MicroRNA Expression Differences in Human Hematopoietic Cell Lineages Enable Regulated Transgene Expression

Blood microRNA (miRNA) levels have been associated with and shown to participate in disease pathophysiology. However, the hematopoietic cell of origin of blood miRNAs and the individual blood cell miRNA profiles are poorly understood. We report the miRNA content of highly purified normal hematopoietic cells from the same individuals. Although T-cells, B-cells and granulocytes had the highest miRNA content per cell, erythrocytes contributed more cellular miRNA to the blood, followed by granulocytes and platelets. miRNA profiling revealed different patterns and different expression levels of miRNA specific for each lineage. miR-30c-5p was determined to be an appropriate reference normalizer for cross-cell qRT-PCR comparisons. miRNA profiling of 5 hematopoietic cell lines revealed differential expression of miR-125a-5p. We demonstrated endogenous levels of miR-125a-5p regulate reporter gene expression in Meg-01 and Jurkat cells by (1) constructs containing binding sites for miR-125a-5p or (2) over-expressing or inhibiting miR-125a-5p. This quantitative analysis of the miRNA profiles of peripheral blood cells identifies the circulating hematopoietic cellular miRNAs, supports the use of miRNA profiles for distinguishing different hematopoietic lineages and suggests that endogenously expressed miRNAs can be exploited to regulate transgene expression in a cell-specific manner.     

Stability analysis of liver cancer-related microRNAs

MicroRNAs (miRNAs) are non-coding, single-stranded RNAs of approximately 22 nt and constitute a novel class of gene regulators that are found in both plants and animals. Several studies have demonstrated that serum miRNAs could serve as potential biomarkers for the detection of various cancers and other diseases. A few documents regarding the stability of liver cancer-related miRNAs in serum are available. A systemic analysis of the stability of miRNA in serum is quite necessary. The purpose of this study was to evaluate the stability of miRNAs from three different sources, cultured liver cancer Huh-7 cell line, clinical liver cancer, and serum under different experimental conditions, including different temperature, time duration, pH values, RNase A digestion, DNase I digestion, and various freeze-thaw cycles. The qRT-PCR analysis demonstrated that liver cancer-related miRNAs were detectable under each of test conditions, indicating that miRNAs were extremely stable and resistant to destruction and degradation under harsh environmental conditions. However, ribosomal RNA was fragile and easily degraded by demonstrating sharp decrease of relative expression under the non-physiological test conditions. We also established a robust procedure for serum RNA extraction, which is greatly important not only for the miRNA profiling studies but also for the disease prognosis based on abnormal miRNA expression.     

Identification of candidate microRNA biomarkers in renal fibrosis: a meta-analysis of profiling studies

Abstract

The prognostic, diagnostic and therapeutic value of microRNA (miRNA) expression aberrations in renal fibrosis has been studied in recent years. However, the miRNA expression profiling efforts have led to inconsistent results between the studies. The aim of this study was to perform a meta-analysis on the renal fibrosis miRNA expression profiling studies to identify candidate diagnostic biomarkers. We performed comprehensive literature searches in several databases to identify miRNA expression studies of renal fibrosis in animal models and humans. The miRNAs expression data were extracted from 20 included studies, and both miRNA vote-counting strategy and Robust Rank Aggregation method were utilized to identify significant miRNA meta-signatures. The predicted and validated targets of miRNA meta-signature were obtained by using MultiMiR package in 11 databases. Then a gene set enrichment analysis (KEGG, PANTHER pathways and GO processes) were carried out with GeneCodis web tool to recognize pathways that are most strongly influenced by modified expressions of these miRNAs. We recognized in both meta-analysis approaches a significant miRNA meta-signature of five up-regulated (miR-142-3p, miR-223-3p, miR-21-5p, miR-142-5p and miR-214-3p) and two down-regulated (miR-29c-3p and miR-200a-3p) miRNAs. Enrichment analysis confirmed that miRNA meta-signature cooperatively target functionally related genes in signalling and developmental pathways in renal fibrosis. This meta-analysis identified seven highly significant and consistently dysregulated miRNAs from 20 datasets, as the focus of future investigations to discover their potential influence to renal fibrosis and their clinical utility as biomarkers and/or as therapeutic mediators against chronic kidney disease..     

A dictionary on microRNAs and their putative target pathways

While in the last decade mRNA expression profiling was among the most popular research areas, over the past years the study of non-coding RNAs, especially microRNAs (miRNAs), has gained increasing interest. For almost 900 known human miRNAs hundreds of pretended targets are known. However, there is only limited knowledge about putative systemic effects of changes in the expression of miRNAs and their regulatory influence. We determined for each known miRNA the biochemical pathways in the KEGG and TRANSPATH database and the Gene Ontology categories that are enriched with respect to its target genes. We refer to these pathways and categories as target pathways of the corresponding miRNA. Investigating target pathways of miRNAs we found a strong relation to disease-related regulatory pathways, including mitogen-activated protein kinase (MAPK) signaling cascade, Transforming growth factor (TGF)-beta signaling pathway or the p53 network. Performing a sophisticated analysis of differentially expressed genes of 13 cancer data sets extracted from gene expression omnibus (GEO) showed that targets of specific miRNAs were significantly deregulated in these sets. The respective miRNA target analysis is also a novel part of our gene set analysis pipeline GeneTrail. Our study represents a comprehensive theoretical analysis of the relationship between miRNAs and their predicted target pathways. Our target pathways analysis provides a ‘miRNA-target pathway’ dictionary, which enables researchers to identify target pathways of differentially regulated miRNAs.     

Strategies for profiling microRNA expression

MicroRNAs (miRNAs) are a class of small RNAs ( approximately 22-nt) that play an important role in the control of different cell processes by negative regulation of protein-coding genes. In the last several years, a number of miRNA profiling strategies have been used to document the miRNA expression changes during physiological and pathological processes. Aberrant expression of miRNAs has been linked to developmental defects, cancer, neurological disorders, and heart diseases. Over 540 human miRNAs have been validated to date; however, computer models suggest there may be thousands more. As bench work continue to verify in silico predictions, miRNA profiling will remain a prominent tool for identification of differential expression miRNAs in normal cellular courses and human disorders. This review focuses on current strategies for miRNA expression profiling and discusses their sensitivity and specificity, as well as advantage and disadvantage.     

miRNA profiling for diagnosis and prognosis of human cancer

MicroRNAs (miRNAs) are a recently discovered class of small (approximately 18-24 nt) nucleic acids that negatively regulate gene expression. This novel class of molecules modulates a wide array of growth and differentiation processes in human cancers. High throughput analyses, utilizing the solid phase, array platform, or liquid phase, bead-based hybridization have variously demonstrated that miRNA expression was commonly dysregulated in human cancer. miRNA expression profiling has shown promise in defining malignant status in retrospective studies. Considerable disagreement remains with respect to the miRNA signature for a specific cancer cell type, which appears to depend largely on the analytical platform. Nonetheless, various internally controlled studies have successfully identified the histotype of tumors of unknown origin according to miRNA expression profile. The evaluation of miRNAs expression may also be of prognostic value, as best exemplified by the correlation of let-7 and mir-155 levels with disease survival in nonsmall cell lung cancer.     

Genomics of microRNA

Discovered just over a decade ago, microRNA (miRNA) is now recognized as one of the major regulatory gene families in eukaryotic cells. Hundreds of miRNAs have been found in animals, plants and viruses, and there are certainly more to come. Through specific base-pairing with mRNAs, these tiny approximately 22-nt RNAs induce mRNA degradation or translational repression, or both. Because a miRNA can target numerous mRNAs, often in combination with other miRNAs, miRNAs operate highly complex regulatory networks. In this article, we summarize the current status of miRNA gene mining and miRNA expression profiling. We also review up-to-date knowledge of miRNA gene structure and the biogenesis mechanism. Our focus is on animal miRNAs.     

Comparison of Microarray Platforms for Measuring Differential MicroRNA Expression in Paired Normal/Cancer Colon Tissues

Background

Microarray technology applied to microRNA (miRNA) profiling is a promising tool in many research fields; nevertheless, independent studies characterizing the same pathology have often reported poorly overlapping results. miRNA analysis methods have only recently been systematically compared but only in few cases using clinical samples.

Methodology/Principal Findings

We investigated the inter-platform reproducibility of four miRNA microarray platforms (Agilent, Exiqon, Illumina, and Miltenyi), comparing nine paired tumor/normal colon tissues. The most concordant and selected discordant miRNAs were further studied by quantitative RT-PCR. Globally, a poor overlap among differentially expressed miRNAs identified by each platform was found. Nevertheless, for eight miRNAs high agreement in differential expression among the four platforms and comparability to qRT-PCR was observed. Furthermore, most of the miRNA sets identified by each platform are coherently enriched in data from the other platforms and the great majority of colon cancer associated miRNA sets derived from the literature were validated in our data, independently from the platform. Computational integration of miRNA and gene expression profiles suggested that anti-correlated predicted target genes of differentially expressed miRNAs are commonly enriched in cancer-related pathways and in genes involved in glycolysis and nutrient transport.

Conclusions

Technical and analytical challenges in measuring miRNAs still remain and further research is required in order to increase consistency between different microarray-based methodologies. However, a better inter-platform agreement was found by looking at miRNA sets instead of single miRNAs and through a miRNAs – gene expression integration approach.     

Comprehensive gene and microRNA expression profiling reveals the crucial role of hsa-let-7i and its target genes in colorectal cancer metastasis

Accumulating evidence has demonstrated that miRNAs play important roles in the occurrence and development of colorectal cancer (CRC). However, whether miRNAs are associated with the metastasis of CRC remains largely unexplored. The aim of the current study is to profile miRNAs in different CRC metastatic cell lines to identify the biomarkers in CRC metastasis. Gene and miRNA expression profiling was performed to analyze the global expression of mRNAs and miRNAs in the four human CRC cell lines (LoVo, SW480, HT29 and Caco-2) with different potential of metastasis. Expression patterns of mRNAs and miRNAs were altered in different CRC cell lines. By developing an integrated bioinformatics analysis of gene and miRNA expression patterns, hsa-let-7i was identified to show the highest degree in the microRNA-GO-network and microRNA-Gene-network. The expression level of hsa-let-7i was further validated by qRT-PCR in CRC cells. In addition, the targets of hsa-let-7i were predicted by two programs TargetScan and PicTar, and target genes were validated by expression profiling in the most epresentative LoVo and Caco-2 cell lines. Eight genes including TRIM41, SOX13, SLC25A4, SEMA4F, RPUSD2, PLEKHG6, CCND2, and BTBD3 were identified as hsa-let-7i targets. Our data showed the power of comprehensive gene and miRNA expression profiling and the application of bioinformatics tools in the identification of novel biomarkers in CRC metastasis.     

The role of upregulated miRNAs and the identification of novel mRNA targets in prostatospheres

TICs are characterized by their ability to self-renew, differentiate and initiate tumor formation. miRNAs are small noncoding RNAs that bind to mRNAs resulting in regulation of gene expression and biological functions. The role of miRNAs and TICs in cancer progression led us to hypothesize that miRNAs may regulate genes involved in TIC maintenance. Using whole genome miRNA and mRNA expression profiling of TICs from primary prostate cancer cells, we identified a set of up-regulated miRNAs and a set of genes down-regulated in PSs. Inhibition of these miRNAs results in a decrease of prostatosphere formation and an increase in target gene expression. This study uses genome-wide miRNA profiling to analyze expression in TICs. We connect aberrant miRNA expression and deregulated gene expression in TICs. These findings can contribute to a better understanding of the molecular mechanisms governing TIC development/maintenance and the role that miRNAs have in the fundamental biology of TICs.     

miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample

Background  

MicroRNAs (miRNAs) are small endogenous non-coding interfering RNA molecules regarded as major regulators in eukaryotic gene expression. Different methods are employed for miRNA expression profiling. For a better understanding of their role in essential biological processes, convenient methods for differential miRNA expression analysis are required.     

Normalization strategies for microRNA profiling experiments: a ‘normal’ way to a hidden layer of complexity?

MicroRNA (miRNA) profiling is a first important step in elucidating miRNA functions. Real time quantitative PCR (RT-qPCR) and microarray hybridization approaches as well as ultra high throughput sequencing of miRNAs (small RNA-seq) are popular and widely used profiling methods. All of these profiling approaches face significant introduction of bias. Normalization, often an underestimated aspect of data processing, can minimize systematic technical or experimental variation and thus has significant impact on the detection of differentially expressed miRNAs. At present, there is no consensus normalization method for any of the three miRNA profiling approach. Several normalization techniques are currently in use, of which some are similar to mRNA profiling normalization methods, while others are specifically modified or developed for miRNA data. The characteristic nature of miRNA molecules, their composition and the resulting data distribution of profiling experiments challenges the selection of adequate normalization techniques. Based on miRNA profiling studies and comparative studies on normalization methods and their performances, this review provides a critical overview of commonly used and newly developed normalization methods for miRNA RT-qPCR, miRNA hybridization microarray, and small RNA-seq datasets. Emphasis is laid on the complexity, the importance and the potential for further optimization of normalization techniques for miRNA profiling datasets.