Probes for LONG

Emerging evidence indicates that long noncoding RNAs (lncRNAs) are actively involved in a number of developmental and tumorigenic processes. Here, the authors describe the first successful use of spherical nucleic acids as an effective nanoparticle platform for regulating lncRNAs in cells; specifically, for the targeted knockdown of the nuclear-retained metastasis associated lung adenocarcinoma transcript 1 (Malat1), a key oncogenic lncRNA involved in metastasis of several cancers. Utilizing the liposomal spherical nucleic acid (LSNA) constructs, the authors first explored the delivery of antisense oligonucleotides to the nucleus. A dose-dependent inhibition of Malat1 upon LSNA treatment as well as the consequent up-regulation of tumor suppressor messenger RNA associated with Malat1 knockdown are shown. These findings reveal the biologic and therapeutic potential of a LSNA-based antisense strategy in targeting disease-associated, nuclear-retained lncRNAs.

Melanoma is one of the most common skin malignancies. Both microRNAs and long non-coding RNAs (lncRNAs) have critical roles in the progression of cancers, including melanoma. However, the underlying molecular mechanism has not been fully characterized. We demonstrated that miR-34a is negatively correlated with MALAT1 in melanoma cells and tumor specimens. Interestingly, MALAT1, which contains functional sequence-specific miR-34a-binding sites, regulates miR-34a stability in melanoma cells and in vivo. Importantly, MALAT1 was significantly enriched in the Ago2 complex, but not when the MALAT1-binding site of miR-34a was mutated. Furthermore, MALAT1 could be shown to regulate c-Myc and Met expression by functioning as a miR-34a sponge. Our results reveal an unexpected mode of action for MALAT1 as an important regulator of miR-34a.

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.

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.

Abstract

BACKGROUND:

Non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), are well-recognized post-transcriptional regulators of gene expression. This study examines the expression of microRNA-21 (miR-21) and lncRNA MALAT1 in medullary thyroid carcinomas (MTCs) and their effects on tumor behavior.

METHODS:

Tissue microarrays (TMAs) were constructed using normal thyroid (n=39), primary tumors (N=39) and metastatic MTCs (N=18) from a total of 42 MTC cases diagnosed between 1987 and 2016. In situ hybridization with probes for miR-21 and MALAT1 was performed. PCR quantification of expression was performed in a subset of normal thyroid (N=10) and primary MTCs (N=32). An MTC-derived cell line (MZ-CRC-1) was transfected with small interfering RNAs (siRNAs) targeting miR-21 and MALAT1 to determine the effects on cell proliferation and invasion.

RESULTS:

In situ hybridization (ISH) showed strong (2+ to 3+) expression of miR-21 in 17 (44%) primary MTCs and strong MALAT1 expression in 37 (95%) primary MTCs. Real-time PCR expression of miR-21 (P<0.001) and MALAT1 (P=0.038) in primary MTCs were significantly higher than in normal thyroid, supporting the ISH findings. Experiments with siRNAs showed inhibition of miR-21 and MALAT1 expression in the MTC-derived cell line, leading to significant decreases in cell proliferation (P<0.05) and invasion (P<0.05).

CONCLUSION:

There is increased expression of miR-21 and MALAT1 in MTCs. This study also showed an in vitro pro-oncogenic effect of MALAT1 and miR-21 in MTCs. The results suggest that overexpression of miR-21 and MALAT1 may regulate MTC progression.

Parathyroid adenomas are slow growing benign neoplasms associated with hypercalcemia, while atypical parathyroid adenomas and parathyroid carcinomas are uncommon tumors and their histologic features may overlap with parathyroid adenomas. LncRNAs participate in transcription and in epigenetic or post-transcriptional regulation of gene expression, and probably contribute to carcinogenesis. We analyzed a group of normal, hyperplastic, and neoplastic parathyroid lesions to determine the best immunohistochemical markers to characterize these lesions and to determine the role of selected lncRNAs in tumor progression. A tissue microarray consisting of 111 cases of normal parathyroid (n = 14), primary hyperplasia (n = 15), secondary hyperplasia (n = 10), tertiary hyperplasia (n = 11), adenomas (n = 50), atypical adenomas (n = 7), and carcinomas (n = 4) was used. Immunohistochemical staining with antibodies against chromogranin A, synaptophysin, parathyroid hormone, and insulinoma-associated protein 1(INSM1) was used. Expression of lncRNAs including metastasis-associated lung adenocarcinoma transcript one (MALAT1), HOX transcript antisense intergenic RNA (HOTAIR), and long intergenic non-protein coding regulator of reprograming (Linc-ROR or ROR) was also analyzed by in situ hybridization and RT-PCR. All of the parathyroid tissues were positive for parathyroid hormone, while most cases were positive for chromogranin A (98%). Synaptophysin was expressed in only 12 cases (11%) and INMS1 was negative in all cases. ROR was significantly downregulated during progression from normal, hyperplastic, and adenomatous parathyroid to parathyroid carcinomas. These results show that parathyroid hormone and chromogranin A are useful markers for parathyroid neoplasms, while synaptophysin and INSM1 are not very sensitive broad-spectrum markers for these neoplasms. LincRNA ROR may function as a tumor suppressor during parathyroid tumor progression.

There is an urgent need for better treatments for chronic pain, which affects more than 1 billion people worldwide. Antisense oligonucleotides (ASOs) have proven successful in treating children with spinal muscular atrophy, a severe infantile neurological disorder, and several compounds based on ASOs are currently being tested in clinical trials for various neurological disorders. Here we characterize the pharmacodynamic activity of ASOs in spinal cord and dorsal root ganglia (DRG), key tissues for pain signaling. We demonstrate that the activity of ASOs lasts up to 2 months after a single intrathecal bolus dose in the spinal cord. Interestingly, comparison of subcutaneous, central intracerebroventricular and intrathecal administration shows DRGs are targetable by systemic and central delivery of ASOs, while target reduction in the spinal cord is achieved only after direct central delivery. Upon detailed characterization of ASO activity in individual cell populations in DRG, we observe robust target suppression in all neuronal populations thereby establishing that ASOs are effective in the cell populations involved in pain propagation. Furthermore, we confirm that ASOs are selective and do not modulate basal pain sensation. We also demonstrate that ASOs targeting the sodium channel Nav1.7 induce sustained analgesia up to 4 weeks. Taken together, our findings support the idea that ASOs possess the required pharmacodynamic properties, along with a long duration of action beneficial for treating pain.