trial registrationThe First Affiliated Hospital of Anhui Medical University Ethics committee approved this study (AHMU20101009).

Long Noncoding RNA WT1-AS Inhibits the Progression of Cervical Cancer by Sponging miR-205.

Background: Cervical cancer (CC) is the second frequent cancer of women in developing countries. Plentiful studies proved that long noncoding RNA antisense of the tumor suppressor gene WT1 (WT1-AS) participated in the progression of CC. However, the role of WT1-AS remains unclear. This study investigated the potential mechanisms of WT1-AS in CC. Methods: The expression of WT1-AS and miR-205 were determined by quantitative real-time polymerase chain reaction. The cellular localization of WT1-AS in CC cells was detected by subcellular fractionation assay. The level of epithelial-mesenchymal transition (EMT)-related proteins of N-cadherin, E-cadherin, MMP9, and MMP2 were measured by Western blot. Moreover, cell cycle, apoptosis, migration, and invasion were detected by flow cytometry and transwell assay, respectively. The interrelation between WT1-AS and miR-205 was verified by dual-luciferase reporter and RNA immunoprecipitation assays. The role of WT1-AS in modifying CC growth was identified using xenograft tumor model. Results: WT1-AS was downregulated in cervical tissues and cell lines. WT1-AS was predominantly located in the cytoplasm of CC cells. Upregulation of WT1-AS promoted cell apoptosis, blocked cell cycle, migration, invasion, and EMT in vitro. Moreover, miR-205, as a target gene of WT1-AS, was increased in cervical tissues and cell lines. Besides, miR-205 mimic reversed the effect of WT1-AS upregulation on cell cycle, apoptosis, migration, invasion, and EMT. Also, WT1-AS caused the curb of xenograft tumor growth in vivo. Conclusion: Upregulation of WT1-AS suppressed CC development through sponging miR-205, providing experimental basis for clinical targeted treatment of CC.

Long Non-coding Wilms Tumor 1 Antisense RNA in the Development and Progression of Malignant Tumors.

A growing number of studies have shown that long non-coding RNAs (lncRNAs) play an important role in tumor development and progression and are key molecules affecting tumor progression. The lncRNA Wilms tumor 1 antisense RNA (WT1-AS) is specifically expressed in various malignant tumors. In particular, WT1-AS expression is upregulated in colon cancer and breast cancer but is significantly downregulated in cervical cancer, liver cancer, and kidney cancer. The level of WT1-AS expression is closely related to the size, stage, and patient survival rate of these cancers. In this article, we review the modes of action, expression, function, and mechanisms of WT1-AS in different tumors to provide new targets for tumor diagnosis and treatment.

LncRNA WT1-AS up-regulates p53 to inhibit the proliferation of cervical squamous carcinoma cells

BackgroundIt has been reported that the development of cervical squamous cell carcinoma (CSCC) requires the involvement of a large number of lncRNAs. In a recent study lncRNA, WT1-AS was been characterized as a tumor-suppressive lncRNA in gastric cancer. In our study we aim to explore the involvement of WT1-AS in CSCC.MethodsSeventy-six CSCC patients (20 to 63 years, 40.1 ± 6.1 year) from the 233 CSCC patients who were admitted by the Affiliated Tumour Hospital of Xinjiang Medical University between august 2010 and January 2014. RT-qPCR, cell proliferation rate measurement, cell transfection, and western blot were carried out to analyze the samples.ResultsWe found that HPV infection failed to affect WT1-AS expression in CSCC tissues, while WT1-AS was down-regulated in CSCC tissues compared with non-cancer tissues. P53 was also down-regulated in CSCC tissues and positively correlated with WT1-AS. Analysis of the survival of CSCC patients revealed that low levels of WT1-AS were accompanied by poor survival. Significantly up-regulated p53 was observed after WT1-AS over-expression in CSCC cells, while p53 over-expression failed to affect WT1-AS. P53 and WT1-AS over-expression resulted in the inhibited proliferation of CSCC cells.ConclusionTherefore, WT1-AS is down-regulated in CSCC and it may inhibit CSCC cell proliferation at least partially by up-regulating p53.

LncRNA WT1-AS inhibits the aggressiveness of cervical cancer cell via regulating p53 expression via sponging miR-330-5p.

BackgroundEmerging evidences have demonstrated that lncRNAs play vital roles in various pathological processes, including cancer. The lncRNA WT1 antisense RNA (WT1-AS) serves as a tumor suppressor in various cancers. Nevertheless, the expression and precise function of WT1-AS in cervical carcinoma still remain not yet investigated. The objective of our study was to explore the expression of WT1-AS and its biological roles in cervical cancer.MethodsDifferences in the lncRNA expression profiles between cervical cancer and adjacent normal tissues were assessed by lncRNA expression microarray analysis. The expression of p53 in cervical cancer cell was assessed by qRT-PCR and immunofluorescence assay. Loss-of-function studies were used to explore the effect of lncRNA WT1-AS on the growth and metastasis of cervical cancer cell in vitro and in vivo.ResultsOur results demonstrated that WT1-AS was remarkably down-regulated in cervical carcinoma. Functional assays proved that up-regulation of WT1-AS significantly suppressed cervical cancer cell proliferation, migration and invasion. In addition, the luciferase reporter assay identified that miR-330-5p was the target of WT1-AS. Moreover, tumor suppressor p53 was identified as the direct target of miR-330-5p and alternation of miR-330-5p/p53 axis reversed the effects of WT1-AS in cervical cancer cell.ConclusionAltogether, our findings suggested that WT1-AS was down-regulated in cervical carcinoma and WT1-AS suppressed cervical carcinoma cell- proliferation, migration and invasion through regulating the miR-330-5p/p53 axis.

Abstract 5435: Role of the WT1 transcription factor in high-grade serous ovarian cancer

Abstract The Wilms Tumor protein 1 (WT1) transcription factor (TF) is a ubiquitous biomarker of high-grade serous ovarian carcinoma (HGSOC), which is the most common and aggressive subtype of epithelial ovarian cancer (EOC). This TF interacts with a host of cellular factors including the tumor suppressor P53, which is mutated in virtually all HGSOC cases. Studies have suggested a role for WT1 in HGSOC development, but little is known about the underlying mechanism. We have generated unique catalogues of epigenomic landscapes (specifically H3K27ac) in primary HGSOC using chromatin immunoprecipitation sequencing (ChIP-seq). Using Bayesian analyses of H3K27ac profiling in HGSOCs, we identified significant tissue specific enrichment of active chromatin at the WT1 locus in HGSOC when compared to all other EOC tumor histotypes. Furthermore, this locus contains a superenhancer in HGSOCs and in normal fallopian tube secretory epithelial cells (FTSECs). This locus features a bidirectional promoter that is shared by WT1-AS lncRNA and that contains a WT1 binding site. LncRNAs are essential in many cellular processes, and have been shown to play pivotal roles in tumorigenicity. The WT1-AS lncRNA is implicated in other diseases but little is known of its function in EOC. We have investigated the mechanisms of interaction between WT1 and WT1-AS. We observed that both protein and transcript expression for WT1 and WT1-AS are positively correlated in HGSOC cell lines. Analysis of data for &amp;gt;400 primary HGSOCs from TCGA further supported this correlation. We hypothesized that WT1 is a critical TF in the development of HGSOC, and that WT1-AS plays a key role in its regulatory pathway. We evaluated the regulatory role of WT1 first by analyzing cellular phenotypes and target gene expression changes in HGSOC cell lines after shRNA mediated knockdown (KD) of WT1. HGSOC cells with stable WT1 knockdown had impaired proliferation and concomitant reduction of WT1-AS expression. We also identified a decrease in expression of SHMT1 in WT1 KD cells, which is a known WT1 target gene. Integrated genome-wide analysis of the WT1 transcriptome by RNA-seq and the WT1 cistrome by ChIP-seq, in parallel with ChIP-MS, is being used to establish the architecture of the WT1 regulome, specifically to establish the regulatory mechanisms and relationships between WT1 and WT1-AS. Ultimately, these studies aim to establish the mechanisms of action for the WT1 TF in neoplastic transformation of HGSOC. Citation Format: Norma I. Rodriguez-Malave, Justyna E. Kanska, Kevin C. Vavra, Dennis Hazelett, Rosario I. Corona, Ji-Heui Seo, Matthew Freedman, Simon R. Knott, Sandra Orsulic, Beth Y. Karlan, Kate Lawrenson, Simon A. Gayther. Role of the WT1 transcription factor in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5435.

WT1-AS promotes cell apoptosis in hepatocellular carcinoma through down-regulating of WT1.

BackgroundThe antisense of the tumor suppressor gene WT1 (WT1-AS) is a long non-coding RNA. The role of WT1-AS in the development of hepatocellular carcinoma (HCC) has not yet been elucidated.MethodsQuantitative real-time PCR and western blot analyses were used to measure levels of WT1-AS and its related genes in tumor and corresponding adjacent tumor tissues of HCC patients. The effect on HCC cell proliferation and apoptosis was assessed by EdU incorporation assays and PI-Annexin-V staining, respectively. ShRNA and dual-luciferase assays were used to investigate the regulatory relationship between WT1-AS and WT1 in cell lines.ResultsWT1-AS expression correlated negatively with WT1 expression in HCC tumor tissue. Kaplan-Meier curve analysis revealed that WT1-AS expression is a reliable indicator of HCC prognosis. The downregulation of WT1 expression by WT1-AS promoted cell apoptosis by suppressing the JAK/STAT3 signaling pathway. Bioinformatics analysis showed that WT1-AS downregulates WT1 by binding to the TATA region of the WT1 promotor. WT1-AS was also able to reverse WT1-mediated resistance to Dox based chemotherapy in HCC cells.ConclusionsWT1-AS downregulates WT1 expression in HCC tumors and promotes apoptosis by binding to the promoter region of WT1. Our findings suggest that WT1-AS may function as a tumor suppressor in HCC by reversing the oncogenic effects of WT1.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0233-7) contains supplementary material, which is available to authorized users.

Decreased expression of long non-coding RNA WT1-AS promotes cell proliferation and invasion in gastric cancer

Tumor recurrence and metastasis remain the major obstacles for the successful treatment of patients diagnosed with gastric cancer. In recent years, long non-coding RNAs (lncRNAs) have been considered as key regulators of tumor behavior. In this study, we investigated the expression and biological role of a newly-identified cancer-related lncRNA, WT1-AS. We found that WT1-AS expression was significantly down-regulated in tumor tissues compared to matched adjacent non-tumor tissues. The WT1-AS expression level was also associated with tumor size and the clinicopathological stage. Cell proliferation, migration, and invasion were inhibited, and the proportion of G0/G1 cells increased when WT1-AS was ectopically-expressed in gastric cancer cells. Furthermore, ectopic expression of WT1-AS was demonstrated to inhibit tumor growth and metastasis in vivo. Finally, we found that WT1-AS overexpression could decrease ERK protein phosphorylation. Our study indicates that WT1-AS is significantly down-regulated in gastric cancers and may be correlated with tumor progression.

Abstract 4579: Variants in long non-coding RNAs are associated with epithelial ovarian cancer risk in a pooled analysis of three genome-wide association studies.

Abstract Since most GWAS hits fall in non-coding regions of the genome and long non-coding RNAs (lncRNAs) are emerging as drivers of carcinogenesis, we hypothesized that SNPs in lncRNAs influence epithelial ovarian cancer (EOC) risk. A comprehensive lncRNA database (lncRNA db) that contained 104 human lncRNAs when downloaded in November 2011 was used to identify 1,737 variants in 63 unique lncRNAs that were represented in three genome-wide association studies from North America, the United Kingdom, and Poland (3,995 EOC cases and 3,277 controls of European background). SNPs with MAF &amp;lt;5% were excluded, and missing genotypes were inferred with Mach using the HapMap CEU population. Unconditional logistic regression treating alternate alleles as an ordinal variable was used to evaluate individual SNP-EOC risk associations. Subgroup analysis was conducted for serous adenocarcinomas (N= 2,066), the main histologic subtype of EOC. Models were adjusted for study and European ancestry using the first two principal components (PCs). We reduced correlated signals (r2 ≥ 0.8) within each lncRNA and kept the most statistically significant variants based on Wald tests, resulting in 651 SNPs for further analyses. Fisher's product method was used to aggregate evidence of multiple variants within each lncRNA using 10,000 permutations. To adjust for multiple comparisons, a false discovery rate (FDR) of 10% was used. A replication phase was carried out for top-ranked SNPs from an additional 9,854 EOC cases (5,802 serous) and 17,633 controls genotyped through an international effort known as the Collaborative Oncological Gene-Environment study (COGS). Four lncRNAs, WT1-AS (p = 0.005, n = 2 SNPs), Hoxa11as (p = 0.013, n = 2), AK082072 (p = 0.016, n = 4) and H19 (p = 0.020, n = 2), were significantly associated with EOC risk among all histologies (FDR ≤ 10%). The SNP in WT1-AS most significantly associated with EOC risk was rs3809061 (T&amp;gt;C) (minor allele frequency= 36%, odds ratio (OR) = 0.91, CI: 0.84-0.97, p = 0.008). Among serous cases, rs3809061 was also protective (OR = 0.92, p = 0.04). These findings were replicated by a correlated SNP rs2301250 (r2 = 0.84) evaluated in COGS among serous cases (OR = 0.94, CI: 0.90-0.99, p = 0.009) and less significant among all histologies (OR = 0.96, CI: 0.93-1.00, p = 0.054). We further interrogated its functional role by performing an expression quantitative trait locus analysis on WTI-AS mRNA expression using the Cancer Genome Atlas (TCGA) data (N = 462 serous cases). WT1-AS expression was significantly higher among carriers of the variant allele (compared to TT homozygotes) using the Mann-Whitney U test (p = 0.001). WT1-AS is the antisense and possible regulator of WT1, a tumor suppressor gene reported to be prognostic in advanced EOC. These findings implicate germline lncRNA variants associated with EOC risk that merit further investigation. Citation Format: Yian Ann Chen, Zhihua Chen, Jennifer Permuth-Wey, Ya-Yu Tsai, Hui-Yi Lin, Xiaotao Qu, Kate Lawrenson, David Fenstermacher, Catherine M. Phelan, Alvaro Monteiro, Simon A. Gayther, Steven A. Narod, Rebecca Sutphen, Michael J. Birrer, Nicolas Wentzensen, Joellen M. Schildkraut, Ellen L. Goode, Paul Pharoah, Thomas Sellers. Variants in long non-coding RNAs are associated with epithelial ovarian cancer risk in a pooled analysis of three genome-wide association studies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4579. doi:10.1158/1538-7445.AM2013-4579

Alternately spliced WT1 antisense transcripts interact with WT1 sense RNA and show epigenetic and splicing defects in cancer.

Many mammalian genes contain overlapping antisense RNAs, but the functions and mechanisms of action of these transcripts are mostly unknown. WT1 is a well-characterized developmental gene that is mutated in Wilms' tumor (WT) and acute myeloid leukaemia (AML) and has an antisense transcript (WT1-AS), which we have previously found to regulate WT1 protein levels. In this study, we show that WT1-AS is present in multiple spliceoforms that are usually expressed in parallel with WT1 RNA in human and mouse tissues. We demonstrate that the expression of WT1-AS correlates with methylation of the antisense regulatory region (ARR) in WT1 intron 1, displaying imprinted monoallelic expression in normal kidney and loss of imprinting in WT. However, we find no evidence for imprinting of mouse Wt1-as. WT1-AS transcripts are exported into the cytoplasm and form heteroduplexes with WT1 mRNA in the overlapping region in WT1 exon 1. In AML, there is often abnormal splicing of WT1-AS, which may play a role in the development of this malignancy. These results show that WT1 encodes conserved antisense RNAs that may have an important regulatory role in WT1 expression via RNA:RNA interactions, and which can become deregulated by a variety of mechanisms in cancer.