Discovery of microRNAs and other small RNAs in solid tumors.

MicroRNAs (miRNAs) are ∼22-nt long, non-coding RNAs that regulate gene silencing. It is known that many human miRNAs are deregulated in numerous types of tumors. Here we report the sequencing of small RNAs (17-25 nt) from 23 breast, bladder, colon and lung tumor samples using high throughput sequencing. We identified 49 novel miRNA and miR-sized small RNAs. We further validated the expression of 10 novel small RNAs in 31 different types of blood, normal and tumor tissue samples using two independent platforms, namely microarray and RT-PCR. Some of the novel sequences show a large difference in expression between tumor and tumor-adjacent tissues, between different tumor stages, or between different tumor types. We also report the identification of novel small RNA classes in human: highly expressed small RNA derived from Y-RNA and endogenous siRNA. Finally, we identified dozens of new miRNA sequence variants that demonstrate the existence of miRNA-related SNP or post-transcriptional modifications. Our work extends the current knowledge of the tumor small RNA transcriptome and provides novel candidates for molecular biomarkers and drug targets.

PKCepsilon mediates glucose-regulated insulin production in pancreatic beta-cells.

Endocrine cells produce large amounts of one or more peptides. The post-translational control of selective production of a single protein is often unknown. We used 3 unrelated approaches to diminish PKCepsilon in rat islets to evaluate its role in preferential glucose-mediated insulin production. Transfection with siRNA (siR-PKCepsilon) or expression of inactive PKCepsilon (PKCepsilon-KD) resulted in a significant reduction in insulin response to glucose (16.7 mmol/l). Glucose stimulation resulted in concentration of PKCepsilon in the perinuclear region, an area known to be rich in ER-Golgi systems, associated with insulin-containing structures. ss'COP1 (RACK2) is the anchoring protein for PKCepsilon. Glucose-stimulated proinsulin production was diminished by 50% in islets expressing PKCepsilon-KD, and 60% in islets expressing RACK2 binding protein (epsilonV1-2); total protein biosynthesis was not affected. In islets expressing epsilonV1-2, a chase period following glucose stimulus resulted in a reduced proinsulin conversion to mature insulin. We propose that PKCepsilon plays a specific role in mediating the glucose-signal into insulin production: binding to ss'COP1 localizes the activated enzyme to the RER where it modulates the shuttling of proinsulin to the TGN. Subsequently the enzyme may be involved in anterograde trafficking of the prohormone or in its processing within the TGN.

Diminished phosphodiesterase-8B potentiates biphasic insulin response to glucose.

cAMP activates multiple signal pathways, crucial for the pancreatic beta-cells function and survival and is a major potentiator of insulin release. A family of phosphodiesterases (PDEs) terminate the cAMP signals. We examined the expression of PDEs in rat beta-cells and their role in the regulation of insulin response. Using RT-PCR and Western blot analyses, we identified PDE3A, PDE3B, PDE4B, PDE4D, and PDE8B in rat islets and in INS-1E cells and several possible splice variants of these PDEs. Specific depletion of PDE3A with small interfering (si) RNA (siPDE3A) led to a small (67%) increase in the insulin response to glucose in INS-1E cells but not rat islets. siPDE3A had no effect on the glucagon-like peptide-1 (10 nmol/liter) potentiated insulin response in rat islets. Depletion in PDE8B levels in rat islets using similar technology (siPDE8B) increased insulin response to glucose by 70%, the potentiation being of similar magnitude during the first and second phase insulin release. The siPDE8B-potentiated insulin response was further increased by 23% when glucagon-like peptide-1 was included during the glucose stimulus. In conclusion, PDE8B is expressed in a small number of tissues unrelated to glucose or fat metabolism. We propose that PDE8B, an 3-isobutyl-1-methylxanthine-insensitive cAMP-specific phosphodiesterase, could prove a novel target for enhanced insulin response, affecting a specific pool of cAMP involved in the control of insulin granule trafficking and exocytosis. Finally, we discuss evidence for functional compartmentation of cAMP in pancreatic beta-cells.

hsa-miR-191 is a candidate oncogene target for hepatocellular carcinoma therapy.

Hepatocellular carcinoma (HCC) is generally a fatal disease due to a paucity of effective treatment options. The identification of oncogenic microRNAs that exert pleiotropic effects in HCC cells may offer new therapeutic targets. In this study, we have identified the human microRNA miR-191 as a potential target for HCC therapy. Inhibition of miR-191 decreased cell proliferation and induced apoptosis in vitro and significantly reduced tumor masses in vivo in an orthotopic xenograft mouse model of HCC. Additionally, miR-191 was found to be upregulated by a dioxin, a known liver carcinogen, and was found to be a regulator of a variety of cancer-related pathways. Our findings offer a preclinical proof of concept for miR-191 targeting as a rational strategy to pursue for improving HCC treatment.

Accurate molecular classification of renal tumors using microRNA expression.

Subtypes of renal tumors have different genetic backgrounds, prognoses, and responses to surgical and medical treatment, and their differential diagnosis is a frequent challenge for pathologists. New biomarkers can help improve the diagnosis and hence the management of renal cancer patients. We extracted RNA from 71 formalin-fixed paraffin-embedded (FFPE) renal tumor samples and measured expression of more than 900 microRNAs using custom microarrays. Clustering revealed similarity in microRNA expression between oncocytoma and chromophobe subtypes as well as between conventional (clear-cell) and papillary tumors. By basing a classification algorithm on this structure, we followed inherent biological correlations and could achieve accurate classification using few microRNAs markers. We defined a two-step decision-tree classifier that uses expression levels of six microRNAs: the first step uses expression levels of hsa-miR-210 and hsa-miR-221 to distinguish between the two pairs of subtypes; the second step uses either hsa-miR-200c with hsa-miR-139-5p to identify oncocytoma from chromophobe, or hsa-miR-31 with hsa-miR-126 to identify conventional from papillary tumors. The classifier was tested on an independent set of FFPE tumor samples from 54 additional patients, and identified correctly 93% of the cases. Validation on qRT-PCR platform demonstrated high correlation with microarray results and accurate classification. MicroRNA expression profiling is a very effective molecular bioassay for classification of renal tumors and can offer a quantitative standardized complement to current methods of tumor classification.

Body size-related variation in Pigment Dispersing Factor-immunoreactivity in the brain of the bumblebee Bombus terrestris (Hymenoptera, Apidae).

Large bumblebee (Bombus terrestris) workers typically visit flowers to collect pollen and nectar during the day and rest in the nest at night. Small workers are less likely to forage, but instead stay in the nest and tend brood around the clock. Because Pigment Dispersing Factor (PDF) has been identified as a neuromodulator in the circadian network of insects, we used an antiserum that recognizes this peptide to compare patterns of PDF-immunoreactivity (PDF-ir) in the brains of large and small workers. Our study provides the first description of PDF distribution in the bumblebee brain, and shows a pattern that is overall similar to that of the honey bee, Apis mellifera. The brains of large bumblebee workers contained a slightly but significantly higher number of PDF-ir neurons than did the brains of small sister bees. Body size was positively correlated with area of the PDF-ir somata and negatively correlated with the maximal staining intensity. These results provide a neuronal correlate to the previously reported body size-associated variation in behavioral circadian rhythmicity. These differences in PDF-ir are consistent with the hypothesis that body size-based division of labor in bumblebees is associated with adaptations of the morphology and function of the brain circadian system.

Diagnostic assay based on hsa-miR-205 expression distinguishes squamous from nonsquamous non-small-cell lung carcinoma.

PURPOSE: Recent advances in treatment of lung cancer require greater accuracy in the subclassification of non-small-cell lung cancer (NSCLC). Targeted therapies which inhibit tumor angiogenesis pose higher risk for adverse response in cases of squamous cell carcinoma. Interobserver variability and the lack of specific, standardized assays limit the current abilities to adequately stratify patients for such treatments. In this study, we set out to identify specific microRNA biomarkers for the identification of squamous cell carcinoma, and to use such markers for the development of a standardized assay. PATIENTS AND METHODS: High-throughput microarray was used to measure microRNA expression levels in 122 adenocarcinoma and squamous NSCLC samples. A quantitative real-time polymerase chain reaction (qRT-PCR) platform was used to verify findings in an independent set of 20 NSCLC formalin-fixed, paraffin-embedded (FFPE) samples, and to develop a diagnostic assay using an additional set of 27 NSCLC FFPE samples. The assay was validated using an independent blinded cohort consisting of 79 NSCLC FFPE samples. RESULTS: We identified hsa-miR-205 as a highly specific marker for squamous cell lung carcinoma. A microRNA-based qRT-PCR assay that measures expression of hsa-miR-205 reached sensitivity of 96% and specificity of 90% in the identification of squamous cell lung carcinomas in an independent blinded validation set. CONCLUSION: Hsa-miR-205 is a highly accurate marker for lung cancer of squamous histology. The standardized diagnostic assay presented here can provide highly accurate subclassification of NSCLC patients.