Beyond the brain-Peripheral kisspeptin signaling is essential for promoting endometrial gland development and function

Uterine growth and endometrial gland formation (adenogenesis) and function, are essential for fertility and are controlled by estrogens and other regulators, whose nature and physiological relevance are yet to be elucidated. Kisspeptin, which signals via Kiss1r, is essential for fertility, primarily through its central control of the hypothalamic-pituitary-ovarian axis, but also likely through peripheral actions. Using genetically modified mice, we addressed the contributions of central and peripheral kisspeptin signaling in regulating uterine growth and adenogenesis. Global ablation of Kiss1 or Kiss1r dramatically suppressed uterine growth and almost fully prevented adenogenesis. However, while uterine growth was fully rescued by E2 treatment of Kiss1(-/-) mice and by genetic restoration of kisspeptin signaling in GnRH neurons in Kiss1r(-/-) mice, functional adenogenesis was only marginally restored. Thus, while uterine growth is largely dependent on ovarian E2-output via central kisspeptin signaling, peripheral kisspeptin signaling is indispensable for endometrial adenogenesis and function, essential aspects of reproductive competence

Small bowel MR enterography: problem solving in Crohn’s disease

Magnetic resonance enterography (MRE) is fast becoming the first-line radiological investigation to evaluate the small bowel in patients with Crohn’s disease. It can demonstrate both mural and extramural complications. The lack of ionizing radiation, together with high-contrast resolution, multiplanar capability and cine-imaging make it an attractive imaging modality in such patients who need prolonged follow-up. A key question in the management of such patients is the assessment of disease activity. Clinical indices, endoscopic and histological findings have traditionally been used as surrogate markers but all have limitations. MRE can help address this question. The purpose of this pictorial review is to (1) detail the MRE protocol used at our institution; (2) describe the rationale for the MR sequences used and their limitations; (3) compare MRE with other small bowel imaging techniques; (4) discuss how MRE can help distinguish between inflammatory, stricturing and penetrating disease, and thus facilitate management of this difficult condition

Quantitative miRNA Expression Analysis Using Fluidigm Microfluidics Dynamic Arrays

MicroRNA (miRNA) is a small non-coding RNA that can regulate gene expression in both plants and animals. Studies showed that miRNAs play a critical role in human cancer by targeting messenger RNAs that are positive or negative regulators of cell proliferation and apoptosis. Here, we evaluated miRNA expression in formalin fixed, paraffin embedded (FFPE) samples and fresh frozen (FF) samples using a high throughput qPCR-based microfluidic dynamic array technology (Fluidigm). We compared the results to hybridization-based microarray platforms using the same samples. We obtained a highly correlated Ct values between multiplex and single-plex RT reactions using standard qPCR assays for miRNA expression. For the same samples, the microfluidic technology (Fluidigm 48.48 dynamic array systems) resulted in a left shift towards lower Ct values compared to those observed by standard TaqMan (ABI 7900HT, mean difference, 3.79). In addition, as little as 10ng total RNA was sufficient to reproducibly detect up to 96 miRNAs at a wide range of expression values using a single 96-multiplexing RT reaction in either FFPE or FF samples. Comparison of miRNAs expression values measured by microfluidic technology with those obtained by other array and Next Generation sequencing platforms showed positive concordance using the same samples but revealed significant differences for a large fraction of miRNA targets. The qPCRarray based microfluidic technology can be used in conjunction with multiplexed RT reactions for miRNA gene expression profiling. This approach is highly reproducible and the results correlate closely with the existing singleplex qPCR platform while achieving much higher throughput at lower sample input and reagent usage. It is a rapid, cost effective, customizable array platform for miRNA expression profiling and validation. However, comparison of miRNA expression using different platforms requires caution and the use of multiple platforms

Identification and analysis of miRNAs in human breast cancer and teratoma samples using deep sequencing

<p>Abstract</p> <p>Background</p> <p>MiRNAs play important roles in cellular control and in various disease states such as cancers, where they may serve as markers or possibly even therapeutics. Identifying the whole repertoire of miRNAs and understanding their expression patterns is therefore an important goal.</p> <p>Methods</p> <p>Here we describe the analysis of 454 pyrosequencing of small RNA from four different tissues: Breast cancer, normal adjacent breast, and two teratoma cell lines. We developed a pipeline for identifying new miRNAs, emphasizing extracting and retaining as much data as possible from even noisy sequencing data. We investigated differential expression of miRNAs in the breast cancer and normal adjacent breast samples, and systematically examined the mature sequence end variability of miRNA compared to non-miRNA loci.</p> <p>Results</p> <p>We identified five novel miRNAs, as well as two putative alternative precursors for known miRNAs. Several miRNAs were differentially expressed between the breast cancer and normal breast samples. The end variability was shown to be significantly different between miRNA and non-miRNA loci.</p> <p>Conclusion</p> <p>Pyrosequencing of small RNAs, together with a computational pipeline, can be used to identify miRNAs in tumor and other tissues. Measures of miRNA end variability may in the future be incorporated into the discovery pipeline as a discriminatory feature. Breast cancer samples show a distinct miRNA expression profile compared to normal adjacent breast.</p

Reduced Expression of Brain-Enriched microRNAs in Glioblastomas Permits Targeted Regulation of a Cell Death Gene

Glioblastoma is a highly aggressive malignant tumor involving glial cells in the human brain. We used high-throughput sequencing to comprehensively profile the small RNAs expressed in glioblastoma and non-tumor brain tissues. MicroRNAs (miRNAs) made up the large majority of small RNAs, and we identified over 400 different cellular pre-miRNAs. No known viral miRNAs were detected in any of the samples analyzed. Cluster analysis revealed several miRNAs that were significantly down-regulated in glioblastomas, including miR-128, miR-124, miR-7, miR-139, miR-95, and miR-873. Post-transcriptional editing was observed for several miRNAs, including the miR-376 family, miR-411, miR-381, and miR-379. Using the deep sequencing information, we designed a lentiviral vector expressing a cell suicide gene, the herpes simplex virus thymidine kinase (HSV-TK) gene, under the regulation of a miRNA, miR-128, that was found to be enriched in non-tumor brain tissue yet down-regulated in glioblastomas, Glioblastoma cells transduced with this vector were selectively killed when cultured in the presence of ganciclovir. Using an in vitro model to recapitulate expression of brain-enriched miRNAs, we demonstrated that neuronally differentiated SH-SY5Y cells transduced with the miRNA-regulated HSV-TK vector are protected from killing by expression of endogenous miR-128. Together, these results provide an in-depth analysis of miRNA dysregulation in glioblastoma and demonstrate the potential utility of these data in the design of miRNA-regulated therapies for the treatment of brain cancers

Prediction of Associations between microRNAs and Gene Expression in Glioma Biology

Despite progress in the determination of miR interactions, their regulatory role in cancer is only beginning to be unraveled. Utilizing gene expression data from 27 glioblastoma samples we found that the mere knowledge of physical interactions between specific mRNAs and miRs can be used to determine associated regulatory interactions, allowing us to identify 626 associated interactions, involving 128 miRs that putatively modulate the expression of 246 mRNAs. Experimentally determining the expression of miRs, we found an over-representation of over(under)-expressed miRs with various predicted mRNA target sequences. Such significantly associated miRs that putatively bind over-expressed genes strongly tend to have binding sites nearby the 3′UTR of the corresponding mRNAs, suggesting that the presence of the miRs near the translation stop site may be a factor in their regulatory ability. Our analysis predicted a significant association between miR-128 and the protein kinase WEE1, which we subsequently validated experimentally by showing that the over-expression of the naturally under-expressed miR-128 in glioma cells resulted in the inhibition of WEE1 in glioblastoma cells

Micro-RNAs as diagnostic or prognostic markers in human epithelial malignancies

Micro-RNAs (miRs) are important regulators of mRNA and protein expression; the ability of miR expression profilings to distinguish different cancer types and classify their sub-types has been well-described. They also represent a novel biological entity with potential value as tumour biomarkers, which can improve diagnosis, prognosis, and monitoring of treatment response for human cancers. This endeavour has been greatly facilitated by the stability of miRs in formalin-fixed paraffin-embedded (FFPE) tissues, and their detection in circulation. This review will summarize some of the key dysregulated miRs described to date in human epithelial malignancies, and their potential value as molecular bio-markers in FFPE tissues and blood samples. There remain many challenges in this domain, however, with the evolution of different platforms, the complexities of normalizing miR profiling data, and the importance of evaluating sufficiently-powered training and validation cohorts. Nonetheless, well-conducted miR profiling studies should contribute important insights into the molecular aberrations driving human cancer development and progression

A Dominant Negative ERβ Splice Variant Determines the Effectiveness of Early or Late Estrogen Therapy after Ovariectomy in Rats

The molecular mechanisms for the discrepancy in outcome of initiating estrogen therapy (ET) around peri-menopause or several years after menopause in women are unknown. We hypothesize that the level of expression of a dominant negative estrogen receptor (ER) β variant, ERβ2, may be a key factor determining the effectiveness of ET in post-menopausal women. We tested this hypothesis in ovariectomized nine month-old (an age when irregular estrous cycles occur) female Sprague Dawley rats. Estradiol treatment was initiated either 6 days (Early ET, analogous to 4 months post-menopause in humans), or 180 days (Late ET, analogous to 11 years post-menopause in humans) after ovariectomy. Although ERβ2 expression increased in all OVX rats, neurogenic and neuroprotective responses to estradiol differed in Early and Late ET. Early ET reduced ERβ2 expression in both hippocampus and white blood cells, increased the hippocampal cell proliferation as assessed by Ki-67 expression, and improved mobility in the forced swim test. Late ET resulted in either no or modest effects on these parameters. There was a close correlation between the degree of ERβ2 expression and the preservation of neural effects by ET after OVX in rats, supporting the hypothesis that persistent elevated levels of ERβ2 are a molecular basis for the diminished effectiveness of ET in late post-menopausal women. The correlation between the expression of ERβ2 in circulating white blood cells and brain cells suggests that ERβ2 expression in peripheral blood cells may be an easily accessible marker to predict the effective window for ET in the brain