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Alcoholic liver disease is a major cause of chronic liver disease worldwide, and cannabinoid receptor type 1 (CB1R) is involved in a diverse metabolic diseases. B-cell translocation gene 2 (BTG2) and yin yang 1 (YY1) are a potent regulator of biological conditions. Melatonin plays a crucial role in regulating diverse physiological functions and metabolic homeostasis. MicroRNAs are key regulators of various biological processes. Herein, we demonstrate that melatonin improves bile acid synthesis in the liver of alcohol-fed mice by controlling miR-497 expression. The level of bile acid and the expression of Cb1r, Btg2, Yy1, and bile acid synthetic enzymes were significantly elevated in the livers of Lieber-DeCarli alcohol-fed mice. The overexpression of Btg2 enhanced Yy1 gene expression and bile acid production, whereas disrupting the CB1R-BTG2-YY1 cascade protected against the bile acid synthesis caused by alcohol challenge. We identified an alcohol-mediated YY1 binding site on the cholesterol 7α-hydroxylase (Cyp7a1) gene promoter using promoter deletion analysis and chromatin immunoprecipitation assays. Notably, melatonin attenuated the alcohol-stimulated induction of Btg2, Yy1 mRNA levels and bile acid production by promoting miR-497. Overexpression of a miR-497 mimic dramatically diminished the increase of Btg2 and Yy1 gene expression as well as bile acid production by alcohol, whereas this phenomenon was reversed by miR-497 inhibitor. These results demonstrate that the upregulation of miR-497 by melatonin represses alcohol-induced bile acid synthesis by attenuating the BTG2-YY1 signaling pathway. The melatonin-miR497 signaling network may provide novel therapeutic targets for the treatment of hepatic metabolic dysfunction caused by the alcohol-dependent pathway., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

With a dataset of more than 600 million small RNAs deeply sequenced from mouse hippocampal and staged sets of mouse cells that underwent reprogramming to induced pluripotent stem cells, we annotated the stem-loop precursors of the known miRNAs to identify isomoRs (miRNA-offset RNAs), loops, non-preferred strands, and guide strands. Products from both strands were readily detectable for most miRNAs. Changes in the dominant isomiR occurred among the cell types, as did switches of the preferred strand. The terminal nucleotide of the dominant isomiR aligned well with the dominant off-set sequence suggesting that Drosha cleavage generates most miRNA reads without terminal modification. Among the terminal modifications detected, most were non-templated mono- or di-nucleotide additions to the 3'-end. Based on the relative enrichment or depletion of specific nucleotide additions in an Ago-IP fraction there may be differential effects of these modifications on RISC loading. Sequence variation of the two strands at their cleavage sites suggested higher fidelity of Drosha than Dicer. These studies demonstrated multiple patterns of miRNA processing and considerable versatility in miRNA target selection.

Objectives: The contribution of overexpressed microRNA-21 and -221 (miR-21 and miR-221) to the malignant phenotype was determined by inhibiting these miRNAs using antisense oligonucleotides., Methods: The effects of antisense to miR-21 and miR-221 on cell proliferation, cell cycle arrest, induction of apoptosis, combinatorial effects with gemcitabine, and effects on target protein levels were studied., Results: Low nanomolar concentrations of both antisense oligonucleotides reduced proliferation of pancreatic cancer cell lines. Reduced proliferation was less pronounced in the normal ductal epithelial cell line human pancreatic Nestin-expressing cell or in pancreatic cancer cell lines exposed to an irrelevant control oligonucleotide. Inhibition of miR-21 and miR-221 increased the amount of apoptosis in HS766T cells by 3- to 6-fold compared with the control oligonucleotide. HS766T cells exposed to miR-21 antisense resulted in cell cycle arrest (G1 phase). Protein levels of tumor suppressor targets of the miRNAs were increased by antisense to miR-21 (PTEN and RECK) and miR-221 (p27). Antisense to miR-21 and miR-221 sensitized the effects of gemcitabine, and the antisense-gemcitabine combinations were synergistic at high fraction affected., Conclusions: We demonstrate that antisense to miR-21 and miR-221 results in significant cell killing under various conditions and that antisense oligonucleotides targeted to miRNA represents a potential new therapy for pancreatic cancer.

We describe a new method for the in situ detection of a mature microRNA (miRNA) in formalin-fixed, paraffin-embedded tissues. The method involves the labeled extension of miRNA hybridized to an approximately 100-nucleotide-long ultramer template containing the complementary sequence of the miRNA at its 3' terminus. Pretreatment of the tissue involves incubation with protease to expose the genomic DNA to DNase digestion, thereby eliminating the ultramer-independent DNA synthesis process inherent in paraffin-embedded tissue. By direct comparison with real-time reverse transcriptase (RT)-PCR, RT in situ PCR, and standard in situ hybridization using a locked nucleic acid (LNA) probe, it was evident that the ultramer extension method detects only the mature miRNA, is easier to optimize, results generally in a stronger signal, and is much less expensive than the LNA probe method currently used.

microRNAs (miRNAs) are challenging molecules to amplify by PCR because the miRNA precursor consists of a stable hairpin and the mature miRNA is roughly the size of a standard PCR primer. Despite these difficulties, successful real-time RT-PCR technologies have been developed to amplify and quantify both the precursor and mature microRNA. An overview of real-time PCR technologies developed by us to detect precursor and mature microRNAs is presented here. Protocols describe presentation of the data using relative (comparative C(T)) and absolute (standard curve) quantification. Real-time PCR assays were used to measure the time course of precursor and mature miR-155 expression in monocytes stimulated by lipopolysaccharide. Protocols are provided to configure the assays as low density PCR arrays for high throughput gene expression profiling. By profiling over 200 precursor and mature miRNAs in HL60 cells induced to differentiate with 12-O-tetradecanoylphorbol-13-acetate, it was possible to identify miRNAs who's processing is regulated during differentiation. Real-time PCR has become the gold standard of nucleic acid quantification due to the specificity and sensitivity of the PCR. Technological advancements have allowed for quantification of miRNA that is of comparable quality to more traditional RNAs.

Real-time PCR is presently the gold standard of gene expression quantification. Configuration of real-time PCR instruments with 384-well reaction blocks, enables the instrument to be used essentially as a low-density array. While PCR will never rival the throughput of microchip arrays, in situations where one is interested in assaying several hundreds of genes, high throughput, real-time PCR is an excellent alternative to microchip arrays. By combining SYBR green detection and 5 microL reaction volume, the associated costs of high-throughput real-time PCR are comparable to microarrays. Described here is a complete protocol to perform real-time PCR in a 384-well configuration. Examples are provided to access numerous PCR primer sequences that may be used for high-throughput real-time PCR. Methods of analysis are described to present real-time PCR data as heat maps and clustered similar to the presentation of cDNA microarray data. An example is provided to profile the expression of over 200 microRNA precursors using high-throughput real-time PCR.

Very little is known regarding regulation of microRNA (miRNA) biogenesis in normal tissues, tumors, and cell lines. Here, we profiled the expression of 225 precursor and mature miRNAs using real-time PCR and compared the expression levels to determine the processing patterns. RNA from 22 different human tissues, 37 human cancer cell lines, and 16 pancreas and liver tissues/tumors was profiled. The relationship between precursor and mature miRNA expression fell into the following four categories: (1) a direct correlation exists between the precursor and mature miRNA expression in all cells/tissues studied; (2) direct correlation of the precursor and mature miRNA exists, yet the expression is restricted to specific cell lines or tissues; (3) there is detectable expression of mature miRNA in certain cells and tissues while the precursor is expressed in all or most cells/tissues; or (4) both precursor and mature miRNA are not expressed. Pearson correlation between the precursor and mature miRNA expression was closer to one for the tissues but was closer to zero for the cell lines, suggesting that processing of precursor miRNAs is reduced in cancer cell lines. By using Northern blotting, we show that many of these miRNAs (e.g., miR-31, miR-105 and miR-128a) are processed to the precursor, but in situ hybridization analysis demonstrates that these miRNA precursors are retained in the nucleus. We provide a database of the levels of precursor and mature miRNA in a variety of cell types. Our data demonstrate that a large number of miRNAs are transcribed but are not processed to the mature miRNA.

Background: MicroRNAs (miRNA) are small non-coding RNAs that regulate translation of mRNA and protein. Loss or enhanced expression of miRNAs is associated with several diseases, including cancer. However, the identification of circulating miRNA in healthy donors is not well characterized. Microvesicles, also known as exosomes or microparticles, circulate in the peripheral blood and can stimulate cellular signaling. In this study, we hypothesized that under normal healthy conditions, microvesicles contain miRNAs, contributing to biological homeostasis., Methodology/principal Findings: Microvesicles were isolated from the plasma of normal healthy individuals. RNA was isolated from both the microvesicles and matched mononuclear cells and profiled for 420 known mature miRNAs by real-time PCR. Hierarchical clustering of the data sets indicated significant differences in miRNA expression between peripheral blood mononuclear cells (PBMC) and plasma microvesicles. We observed 71 miRNAs co-expressed between microvesicles and PBMC. Notably, we found 33 and 4 significantly differentially expressed miRNAs in the plasma microvesicles and mononuclear cells, respectively. Prediction of the gene targets and associated biological pathways regulated by the detected miRNAs was performed. The majority of the miRNAs expressed in the microvesicles from the blood were predicted to regulate cellular differentiation of blood cells and metabolic pathways. Interestingly, a select few miRNAs were also predicted to be important modulators of immune function., Conclusions: This study is the first to identify and define miRNA expression in circulating plasma microvesicles of normal subjects. The data generated from this study provides a basis for future studies to determine the predictive role of peripheral blood miRNA signatures in human disease and will enable the definition of the biological processes regulated by these miRNA.

microRNAs are functional, 22 nt, noncoding RNAs that negatively regulate gene expression. Disturbance of microRNA expression may play a role in the initiation and progression of certain diseases. A microRNA expression signature has been identified that is associated with pancreatic cancer. This has been accomplished with the application of real-time PCR profiling of over 200 microRNA precursors on specimens of human pancreatic adenocarcinoma, paired benign tissue, normal pancreas, chronic pancreatitis and nine pancreatic cancer cell lines. Hierarchical clustering was able to distinguish tumor from normal pancreas, pancreatitis and cell lines. The PAM algorithm correctly classified 28 of 28 tumors, 6 of 6 normal pancreas and 11 of 15 adjacent benign tissues. One hundred microRNA precursors were aberrantly expressed in pancreatic cancer or desmoplasia (p < 0.01), including microRNAs previously reported as differentially expressed in other human cancers (miR-155, miR-21, miR-221 and miR-222) as well as those not previously reported in cancer (miR-376a and miR-301). Most of the top aberrantly expressed miRNAs displayed increased expression in the tumor. Expression of the active, mature microRNA was validated using a real-time PCR assay to quantify the mature microRNA and Northern blotting. Reverse transcription in situ PCR showed that three of the top differentially expressed miRNAs (miR-221, -376a and -301) were localized to tumor cells and not to stroma or normal acini or ducts. Aberrant microRNA expression may offer new clues to pancreatic tumorigenesis and may provide diagnostic biomarkers for pancreatic adenocarcinoma., (Copyright 2006 Wiley-Liss, Inc.)

A large number of studies have demonstrated that the expression of the angiotensin II type 1 receptor (AT(1)R) is regulated predominantly by post-transcriptional mechanisms. Recently, it has been suggested that 10% of human genes may be regulated, in part, by a novel post-transcriptional mechanism involving microRNAs (miRNAs). miRNAs are small RNAs that regulate gene expression primarily through translational repression. The aim of this study was to determine whether miRNAs could regulate human AT(1)R expression. Luciferase reporter assays demonstrated that miR-155 could directly interact with the 3'-untranslated region of the hAT(1)R mRNA. Functional studies demonstrated that transfection of miR-155 into human primary lung fibroblasts (hPFBs) reduced the endogenous expression of the hAT(1)R compared with non-transfected cells. Additionally, miR-155 transfected cells showed a significant reduction in angiotensin II-induced extracellular signal-related kinase 1/2 (ERK1/2) activation. Furthermore, when hPFBs were transfected with an antisense miR-155 inhibitor, anti-miR-155, endogenous hAT(1)R expression and angiotensin II-induced ERK1/2 activation were significantly increased. Finally, transforming growth factor-beta(1) treatment of hPFBs resulted in the decreased expression of miR-155 and the increased expression of the hAT(1)R. In summary, our studies suggest that miR-155 can bind to the 3'-untranslated region (UTR) of hAT(1)R mRNAs and translationally repress the expression of this protein in vivo. Importantly, the translational repression mediated by miR-155 can be regulated by physiological stimuli.

Our previous study described a real-time PCR method to quantify microRNA (miRNA) precursors using SYBR green detection [T. D. Schmittgen, J. Jiang, Q. Liu and L. Yang (2004) Nucleic Acids Res., 32, e43]. The present study adapted the assay to a 384-well format and expanded it to include primers to 222 human miRNA precursors. TaqMan minor groove binder probes were used to discriminate nearly identical members of the let-7 family of miRNA isoforms. The miRNA precursor expression was profiled in 32 human cell lines from lung, breast, colorectal, hematologic, prostate, pancreatic, and head and neck cancers. Some miRNA precursors were expressed at similar levels in many of the cell lines, while others were differentially expressed. Clustering analysis of the miRNA precursor expression data revealed that most of the cell lines clustered into their respective tissues from which each cell line was ostensibly derived. miRNA precursor expression by PCR paralleled the mature miRNA expression by northern blotting for most of the conditions studied. Our study provides PCR primer sequences to all of the known human miRNA precursors as of December 2004 and provides a database of the miRNA precursor expression in many commonly used human cancer cell lines.