BAP1 dependent expression of long non-coding RNA NEAT-1 contributes to sensitivity to gemcitabine in cholangiocarcinoma.

BACKGROUND: Genetic alterations in chromatin modulators such as BRCA-1 associated protein-1 (BAP1) are the most frequent genetic alteration in intrahepatic cholangiocarcinomas (CCA). We evaluated the contribution of BAP1 expression on tumor cell behavior and therapeutic sensitivity to identify rationale therapeutic strategies. METHODS: The impact of BAP1 expression on sensitivity to therapeutic agents was evaluated in CCA cells with a 7-fold difference in BAP1 expression (KMBC-low, HuCCT1-high) and genetically engineered haplo-insufficient BAP1 knockout cells. We also identified long non-coding RNA genes associated with loss of BAP1 and their role in therapeutic sensitivity. RESULTS: Sensitivity to gemcitabine was greater in low BAP1 expressing or BAP1 knockout cells compared with the high BAP1 expressing cells or control haplo-insufficient cells respectively. Similar results were observed with TSA, olaparib, b-AP15 but not with GSK126. A differential synergistic effect was observed in combinations of gemcitabine with olaparib or GSK126 in KMBC cells and TSA or bAP15 in HuCCT1 cells, indicating BAP1 dependent target-specific synergism and sensitivity to gemcitabine. A BAP1 dependent alteration in expression of lncRNA NEAT-1 was identified by RT-PCR based lncRNA expression profiling, and an inverse relationship between this lncRNA and BAP1 was observed in analysis of the Tumor Cancer Genome Atlas cholangiocarcinoma dataset. Exogenous modulation of NEAT-1 and/or BAP1 expression altered tumor cell phenotype and modulated sensitivity to gemcitabine. CONCLUSIONS: NEAT-1 is a downstream effector of gemcitabine sensitivity in CCA. The expression of BAP1 is a determinant of sensitivity to therapeutic drugs that can be exploited to enhance responses through combination strategies.

Features of CFTR mRNA and implications for therapeutics development.

Cystic fibrosis (CF) is an autosomal recessive disease impacting ∼100,000 people worldwide. This lethal disorder is caused by mutation of the CF transmembrane conductance regulator (CFTR) gene, which encodes an ATP-binding cassette-class C protein. More than 2,100 variants have been identified throughout the length of CFTR. These defects confer differing levels of severity in mRNA and/or protein synthesis, folding, gating, and turnover. Drug discovery efforts have resulted in recent development of modulator therapies that improve clinical outcomes for people living with CF. However, a significant portion of the CF population has demonstrated either no response and/or adverse reactions to small molecules. Additional therapeutic options are needed to restore underlying genetic defects for all patients, particularly individuals carrying rare or refractory CFTR variants. Concerted focus has been placed on rescuing variants that encode truncated CFTR protein, which also harbor abnormalities in mRNA synthesis and stability. The current mini-review provides an overview of CFTR mRNA features known to elicit functional consequences on final protein conformation and function, including considerations for RNA-directed therapies under investigation. Alternative exon usage in the 5'-untranslated region, polypyrimidine tracts, and other sequence elements that influence splicing are discussed. Additionally, we describe mechanisms of CFTR mRNA decay and post-transcriptional regulation mediated through interactions with the 3'-untranslated region (e.g. poly-uracil sequences, microRNAs). Contributions of synonymous single nucleotide polymorphisms to CFTR transcript utilization are also examined. Comprehensive understanding of CFTR RNA biology will be imperative for optimizing future therapeutic endeavors intended to address presently untreatable forms of CF.

Comparison of miRNA quantitation by Nanostring in serum and plasma samples.

Circulating microRNAs that are associated with specific diseases have garnered much attention for use in diagnostic assays. However, detection of disease-associated miRNA can be affected by several factors such as release of contaminating cellular miRNA during sample collection, variations due to amplification of transcript for detection, or controls used for normalization for accurate quantitation. We analyzed circulating miRNA in serum and plasma samples obtained concurrently from 28 patients, using a Nanostring quantitative assay platform. Total RNA concentration ranged from 32-125 µg/ml from serum and 30-220 µg/ml from plasma. Of 798 miRNAs, 371 miRNAs were not detected in either serum or plasma samples. 427 were detected in either serum or plasma but not both, whereas 151 miRNA were detected in both serum and plasma samples. The diversity of miRNA detected was greater in plasma than in serum samples. In serum samples, the number of detected miRNA ranged from 3 to 82 with a median of 17, whereas in plasma samples, the number of miRNA detected ranged from 25 to 221 with a median of 91. Several miRNA such as miR451a, miR 16-5p, miR-223-3p, and mir25-3p were highly abundant and differentially expressed between serum and plasma. The detection of endogenous and exogenous control miRNAs varied in serum and plasma, with higher levels observed in plasma. Gene expression stability identified candidate invariant microRNA that were highly stable across all samples, and could be used for normalization. In conclusion, there are significant differences in both the number of miRNA detected and the amount of miRNA detected between serum and plasma. Normalization using miRNA with constant expression is essential to minimize the impact of technical variations. Given the challenges involved, ideal candidates for blood based biomarkers would be those that are indifferent to type of body fluid, are detectable and can be reliably quantitated.

MicroRNAs as paracrine signaling mediators in cancers and metabolic diseases.

The contribution of microRNAs to the regulation of mRNA expression during physiological and developmental processes are well-recognized. These roles are being expanded by recent observations that emphasize the capability of miRNA to participate in inter-cellular signaling and communication. Several factors support a functional role for miRNA as mediators of cell-to-cell signaling. miRNA are able to exist within the extracellular milieu or circulation, and their stability and integrity maintained through association with binding proteins or lipoproteins, or through encapsulation within cell-derived membrane vesicles. Furthermore, miRNA can retain functionality and regulate target gene expression following their uptake by recipient cells. In this overview, we review specific examples that will highlight the potential of miRNA to serve as paracrine signaling mediators in metabolic diseases and cancers. Elucidating the mechanisms involved in inter-cellular communication involving miRNA will provide new insights into disease pathogenesis and potential therapeutic opportunities.