HOTAIR Knockdown Research Articles

Knockdown of long non-coding RNA HOTAIR increases miR-454-3p by targeting Stat3 and Atg12 to inhibit chondrosarcoma growth

Current practices for the therapy of chondrosarcoma, including wide-margin surgical resection and chemotherapy, are less than satisfactory. Recently, emerging evidence has demonstrated that long non-coding RNAs (lncRNAs) have an essential role in the initiation and progression of tumors. As a typical lncRNA, HOTAIR is significantly overexpressed in various tumors. However, the function and potential biological mechanisms of HOTAIR in human chondrosarcoma remain unknown. Quantitative RT-PCR demonstrated that HOTAIR expression was upregulated in chondrosarcoma tissues and cell lines. High HOTAIR expression is correlated with tumor stage and poor prognosis. Functional experiments reveal that HOTAIR knockdown leads to growth inhibition of human chondrosarcoma cells in vitro and in vivo. In addition to cycle arrest and apoptosis, knockdown of HOTAIR inhibits autophagy, which favors cell death. Mechanistically, we demonstrated that HOTAIR induced DNA methylation of miR-454-3p by recruiting EZH2 and DNMT1 to the miR-454-3p promoter regions, which markedly silences miR-454-3p expression. Further analysis revealed that STAT3 and ATG12 are targets of miR-454-3p, initiate HOTAIR deficiency-induced apoptosis and reduce autophagy. Collectively, our data reveal the roles and functional mechanisms of HOTAIR in human chondrosarcoma and suggest that HOTAIR may act as a prognostic biomarker and potential therapeutic target for chondrosarcoma.

Up-regulated HOTAIR induced by fatty acids inhibits PTEN expression and increases triglycerides accumulation in HepG2 cells

ABSTRACTNon-alcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation unrelated to excess alcohol intake, in which the hepatic expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is inhibited. Long non-coding RNA HOTAIR suppresses PTEN expression in hepatic stellate cells during liver fibrosis, and it is involved in liver lipid dysregulation. In this study, we evaluated whether the PTEN down-regulation and lipid accumulation in hepatic cells might be mediated by HOTAIR during the development of NAFLD. Free fatty acids (FFAs) treatment promoted triglyceride accumulation in HepG2 cells, significantly increasing HOTAIR expression and inhibiting PTEN expression (both at mRNA and protein levels).The effects on HOTAIR and PTEN expressions disappeared after withdrawal of the FFAs treatment. SiRNA-mediated HOTAIR knockdown prevented PTEN down-regulation and triglyceride accumulation in HepG2 cells treated with FFAs; and CAPE (an NF-κBp65 inhibitor) treatment prevented the HOTAIR up-regulation and PTEN down-regulation. FFAs could induce the up-regulation of HOTAIR in HepG2 cells probably dependent on the NF-κB signaling, and consequently suppress PTEN expression and promote triglyceride accumulation. Aberrant up-regulation of HOTAIR mediated by excessive circulating FFAs levels may be a crucial mechanism associated with liver steatosis.

The role of long noncoding RNA HOTAIR in the acquired multidrug resistance to imatinib in chronic myeloid leukemia cells

ABSTRACTObjectives: Imatinib, a breakpoint cluster region-Abelson murine leukemia tyrosine kinase inhibitor, has revolutionized the treatment of chronic myelogenous leukemia (CML). However, the development of multidrug resistance (MDR) limits the clinical application of imatinib. In this study, we aimed to investigate the mechanisms of long noncoding RNA (lncRNA) HOTAIR in CML resistance to imatinib.Methods: Thirty-four CML patients were divided into multidrug resistance protein 1 (MRP1)-low and MRP1-high groups according to the median expression. Real-time PCR (qPCR) was used to detect the expression of lncRNA HOTAIR in CML patients, and MTT assay and flow cytometry assay were employed to detect the biological function of silencing lncRNA HOTAIR on the cell survival rate and apoptotic rate. An imatinib-resistant human CML cell line K562 (K562-R) was established, and western blot was used to detect the impact of lncRNA HOTAIR on the activation of PI3K/Akt signaling pathway.Results: Our results showed that lncRNA HOTAIR was greatly upregulated in the MRP1-high patients as well as in the K562-imatinib-resistant cells compared with control. Knockdown of HOTAIR expression downregulated the MRP1 expression levels in the K562-imatinib cells and resulted in higher sensitivity to the imatinib treatment. In addition, the activation of PI3K/Akt was greatly attenuated when HOTAIR was knocked down in K562-imatinib cells.Discussions: These data suggest that the knockdown of HOTAIR may play a crucial role in improving acquired resistance to imatinib in CML K562-R cells via PI3K/Akt pathway.Conclusions: LncRNA HOTAIR modulates CML cell MDR in a PI3K/Akt-dependent way.

Downregulation of HOTAIR Expression Mediated Anti-Metastatic Effect of Artesunate on Cervical Cancer by Inhibiting COX-2 Expression.

Artesunate (ART) has anti-cancer activities for a variety of solid tumors. The aim of this study was to investigate the anti-metastatic effect of ART on cervical cancer cells. In vivo anti-metastatic effect of ART was investigated in mice with the lung metastasis model by the subcutaneous injection of ART. The interaction of HOTAIR and COX-2 was measured by RNA immunoprecipitation and RNA pull-down assay. The effect of ART on metastasis of CaSki and Hela cells was evaluated by invasion and migration assay. We found that ART inhibited cervical cancer metastasis and HOTAIR expression. HOTAIR overexpression partially abolished the anti-metastatic effect of ART on cervical cancer cells. In addition, HOTAIR can interact with COX-2 to positively regulate COX-2 expression and catalytic activity. Finally, overexpression of COX-2 reversed the effect of HOTAIR knockdown on Hela cell migration and invasion. Taken together, our data revealed that ART may elicit anti-metastatic effect against cervical cancer by inhibition of HOTAIR expression, which resulted in the decrease of COX-2 expression.

Dioscin inhibits gastric tumor growth through regulating the expression level of lncRNA HOTAIR.

BackgroundAs a member of non-coding RNAs family, long non-coding RNAs’ functions in cancer needs to be further investigated. It has been indicated that the functions of Hox transcript antisense intergenic RNA (lncRNA: HOTAIR) include reprogramming chromatin organization and promoting tumor metastasis such as breast and colorectal tumor. The aim of this study is to investigate the functions of Hox in gastric cancer.MethodsIn the present study, the expression level of HOTAIR was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), 20 gastric cancer tissues and 20 normal tissues was included. All clinical data were analyzed retrospectively. The CCK-8 and colony formation assay was used to identify if the knockdown of HOTAIR have an influence on gastric cancer cell lines.ResultsCompared with normal tissues, higher expression level of HOTAIR was found in gastric cancer tissues. Dioscin inhibits proliferation of the three gastric cancer cell lines and decrease HOTAIR expression.ConclusionsThe expression of HOTAIR is up regulated in gastric cancer and gastric cancer cell lines, dioscin inhibits the proliferation of three gastric cancer cell lines and the anti-tumor effect of dioscin may partly depend on the down regulation of HOTAIR.

Long non-coding RNA HOTAIR regulates proliferation and invasion via activating Notch signalling pathway in retinoblastoma.

Retinoblastoma is the most frequently occurring tumour in the eyes in early childhood. Novel targets that are important for the diagnosis or treatment of retinoblastoma could be valuable in increasing the survival rate of patients affected by this disease. Long non-coding RNAs (lncRNAs) are a recently discovered type of RNAs with no proteincoding function; yet it has become increasingly clear that lncRNAs are responsible for important gene regulatory functions in various diseases. In this study, the expression of lncRNA HOTAIR was measured by qRT-PCR, and HOTAIR expression was found to be significantly upregulated in human retinoblastomas tissues as compared with that in paracancerous tissues. Knockdown of HOTAIR restricted the proliferation and invasion of the more invasive retinoblastoma Y79 cells, and led to G0/G1 arrest, possibly through inhibiting phospho-RB1, RB1 and CCNE. Furthermore, we found that the Notch signalling pathway was activated abnormally in retinoblastoma cell lines, while knockdown of HOTAIR attenuated the endogenous Notch signalling pathway in vitro and in vivo. In addition, knockdown of HOTAIR could inhibit the tumour progression in a xenograft model of retinoblastoma. In summary, our findings indicate that HOTAIR may play important roles in retinoblastoma progression via Notch pathway. HOTAIR has the potential to enhance the development of novel targeted diagnostic and therapeutic approaches for retinoblastoma.

EZH2 coupled with HOTAIR to silence MicroRNA‐34a by the induction of heterochromatin formation in human pancreatic ductal adenocarcinoma

MicroRNA-34a (miR-34a) is frequently downregulated in pancreatic ductal adenocarcinoma (PDAC) cells, however, the silencing mechanism remains unclear. Enhancer of zeste homolog 2 (EZH2) is overexpressed in PDAC, and our previous miRNA profiling showed that inhibition of EZH2 in PDAC cells led to the re-expression of a group of tumor suppressor miRNAs including miR-34a. Here, we studied the effect of ectopic EZH2 expression to the silencing of miR-34a, and identified HOTAIR as an interacting partner to induce heterochromatin formation during miR-34a repression. We identified EZH2 as a major player in silencing miR-34a. Inhibition of EZH2 upregulated miR-34a expression in PDAC cells, while EZH2 overexpression in human pancreatic ductal epithelial (HPDE) cells repressed miR-34a expression and decreased the miR-34a promoter activity. We then showed that HOTAIR played a critical role in EZH2-mediated repression of miR-34a, as knockdown of HOTAIR attenuated the miR-34a inhibition effect in EZH2-overexpressing HPDE cells. HOTAIR physically interacted with miR-34a promoter, and the EZH2-interacting region located at 5' HOTAIR RNA was essential in repressing miR-34a and promoting cell proliferation. More importantly, we showed that the interaction between EZH2 and HOTAIR underlay the silencing of miR-34a through induction of heterochromatin formation. We first showed that manipulation of EZH2 level interfered the occupancy of heterochromatin markers H3K9me2, heterochromatin associated protein 1α and 1γ in PDAC cells. In turn, we showed that knockdown of HOTAIR reduced the occupancy of EZH2 at miR-34a promoter. The identification of HOTAIR-guided miR-34a silencing opened a new avenue in miR-34a-oriented therapy against PDAC.

The lncRNA HOTAIR impacts on mesenchymal stem cells via triple helix formation.

There is a growing perception that long non-coding RNAs (lncRNAs) modulate cellular function. In this study, we analyzed the role of the lncRNA HOTAIR in mesenchymal stem cells (MSCs) with particular focus on senescence-associated changes in gene expression and DNA-methylation (DNAm). HOTAIR binding sites were enriched at genomic regions that become hypermethylated with increasing cell culture passage. Overexpression and knockdown of HOTAIR inhibited or stimulated adipogenic differentiation of MSCs, respectively. Modification of HOTAIR expression evoked only very moderate effects on gene expression, particularly of polycomb group target genes. Furthermore, overexpression and knockdown of HOTAIR resulted in DNAm changes at HOTAIR binding sites. Five potential triple helix forming domains were predicted within the HOTAIR sequence based on reverse Hoogsteen hydrogen bonds. Notably, the predicted triple helix target sites for these HOTAIR domains were also enriched in differentially expressed genes and close to DNAm changes upon modulation of HOTAIR. Electrophoretic mobility shift assays provided further evidence that HOTAIR domains form RNA–DNA–DNA triplexes with predicted target sites. Our results demonstrate that HOTAIR impacts on differentiation of MSCs and that it is associated with senescence-associated DNAm. Targeting of epigenetic modifiers to relevant loci in the genome may involve triple helix formation with HOTAIR.

The long non-coding RNA HOTAIR increases tumour growth and invasion in cervical cancer by targeting the Notch pathway.

Evidence suggests that the long non-coding RNA (lncRNA), HOTAIR, is involved in cervical cancer pathogenesis. We examined serum HOTAIR expression levels in cervical cancer patients and determined the relationships between HOTAIR expression and several clinicopathological factors, including survival. We also examined the functional consequences of HOTAIR overexpression both in vitro and in vivo. Compared with control patients, HOTAIR expression was significantly greater in the serum of cervical cancer patients (P < 0.001). The results indicated that this increase was significantly associated with tumour size (P = 0.030), lymphovascular space invasion (P = 0.037), and lymph node metastasis (P = 0.043). Univariate analysis revealed that disease-free survival and overall survival times were significantly shorter in cervical cancer patients with high HOTAIR expression (hazard ratio [HR] = 4.27, 4.68 and P = 0.039, 0.031, respectively). Cell proliferation and invasion in vitro increased as a result of lentiviral-mediated HOTAIR overexpression in cervical cancer cell lines. HOTAIR knockdown inhibited these properties and increased apoptosis. In vivo xenograft experiments using the HOTAIR-overexpressing SiHa cell line revealed that HOTAIR was a strong inducer of tumour growth and modulated the expression of epithelial-mesenchymal transition and Notch-Wnt signalling pathway-related genes. This result suggested that HOTAIR overexpression promoted cell proliferation and invasion. In conclusion, increased HOTAIR expression was associated with decreased patient survival times. HOTAIR may be a useful target for treatment of cervical cancer patients.

Epigenetic modification of miR-141 regulates SKA2 by an endogenous 'sponge' HOTAIR in glioma.

Aberrant expression of miR-141 has recently implicated in the occurrence and development of various types of malignant tumors. However whether the involvement of miR-141 in the pathogenesis of glioma remains unknown. Here, we showed that miR-141 was markedly downregulated in glioma tissues and cell lines compared with normal brain tissues, and its expression correlated with the pathological grading. Enforced expression of miR-141 in glioma cells significantly inhibited cell proliferation, migration and invasion, whereas knockdown of miR-141 exerted opposite effect. Mechanistic investigations revealed that HOTAIR might act as an endogenous ‘sponge’ of miR-141, thereby regulating the derepression of SKA2. Further, we explored the molecular mechanism by which miR-141 expression was regulated, and found that the miR-141 promoter was hypermethylated and that promoter methylation of miR-141 was mediated by DNMT1 in glioma cells. Finally, both overexpression of miR-141 and knockdown of HOTAIR in a mouse model of human glioma resulted in significant reduction of tumor growth in vivo. Collectively, these results suggest that epigenetic modification of miR-141 and the interaction of ceRNA regulatory network will provide a new approach for therapeutics against glioma.

Upregulation of lncRNA HOTAIR contributes to IL-1β-induced MMP overexpression and chondrocytes apoptosis in temporomandibular joint osteoarthritis

Temporomandibular joint osteoarthritis (TMJ OA) is a common and heterogeneous disease that causes painful and progressive joint degeneration, which restricts daily activities, including talking and chewing. Long noncoding RNAs (lncRNAs) are an important class of genes involved in various physiological and pathological functions, including osteoarthritis (OA).The present study aimed to identify the lncRNAs that are important in TMJ OA and their potential functions. Here, we found that HOTAIR was significantly upregulated in the synovial fluid of TMJ OA patients compared with that of normal controls. Increased HOTAIR was similarly observed in the synovial fluid of TMJ OA rabbits as compared to control rabbits. Furthermore, in interleukin-1β (IL-1β)-induced TMJ OA in vitro model (primary rabbit condylar chondrocytes), the expressions of matrix metalloproteinase (MMP)-1, MMP3, MMP9 and HOTAIR were all dramatically increased. Most importantly, knockdown of HOTAIR in IL-1β-induced TMJ OA in vitro model could not only reverse the IL-1β-stimulated expressions of MMP1, MMP3 and MMP9, but also significantly decrease the apoptosis rate induced by IL-1β in primary rabbit condylar chondrocytes. Our data provides new insight into the mechanisms of chondrocytes destruction in TMJ OA.

PD38-10 BLADDER CANCER EXOSOMES FROM HIGH-GRADE MUSCLE INVASIVE BLADDER CANCER CONTAIN LONG NON-CODING RNA AND MESSENGER RNA

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology III1 Apr 2016PD38-10 BLADDER CANCER EXOSOMES FROM HIGH-GRADE MUSCLE INVASIVE BLADDER CANCER CONTAIN LONG NON-CODING RNA AND MESSENGER RNA Claudia Berrondo, Jonathan Flax, Victor Kucherov, Aisha Siebert, Thomas Osinski, Alex Rosenberg, Christopher Fucile, and Carla Beckham Claudia BerrondoClaudia Berrondo More articles by this author , Jonathan FlaxJonathan Flax More articles by this author , Victor KucherovVictor Kucherov More articles by this author , Aisha SiebertAisha Siebert More articles by this author , Thomas OsinskiThomas Osinski More articles by this author , Alex RosenbergAlex Rosenberg More articles by this author , Christopher FucileChristopher Fucile More articles by this author , and Carla BeckhamCarla Beckham More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1490AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Many long non-coding RNAs (lncRNA) play critical roles in tumor biology. The lncRNA HOX Antisense Intergenic RNA (HOTAIR) is overexpressed in many solid tumors and regulates genes involved in epithelial-to-mesenchyme transition (EMT). There is a need to incorporate lncRNA into biomarker and therapeutic target discovery algorithms. Exosomes may be a good source for biomarkers for UBC because they can be isolated from urine and their contents are protected from degradation. To determine the role of HOTAIR in UBC progression and to identify novel mRNA and lncRNA in urinary exosomes of patients with high-grade muscle invasive (HGMI) UBC. METHODS HOTAIR was knocked down using lentiviral shRNA. Migration was measured with a wound-scratch assay. Invasion was measured with trans-well assay and 3D culture system. Tumor, distal normal tissue and urine from patients with HGMI UBC and healthy volunteers (HVs) was obtained with IRB approval. mRNA and lncRNA levels were measured by qRT-PCR. Exosomes were isolated by ultracentrifugation and confirmed with electron microscopy, nanoparticle tracing and Western blot. RNA sequencing was completed using Illumina HiSeq2500 platform with 20,000,000 pair-ended reads and 125 base pair length. RESULTS UBC cell lines express elevated levels of tumor-associated mRNA and lncRNA (including HOTAIR). The knockdown of HOTAIR in UBC cell lines reduces migration and invasion and correlates with the loss of expression of EMT factors. Tumor from patients with HGMI UBC express elevated levels of tumor-associated mRNA and lncRNA (including HOTAIR). Exosomes from UBC cell lines and urinary exosomes from patients with HGMI UBC have elevated levels of tumor-associated mRNA and lncRNA (including HOTAIR). RNA sequencing revealed elevated levels of 22 novel mRNA and 4 novel lncRNA in the urinary exosomes of patients with HGMI UBC compared to HVs. qRT-PCR confirmed the differential enrichment of mRNA PDX1, TEDDM1, ZBTB4 and lncRNA HYMA1, LOC100506688 and OXT2-AS1. CONCLUSIONS UBC cell lines, tumors from patients with HGMI UBC and the exosomes they produce have elevated expression of tumor-associated mRNA and lncRNA (including HOTAIR). HOTAIR is overexpressed in UBC cell lines and UBC patient tumors and is important in UBC progression in vitro through its effects on EMT factor expression, cell migration and invasion. Urinary exosomes from patients with HGMI UBC contain elevated levels of novel mRNA and lncRNA. Taken together, these data suggest that urinary exosomes may contain mRNA and lncRNA that can serve as biomarkers for UBC. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e928 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Claudia Berrondo More articles by this author Jonathan Flax More articles by this author Victor Kucherov More articles by this author Aisha Siebert More articles by this author Thomas Osinski More articles by this author Alex Rosenberg More articles by this author Christopher Fucile More articles by this author Carla Beckham More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MiR-125a-5p decreases after long non-coding RNA HOTAIR knockdown to promote cancer cell apoptosis by releasing caspase 2.

HOTAIR (homeobox transcript antisense RNA), one of the prototypical long non-coding RNAs, has been verified overexpressed in multiple carcinomas and has emerged as a promising novel anticancer target. Its well-established role is acting as a predictor of poor prognosis and promoting cancer cell metastasis. Recently, another important mission of HOTAIR was uncovered that targeting HOTAIR caused cancer cell apoptosis. Nevertheless, so far there is no published data elaborating the mechanism. Here, we report that microRNA miR-125a-5p decreases and releases caspase 2 to promote cancer cell apoptosis after HOTAIR knockdown. We applied siRNAs targeting HOTAIR to various cancer cells, and observed apoptosis in all of these cell lines. RNA sequencing detected that miR-125a-5p was decreased after HOTAIR knockdown and miR-125a-5p mimics could rescue the apoptosis induced by HOTAIR deficiency. Luciferase assays identified caspase 2, an initiator caspase, to be a new target of miR-125a-5p. Elevated expression and subsequent cleavage of caspase 2 was observed after HOTAIR knockdown or inhibition of miR-125a-5p. RNAi of caspase 2 could attenuate the apoptosis induced by HOTAIR knockdown. In 80 clinical colon cancer tissues, HOTAIR and miR-125a-5p levels were higher than adjacent tissues, whereas caspase 2 was lower. MiR-125a-5p expression level was significantly correlated with colon tumor size, lymph node metastasis and clinical stage. These findings indicate that miR-125a-5p decreases after HOTAIR knockdown to promote cancer cell apoptosis by releasing caspase 2. Our work reveals a previously unidentified apoptotic mechanism, which might be exploitable in anticancer drug development.

Long noncoding RNA Hotair mediated angiogenesis in nasopharyngeal carcinoma by direct and indirect signaling pathways.

Nasopharyngeal carcinoma (NPC), as a unique head and neck cancer type, is particularly prevalent in certain geographic areas such as eastern Asia. Until now, the therapeutic options have been restricted mainly to radiotherapy or chemotherapy. However, the clinical treatment effect remains unsatisfactory even if the combined radio-chemotherapies. Therefore, it is urgently needed to develop effective novel therapies against NPC. In this study, we discovered that lncRNA Hotair was extremely abundant in NPC cells and clinical NPC samples. Further studies showed that Hotair knockdown significantly attenuated both in vitro and in vivo tumor cell growth and angiogenesis. Our study also demonstrated that Hotair promoted angiogenesis through directly activating the transcription of angiogenic factor VEGFA as well as through GRP78-mediated upregulation of VEGFA and Ang2 expression. Therefore, Hotair may serve as a promising diagnostic marker and therapeutic target for NPC patients.

The ratio of FoxA1 to FoxA2 in lung adenocarcinoma is regulated by LncRNA HOTAIR and chromatin remodeling factor LSH.

The lncRNA HOTAIR is a critical regulator of cancer progression. Chromatin remodeling factor LSH is critical for normal development of plants and mammals. However, the underlying mechanisms causing this in cancer are not entirely clear. The functional diversification of the FOXA1 and FOXA2 contributes to the target genes during evolution and carcinogenesis. Little is known about the ratio of FOXA1 to FOXA2 in cancer. We here found that both HOTAIR and LSH overexpression was significantly correlated with poor survival in patients with lung adenocarcinoma cancer (ADC). Also, the ratio of FOXA1 and FOXA2 is linked with poor survival in patients with lung ADC. HOTAIR regulates the ratio of FOXA1 to FOXA2 and migration and invasion. HOTAIR and the ratio of FOXA1 to FOXA2 are negatively correlated. HOTAIR knockdown inhibits migration and invasion. HOTAIR is associated with LSH, and this association linked with the binding of LSH in the promoter of FOXA1, not FOXA2. Targeted inhibition of HOTAIR suppresses the migratory and invasive properties. These data suggest that HOTAIR is an important mediator of the ratio of FOXA1 and FOXA2 and LSH involves in, and suggest that HOTAIR inhibition may represent a promising therapeutic option for suppressing lung ADC progression.

Knockdown of long non-coding RNA HOTAIR inhibits proliferation and invasiveness and improves radiosensitivity in colorectal cancer.

Colorectal cancer (CRC) is still one of the most important neoplasias causing human death. Multidisciplinary therapy has won consensus in the management of CRC, of which, radiotherapy occupies an important position. However, radioresistance is still a major obstacle in local control of CRC. Overexpression of long non-coding RNA HOTAIR has been found to correlate with tumorigenesis and poor prognosis in several types of cancer. In the present study, we analyzed HOTAIR expression levels of 53 CRC patients in tumor and adjacent normal tissue by real-time quantitative PCR. Knockdown of HOTAIR by RNA interference was performed to explore its roles in cell proliferation, migration, invasion, apoptosis and radiosensitivity. Results showed that CRC patients had higher HOTAIR expression in tumor tissues compared with adjacent normal tissues. In vitro, downregulation of HOTAIR reduced proliferation, migration and invasiveness while enhanced apoptosis and radio-sensitivity of CRC cells. Taken together, our findings suggest that long non-coding RNA HOTAIR expression is closely associated with tumor invasion and radiosensitivity, indicating the potential role in diagnostics and therapeutics of CRC.

P152: Long non-coding RNA HOTAIR affects DNA methylation patterns in gastrointestinal stromal tumours (GIST)

Aberrant alterations of DNA methylation patterns have been recognized as early and common events in oncogenesis emphasizing high therapeutic and preventive potential. Concurrent epigenetic silencing of multiple tumour suppressor genes has been reported in different tumours suggesting the existence of a directed program, whereby groups of genes are regulated by promoter methylation. The molecular bases of such a program remain largely unclear. Certain long non-coding RNAs (lncRNAs) have been recently identified that are responsible for target specificity of the histone modification complexes. It remains incompletely understood, however, if lncRNAs may play a role in patterning DNA methylation. In this study, we asked by using gastrointestinal stromal tumours as a model if an epigenetic related HOX Antisense Intergenic RNA , or HOTAIR , is involved in establishment of DNA methylation patterns. To this end, we first showed high up-regulation of HOTAIR in patient samples of high risk compared to low and intermediate risk groups ( n = 66, p = 6.7 × 10 −6 ), what is supported by the earlier reports on common carcinomas. Highest levels of HOTAIR endogenous expression were next detected also in cell lines GIST T1, GIST48b and GIST882. Stable knockdown of HOTAIR was achieved in GIST T1 and GIST48b cells by RNAi using lentiviral transduction. As expected epigenetic alterations due to HOTAIR knockdown could develop with a delay, genome-wide DNA methylation profiling was performed at passage 12 after the transduction on the Infinium HumanMethylation450 BeadChip platform in triplicates. DNA methylation data were analysed by an R package RnBeads. A total of 218 CpG sites got hypomethylated upon HOTAIR knockdown in GIST T1 and GIST48b cells (Δ β > 0.3, FDR RASSF1 was almost entirely erased upon HOTAIR knockdown in GIST T1 cell line with concomitant up-regulation detected by qRT-PCR. Concordant with earlier reports, HOTAIR knockdown led to reduced migration potential in GIST T1 cells. Taken together, the results suggest that HOTAIR is one of the factors involved in establishment of specific DNA methylation patterns in GIST. While the molecular mechanism remains to be determined, it is plausible to assume a recruitment of DNA methyltransferases by the Polycomb repressive complex 2, which target specificity is determined by HOTAIR . The results further suggest the feasibility of manipulating DNA methylation patterns in a targeted manner and are of potential interest in context of developing epigenetic cancer therapy. The study is supported by the Interdisciplinary Centre for Clinical Research (IZKF) at the University of Erlangen-Nuremberg.

LncRNA HOTAIR Enhances the Androgen-Receptor-Mediated Transcriptional Program and Drives Castration-Resistant Prostate Cancer.

SUMMARYUnderstanding the mechanisms of androgen receptor (AR) activation in the milieu of low androgen is critical to effective treatment of castration-resistant prostate cancer (CRPC). Here, we report HOTAIR as an androgen-repressed lncRNA, and, as such, it is markedly upregulated following androgen deprivation therapies and in CRPC. We further demonstrate a distinct mode of lncRNA-mediated gene regulation, wherein HOTAIR binds to the AR protein to block its interaction with the E3 ubiquitin ligase MDM2, thereby preventing AR ubiquitination and protein degradation. Consequently, HOTAIR expression is sufficient to induce androgen-independent AR activation and drive the AR-mediated transcriptional program in the absence of androgen. Functionally, HOTAIR overexpression increases, whereas HOTAIR knockdown decreases, prostate cancer cell growth and invasion. Taken together, our results provide compelling evidence of lncRNAs as drivers of androgen-independent AR activity and CRPC progression, and they support the potential of lncRNAs as therapeutic targets.

Overexpression of long non-coding RNA HOTAIR leads to chemoresistance by activating the Wnt/β-catenin pathway in human ovarian cancer.

Overexpression of HOTAIR (HOX antisense intergenic RNA) is significantly correlated with tumor progression and poor prognosis in human ovarian cancer. However, the underlying mechanisms are largely unknown. In the present study, we investigated the roles of HOTAIR in the initiation and chemoresistance of ovarian cancer. As our data show, HOTAIR overexpression promoted cell cycle progression (and thus cell proliferation) by activating the wnt/β-catenin signaling pathway. Likewise, knockdown of HOTAIR suppressed cell proliferation and arrested cell cycle at G1 phase via inhibition of wnt/β-catenin signaling. Moreover, the results of primary culture demonstrated that elevated HOTAIR expression correlated positively with chemoresistance in ovarian cancer. In vitro and in vivo, HOTAIR induced cellular resistance to cisplatin by activating the wnt/β-catenin pathway, which could be reversed by pre-treatment with the wnt/β-catenin inhibitor, XAV939. In conclusion, HOTAIR promotes the initiation and chemoresistance of ovarian cancer by activating wnt/β-catenin signaling, suggesting that HOTAIR might be a potent therapeutic target for ovarian cancer treatment.

Abstract 152: The long non-coding RNA HOTAIR affects exosome-mediated bladder cancer progression

Abstract Exosomes are 30-100nM membrane-bound vesicles that participate in intercellular communication and facilitate tumor microenvironments. The mechanisms by which exosomes modify tumor niches are not fully understood. One possibility is that exosomes deliver biologically active molecules like mRNA, protein, miRNA and long non coding RNA (lncRNA) that alter the metabolism of recipient cells to produce pathological phenotypes. Exosomes may also activate the expression of genes involved in tumor progression in recipient cells. Overexpression of the lncRNA HOTAIR is associated with poor prognosis and affects tumor progression, in part, by recruiting PRC2 and LSD1 chromatin modifying complexes to thousands of target genes involved in tumor progression processes such as epithelial-to-mesenchyme transition (EMT). We show that HOTAIR is enriched in bladder cancer (BC) patient tumors and urinary exosomes. BC cell lines also express elevated levels of HOTAIR. Knockdown of HOTAIR in BC cell lines significantly reduces migration and invasion in trans-well and 3D culture, which correlates with EMT gene expression changes in these cells. Exosomes isolated from shHOTAIR vs shScramble cells fail to facilitate migration and invasion in 3D culture of a recipient BC cell line. Selected bands were isolated from a coomassie gel of shHOTAIR and shScramble exosomes and sent for mass spectrometry. Hundreds of putative proteins were identified including proteins involved in RNA binding, apoptosis, vesicle trafficking and integrins. These data support HOTAIR as an important player in exosome-mediated BC tumor progression possibly through affects on gene expression in exosome producer cells. For example, in shHOTAIR cells the loss of PRC2 and/or LSD1 complexes from key genes may ultimately affect the proteins available for packaging into exosomes. Exosomes have important roles in the tumor microenvironment and these data support a role for HOTAIR in the modulation of exosome-mediated tumor progression. Citation Format: Claudia Berrondo, Jonathan Flax, Edward M. Messing, Carla Beckham. The long non-coding RNA HOTAIR affects exosome-mediated bladder cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 152. doi:10.1158/1538-7445.AM2015-152