Probes for LONG

Abstract

AIM:
Long non-coding RNAs (lncRNAs) are potential biomarkers for breast cancer risk stratification. LncRNA expression has been investigated primarily by RNA sequencing, quantitative reverse transcription PCR or microarray techniques. In this study, six breast cancer-implicated lncRNAs were investigated by chromogenic in situ hybridisation (CISH).

METHODS:
Invasive breast carcinoma (IBC), ductal carcinoma in situ (DCIS) and normal adjacent (NA) breast tissues from 52 patients were screened by CISH. Staining was graded by modified Allred scoring.

RESULTS:
HOTAIR, H19 and KCNQ1OT1 had significantly higher expression levels in IBC and DCIS than NA (p<0.05), and HOTAIR and H19 were expressed more strongly in IBC than in DCIS tissues (p<0.05). HOTAIR and KCNQ101T were expressed in tumour cells; H19 and MEG3 were expressed in stromal microenvironment cells; MALAT1 was expressed in all cells strongly and ZFAS1 was negative or weakly expressed in all specimens.

CONCLUSION:
These data corroborate the involvement of three lncRNAs (HOTAIR, H19 and KCNQ1OT1) in breast tumourigenesis and support lncRNA CISH as a potential clinical assay. Importantly, CISH allows identification of the tissue compartment expressing lncRNA.

Prostate cancers remain indolent in the majority of individuals but behave aggressively in a minority. The molecular basis for this clinical heterogeneity remains incompletely understood. Here we characterize a long noncoding RNA termed SChLAP1 (second chromosome locus associated with prostate-1; also called LINC00913) that is overexpressed in a subset of prostate cancers. SChLAP1 levels independently predict poor outcomes, including metastasis and prostate cancer-specific mortality. In vitro and in vivo gain-of-function and loss-of-function experiments indicate that SChLAP1 is critical for cancer cell invasiveness and metastasis. Mechanistically, SChLAP1 antagonizes the genome-wide localization and regulatory functions of the SWI/SNF chromatin-modifying complex. These results suggest that SChLAP1 contributes to the development of lethal cancer at least in part by antagonizing the tumor-suppressive functions of the SWI/SNF complex.

The long noncoding RNA SChLAP1 is overexpressed in a subset of prostate cancers (PCa), and high SChLAP1 expression by in situ hybridization (ISH) independently predicts biochemical recurrence after radical prostatectomy. Importantly, although biochemical recurrence is a significant clinical outcome, it is not a validated surrogate for PCa-related mortality. Thus, we evaluated the association between SChLAP1 expression and development of lethal PCa in a large cohort of American men with PCa and long-term follow-up. SChLAP1 ISH was performed on tissue microarrays containing representative formalin-fixed, paraffin-embedded PCa tissue from all patients and scored using a semiquantitative method (ISH score range 0-400). Hazard ratios (HRs) for the association between SChLAP1 expression and time to development of lethal PCa were estimated using multivariable Cox regression analysis. Of the 937 patients evaluated, 89 (9.5%) had high SChLAP1 expression (ISH score ≥100), which in patients treated with radical prostatectomy was strongly associated with development of lethal PCa independent of age, Gleason score, pathologic stage, and PTEN status (HR 2.2, 95% confidence interval 1.1-4.1). These results suggest that SChLAP1 may be a useful tissue-based biomarker for identifying PCa patients at higher risk of lethal progression.

PATIENT SUMMARY:
We examined expression of the RNA molecule SChLAP1 in a large group of prostate cancer patients with long-term follow-up and found that patients with high SChLAP1 expression had a significantly higher chance of developing lethal disease.

Recent studies have implicated long noncoding RNAs (lncRNAs) as regulators of many important biological processes. Here we report on the identification and characterization of a lncRNA, lnc13, that harbors a celiac disease-associated haplotype block and represses expression of certain inflammatory genes under homeostatic conditions. Lnc13 regulates gene expression by binding to hnRNPD, a member of a family of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs). Upon stimulation, lnc13 levels are reduced, thereby allowing increased expression of the repressed genes. Lnc13 levels are significantly decreased in small intestinal biopsy samples from patients with celiac disease, which suggests that down-regulation of lnc13 may contribute to the inflammation seen in this disease. Furthermore, the lnc13 disease-associated variant binds hnRNPD less efficiently than its wild-type counterpart, thus helping to explain how these single-nucleotide polymorphisms contribute to celiac disease.

Long non-coding RNAs (lncRNAs) are important for transcription and for epigenetic or posttranscriptional regulation of gene expression and may contribute to carcinogenesis. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), an lncRNA involved in the regulation of the cell cycle, cell proliferation, and cell migration, is known to be deregulated in multiple cancers. Here, we analyzed the expression of MALAT1 on 195 cases of benign and malignant thyroid neoplasms by using tissue microarrays for RNA in situ hybridization (ISH) and real-time PCR. MALAT1 is highly expressed in normal thyroid (NT) tissues and thyroid tumors, with increased expression during progression from NT to papillary thyroid carcinomas (PTCs) but is downregulated in poorly differentiated thyroid cancers (PDCs) and anaplastic thyroid carcinomas (ATCs) compared to NT. Induction of epithelial to mesenchymal transition (EMT) by transforming growth factor (TGF)-beta in a PTC cell line (TPC1) led to increased MALAT1 expression, supporting a role for MALAT1 in EMT in thyroid tumors. This is the first ISH study of MALAT1 expression in thyroid tissues. It also provides the first piece of evidence suggesting MALAT1 downregulation in certain thyroid malignancies. Our findings support the notion that ATCs may be molecularly distinct from low-grade thyroid malignancies and suggest that MALAT1 may function both as an oncogene and as a tumor suppressor in different types of thyroid tumors.

Tetraspanins (Tspan) are transmembrane proteins with important scaffold and signalling functions. Deletions of Tetraspanin 6 (Tspan6) gene, a member of the tetraspanin family, have been reported in patients with Epilepsy Female-restricted with Mental Retardation (EFMR). Interestingly, mutations in Tspan7, highly homologous to Tspan6, are associated with X-linked intellectual disability, suggesting that these two proteins are important for cognition. Considering recent evidences showing that Tspan7 plays a key role in synapse development and AMPAR trafficking, we initiated the study of Tspan6 in synaptic function using a Tspan6 knock out mouse model. Here we report that hippocampal field recordings from Tspan6 knock out mice show an enhanced basal synaptic transmission and impaired long term potentiation (LTP). A normal paired-pulse facilitation response suggests that Tspan6 affects the properties of the postsynaptic rather than the presynaptic terminal. However, no changes in spine morphology or postsynaptic markers could be detected in Tspan6 KO mice compared with wild types. In addition, Tspan6 KO mice show normal locomotor behaviour and no defects in hippocampus-dependent memory tests.

Long non-coding RNA RAD51 antisense RNA 1 (RAD51-AS1, also known as TODRA) has been shown to be down-regulated by E2F1, a key cell cycle and apoptosis regulator, in breast cancer. Little is known regarding the role of RAD51-AS1 in disease. Here, we investigate the role of RAD51-AS1 in epithelial ovarian cancer (EOC). Using luciferase reporter and chromatin immunoprecipitation experiments, we verified RAD51-AS1 as a target of E2F1 under negative regulation in EOC. We then examined RAD51-AS1 expression in EOC samples using in situ hybridization (ISH). RAD51-AS1 was localized to the nucleus and found to be a critical marker for clinical features that significantly correlated with poor survival in EOC patients. RAD51-AS1 was also an independent prognostic factor for EOC. Overexpression of RAD51-AS1promoted EOC cell proliferation, while silencing of RAD51-AS1 inhibited EOC cell proliferation, delayed cell cycle progression and promoted apoptosis in vitro and in vivo. RAD51-AS1 may participate in carcinogenesis via regulation of p53 and p53-related genes. Our study highlights the role of RAD51-AS1 as a prognostic marker of EOC. Based on its regulation of the tumor suppressor p53, RAD51-AS1-based therapy may represent a viable therapeutic option for EOC in the near future.

Conventional therapies for breast cancer brain metastases (BCBMs) have been largely ineffective because of chemoresistance and impermeability of the blood-brain barrier. A comprehensive understanding of the underlying mechanism that allows breast cancer cells to infiltrate the brain is necessary to circumvent treatment resistance of BCBMs. Here, we determined that expression of a long noncoding RNA (lncRNA) that we have named lncRNA associated with BCBM (Lnc-BM) is prognostic of the progression of brain metastasis in breast cancer patients. In preclinical murine models, elevated Lnc-BM expression drove BCBM, while depletion of Lnc-BM with nanoparticle-encapsulated siRNAs effectively treated BCBM. Lnc-BM increased JAK2 kinase activity to mediate oncostatin M- and IL-6-triggered STAT3 phosphorylation. In breast cancer cells, Lnc-BM promoted STAT3-dependent expression of ICAM1 and CCL2, which mediated vascular co-option and recruitment of macrophages in the brain, respectively. Recruited macrophages in turn produced oncostatin M and IL-6, thereby further activating the Lnc-BM/JAK2/STAT3 pathway and enhancing BCBM. Collectively, our results show that Lnc-BM and JAK2 promote BCBMs by mediating communication between breast cancer cells and the brain microenvironment. Moreover, these results suggest targeting Lnc-BM as a potential strategy for fighting this difficult disease.

Large-scale transcriptome sequencing efforts have vastly expanded the catalog of long non-coding RNAs (lncRNAs) with varying evolutionary conservation, lineage expression, and cancer specificity. Here, we functionally characterize a novel ultraconserved lncRNA, THOR (ENSG00000226856), which exhibits expression exclusively in testis and a broad range of human cancers. THOR knockdown and overexpression in multiple cell lines and animal models alters cell or tumor growth supporting an oncogenic role. We discovered a conserved interaction of THOR with IGF2BP1 and show that THOR contributes to the mRNA stabilization activities of IGF2BP1. Notably, transgenic THOR knockout produced fertilization defects in zebrafish and also conferred a resistance to melanoma onset. Likewise, ectopic expression of human THOR in zebrafish accelerated the onset of melanoma. THOR represents a novel class of functionally important cancer/testis lncRNAs whose structure and function have undergone positive evolutionary selection.

Nuclear receptors regulate gene expression in response to environmental cues, but the molecular events governing the cell type specificity of nuclear receptors remain poorly understood. Here we outline a role for a long noncoding RNA (lncRNA) in modulating the cell type-specific actions of liver X receptors (LXRs), sterol-activated nuclear receptors that regulate the expression of genes involved in cholesterol homeostasis and that have been causally linked to the pathogenesis of atherosclerosis. We identify the lncRNA MeXis as an amplifier of LXR-dependent transcription of the gene Abca1, which is critical for regulation of cholesterol efflux. Mice lacking the MeXis gene show reduced Abca1 expression in a tissue-selective manner. Furthermore, loss of MeXis in mouse bone marrow cells alters chromosome architecture at the Abca1 locus, impairs cellular responses to cholesterol overload, and accelerates the development of atherosclerosis. Mechanistic studies reveal that MeXis interacts with and guides promoter binding of the transcriptional coactivator DDX17. The identification of MeXis as a lncRNA modulator of LXR-dependent gene expression expands understanding of the mechanisms underlying cell type-selective actions of nuclear receptors in physiology and disease.

Abstract: Long noncoding RNA HOXA11 antisense RNA (HOXA11-AS) is involved in tumorigenesis and development of some human cancers. However, the role of HOXA11-AS in human laryngeal squamous cell cancer (LSCC) is yet
unclear. In this study, we firstly investigated the expression of HOXA11-AS in LSCC. Microarray and qRT-PCR showed that the level of HOXA11-AS was significantly higher in LSCC than that in the corresponding adjacent non-neoplastic
tissues. ISH revealed that HOXA11-AS was strongly expressed in the nucleus and closely related to the T grade, neck nodal metastasis, and clinical stage. Patients with T3-4 grade, neck nodal metastasis, or advanced clinical
stage presented a high HOXA11-AS expression. Kaplan-Meier analysis showed that high HOXA11-AS expression could predict a poor prognosis in LSCC patients. Furthermore, HOXA11-AS knockdown significantly inhibited the
growth, migration, and invasion of LSCC cells. Taken together, the current data indicated that HOXA11-AS plays an oncogenic role in the cellular processes of LSCC and serve as a novel marker and a potential therapeutic target in
LSCC patients.