Probes for INS

Abstract

Long noncoding RNA (lncRNA) H19 is abundantly expressed in fetal liver. Its expression is significantly diminished in adult healthy liver but is re‐induced in chronic liver diseases, including cholestasis. In this study, we developed a new method with combined in situhybridization (ISH) and immunofluorescence (IF) colabeling to establish an H19 expression profile with both parenchymal and nonparenchymal cell‐specific markers in the livers of cholestatic mouse models and patients with cholestasis. H19RNA+ cells showed no colocalization with biliary epithelial cell marker cytokeratin 19 (CK19)+cholangiocytes but were immediately adjacent to biliary structures in bile duct ligation (BDL), 3,5‐diethoxycarbony1‐1,4‐dihydrocollidine (DDC), and multidrug‐resistant gene 2 knockout (Mdr2–/–) mouse models and in human primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) liver specimens. In contrast, double‐positive H19RNA+/sex‐determining region Y (SRY)‐box 9 (SOX9)+ ductal progenitor cells, H19RNA+/hepatocyte nuclear factor 4α (HNF4α)+ hepatocytes, H19RNA+/F4/80+ Kupffer cells, HNF4α+/SOX9+ hybrid hepatocytes, as well as triple‐positive H19RNA+/HNF4α+/SOX9+ periportal hepatocytes were identified. In addition, H19RNA could not be detected in mesenchymal cell marker desmin+ cells. Furthermore, H19RNA was predominately detected in cytoplasm with a small amount at the interspace with neighboring cells. Conclusion: H19RNA is localized in HNF4α+ periportal hepatocytes, SOX9+ ductal progenitor cells, and F4/80+ Kupffer cells but not in CK19+ cholangiocytes and desmin+ stellate cells in cholestatic livers.

Melatonin is the primary pineal hormone that relays light/dark cycle information to the circadian system. It was recently reported to exert intrinsic antitumor activity in various cancers. However, the regulatory mechanisms underlying the antitumor activity of melatonin are poorly understood. Moreover, a limited number of studies have addressed the role of melatonin in hepatocellular carcinoma (HCC), a major life-threatening malignancy in both sexes in Taiwan. In this study, we investigated the antitumor effects of melatonin in HCC and explored the regulatory mechanisms underlying these effects. We observed that melatonin significantly inhibited the proliferation, migration, and invasion of HCC cells and significantly induced the expression of the transcription factor FOXA2 in HCC cells. This increase in FOXA2 expression resulted in upregulation of lncRNA-CPS1 intronic transcript 1 (CPS1-IT1), which reduced HIF-1α activity and consequently resulted in the suppression of epithelial-mesenchymal transition (EMT) progression and HCC metastasis. Furthermore, the results of the in vivo experiments confirmed that melatonin exerts tumor suppressive effects by reducing tumor growth. In conclusion, our findings suggested that melatonin inhibited HCC progression by reducing lncRNA-CPS1-IT1-mediated EMT suppression and indicated that melatonin could be a promising treatment for HCC.

Oncocytic (Hürthle cell) and follicular neoplasms are related thyroid tumors with distinct molecular profiles. Diagnostic criteria separating adenomas and carcinomas for these two types of neoplasms are similar, but there may be some differences in the biological behavior of Hürthle cell and follicular carcinomas. Recent studies have shown that noncoding RNAs may have diagnostic and prognostic utility in separating benign and malignant Hürthle cell and follicular neoplasms. In this study, we examined expression of various noncoding RNAs including metastasis associated lung adenocarcinoma transcript 1 (MALAT1) and miR-RNA-885-5p (miR-885) in distinguishing between benign and malignant neoplasms. In addition, the expression of phosphorylated mechanistic receptor of rapamycin (p-mTOR) was also analyzed in these two groups of tumors. Tissue microarrays (TMAs) with archived tissue samples were analyzed using in situ hybridization (ISH) for MALAT1 and miR-885 and immunohistochemistry (IHC) for p-mTOR. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was also performed on a subset of the cases.MALAT1 and miR-885 were increased in all neoplastic groups compared to the normal thyroid tissues (p < 0.05). MALAT1 was more highly expressed in HCCs compared to FTCs, although the differences were not statistically significant (p = 0.06). MiR-885 was expressed at similar levels in FTCs and HCCs. P-mTOR protein was more highly expressed in FTCs than in HCCs (p<0.001). qRT-PCR analysis of noncoding RNAs supported the ISH findings. These results indicate that the noncoding RNAs MALAT1 and miR-885 show increased expression in neoplastic follicular and Hürthle cell thyroid neoplasms compared to normal thyroid tissues. P-mTOR was most highly expressed in FTC but was also increased in HCC, suggesting that drugs targeting this pathway may be useful for treatment of tumors unresponsive to conventional therapies.

Mucoepidermoid carcinoma (MEC) arises in many glandular tissues and contributes to the most common malignant salivary gland cancers. MEC is specifically associated with a unique t(11;19) translocation and the resulting CRTC1-MAML2 fusion is a major oncogenic driver for MEC initiation and maintenance. However, the molecular basis underlying the CRTC1-MAML2 oncogenic functions remains elusive. Through gene expression profiling analysis, we observed that LINC00473, a long non-coding RNA (lncRNA), was the top down-regulated target in CRTC1-MAML2-depleted human MEC cells. LncRNAs belong to a new class of non-coding RNAs with emerging roles in tumorigenesis and progression, but remain poorly characterized. In this study, we investigated the role of LINC00473 in mediating CRTC1-MAML2 oncogenic activity in human MEC. We found that LINC00473 transcription was significantly induced in human CRTC1-MAML2-positive MEC cell lines and primary MEC tumors, and was tightly correlated with the CRTC1-MAML2 RNA level. LINC00473 induction was dependent on the ability of CRTC1-MAML2 to activate CREB-mediated transcription. Depletion of LINC00473 significantly reduced the proliferation and survival of human MEC cells in vitro and blocked the in vivo tumor growth in a human MEC xenograft model. RNA in situ hybridization analysis demonstrated a predominantly nuclear localization pattern for LINC00473 in human MEC cells. Furthermore, gene expression profiling revealed that LINC00473 depletion resulted in differential expression of genes important in cancer cell growth and survival. LINC00473 likely regulates gene expression in part through its ability to bind to a cAMP signaling pathway component NONO, enhancing the ability of CRTC1-MAML2 to activate CREB-mediated transcription. Our overall results demonstrate that LINC00473 is a downstream target and an important mediator of the CRTC1-MAML2 oncoprotein. Therefore, LINC00473 acts as a promising biomarker and therapeutic target for human CRTC1-MAML2-positive MECs.

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

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.

Renal cell carcinoma (RCC) is one of the most lethal urologic cancers. About one-third of RCC patients already have distal metastasis at the time of diagnosis. There is growing evidence that Hox antisense intergenic RNA (HOTAIR) plays essential roles in metastasis in several types of cancers. However, the precise mechanism by which HOTAIR enhances malignancy remains unclear, especially in RCC. Here, we demonstrated that HOTAIR enhances RCC-cell migration by regulating the insulin growth factor-binding protein 2 (IGFBP2) expression. HOTAIR expression in tumors was significantly correlated with nuclear grade, lymph-node metastasis, and lung metastasis. High HOTAIR expression was associated with a poor prognosis in both our dataset and The Cancer Genome Atlas dataset. Migratory capacity was enhanced in RCC cell lines in a HOTAIR-dependent manner. HOTAIR overexpression accelerated tumorigenicity and lung metastasis in immunodeficient mice. Microarray analysis revealed that IGFBP2 expression was upregulated in HOTAIR-overexpressing cells compared with control cells. The enhanced migration activity of HOTAIR-overexpressing cells was attenuated by IGFBP2 knockdown. IGFBP2 and HOTAIR were co-expressed in clinical RCC samples. Our findings suggest that the HOTAIR-IGFBP2 axis plays critical roles in RCC metastasis and may serve as a novel therapeutic target for advanced RCC.

Aggressive breast cancer is difficult to treat as it is unresponsive to many hormone-based therapies; therefore, it is imperative to identify novel, targetable regulators of progression. Long non-coding RNAs (lncRNAs) are important regulators in breast cancer and have great potential as therapeutic targets; however, little is known about how the majority of lncRNAs function within breast cancer. This study, characterizes a novel lncRNA, MANCR (mitotically-associated long non-coding RNA; LINC00704), which is upregulated in breast cancer patient specimens and cells. Depletion of MANCR in triple-negative breast cancer (TNBC) cells significantly decreases cell proliferation and viability, with concomitant increases in DNA damage. Transcriptome analysis, based on RNA sequencing (RNA-seq), following MANCR knockdown reveals significant differences in the expression of >2000 transcripts, and gene set enrichment analysis (GSEA) identifies changes in multiple categories related to cell cycle regulation. Furthermore, MANCR expression is highest in mitotic cells by both RT-qPCR and RNA in situ hybridization. Consistent with a role in cell cycle regulation, MANCR-depleted cells have a lower mitotic index and higher incidences of defective cytokinesis and cell death. Taken together, these data reveal a role for the novel lncRNA, MANCR, in genomic stability of aggressive breast cancer, and identify it as a potential therapeutic target.

IMPLICATIONS:

The novel lncRNA, MANCR (LINC00704), is upregulated in breast cancer and is functionally linked with cell proliferation, viability, and genomic stability.

Long non-coding RNAs (lncRNAs) are a diverse and poorly conserved category of transcripts that have expanded greatly in primates, particularly in the brain. We identified an lncRNA, which has acquired 16 microRNA response elements for miR-143-3p in the Catarrhini branch of primates. This lncRNA, termed LncND (neurodevelopment), is expressed in neural progenitor cells and then declines in neurons. Binding and release of miR-143-3p by LncND control the expression of Notch receptors. LncND expression is enriched in radial glia cells (RGCs) in the ventricular and subventricular zones of developing human brain. Downregulation in neuroblastoma cells reduced cell proliferation and induced neuronal differentiation, an effect phenocopied by miR-143-3p overexpression. Gain of function of LncND in developing mouse cortex led to an expansion of PAX6+ RGCs. These findings support a role for LncND in miRNA-mediated regulation of Notch signaling within the neural progenitor pool in primates that may have contributed to the expansion of cerebral cortex.