Genome-wide screen for aberrantly expressed miRNAs reveals miRNA profile signature in breast cancer

Dysregulation in the expression of miRNAs contributes to the occurrence and development of many human cancers. We herein attempted to obtain the potential association between miRNA expression profile and breast cancer by applying high-throughput sequencing technology. Small RNAs from seven paired tumor and adjacent normal tissue samples were sequenced. To determine the miRNA expression profiles in tissues and sera, another five equally pooled serum samples from 20 patients and 30 normal women were sequenced. Despite a similar number in abundantly expressed miRNAs across samples, we detected varying miRNA expression profiles. Some miRNAs showed inconsistent or opposite dysregulation trends across different tumor tissues, including some abundantly expressed miRNA gene clusters and gene families. Wilcoxon sign-rank test for paired samples analysis revealed that abnormal miRNAs showed a higher level of variation across the seven tumor samples. We also completely surveyed abnormal miRNAs expressed in tumor and serum tissues in the mixed datasets based on the relative expression levels. Most of these miRNAs were significantly down-regulated in tumor samples, but nine abnormal miRNAs (miR-18a, 19a, 20a, 30a, 103b, 126, 126*, 192, 1287) were consistently expressed in tumor tissues and serum samples. Based on experimentally validated target mRNAs, functional enrichment analysis indicated that these abnormal miRNAs and miRNA groups (miRNA gene clusters and gene families) have important roles in multiple biological processes. Dynamic miRNA expression profiles, various abnormal miRNA profiles and complexity of the miRNA regulatory network reveal that the miRNA expression profile is a potential biomarker for classifying or detecting human disease.     

Expression profile analysis of microRNA (miRNA) in mouse central nervous system using a new miRNA detection system that examines hybridization signals at every step of washing

MicroRNAs (miRNAs) are small noncoding RNAs, with a length of 19 to 23 nucleotides, which appear to be involved in the regulation of gene expression by inhibiting the translation of messenger RNA. Expression profile analysis of miRNAs is necessary to understand their complex role in the regulation of gene expression during the development and differentiation of cells and in various tissues. We describe here a detection system for miRNA expression profiles, using a new type of DNA chip and fluorescent labeled cellular RNAs, which allows real-time detection of hybridization signals at every step of washing and results in highly reproducible miRNA expression profiles. Using the system, we investigated the expression profiles of miRNA in the mouse central nervous system (CNS), namely the spinal cord, medulla oblongata, pons, cerebellum, midbrain, diencephalons, and cerebral hemispheres. The results indicated that although the CNS subregions expressed similar miRNA genes, the expression levels of the miRNAs varied among the subregions, suggesting that the CNS subregions specialized for different functions possess different expression profiles of miRNAs.     

Differential expression of microRNA patterns in planarian normal and regenerative tissues

MicroRNAs (miRNAs) are ~22-nt small non-coding RNAs that regulate the expression of specific target genes in many eukaryotes. miRNAs have been shown to play important roles in stem cell maintenance, cell fate determination, and differentiation. Planarians are capable of regenerating entire body plans from tiny fragments; this regenerative capacity is facilitated by a population of pluripotent stem cells known as neoblasts. Planarians have been a classic model system for the study of many aspects of stem cell biology. However, very limited knowledge on miRNA involved in this regulatory mechanism exists. This study profiles the expression of miRNAs in the normal and regenerative tissues of planarians using miRCURY LNA array technology. Thirteen miRNAs showed significant differences in expression between these two tissues. To further confirm our results, we examined the expression of two miRNAs by qRT-PCR. Results show that some known miRNAs may play key roles in the regulatory mechanisms of regeneration. Our findings can be utilized in future research on miRNA function.     

Focus on RNA isolation: obtaining RNA for microRNA (miRNA) expression profiling analyses of neural tissue

MicroRNAs (miRNAs) are present in all known plant and animal tissues and appear to be somewhat concentrated in the mammalian nervous system. Many different miRNA expression profiling platforms have been described. However, relatively little research has been published to establish the importance of 'upstream' variables in RNA isolation for neural miRNA expression profiling. We tested whether apparent changes in miRNA expression profiles may be associated with tissue processing, RNA isolation techniques, or different cell types in the sample. RNA isolation was performed on a single brain sample using eight different RNA isolation methods, and results were correlated using a conventional miRNA microarray and then cross-referenced to Northern blots. Differing results were seen between samples obtained using different RNA isolation techniques and between microarray and Northern blot results. Another complication of miRNA microarrays is tissue-level heterogeneity of cellular composition. To investigate this phenomenon, miRNA expression profiles were determined and compared between highly-purified primary cerebral cortical cell preparations of rat primary E15-E18 neurons versus rat primary E15-E18 astrocytes. Finally, to assess the importance of dissecting human brain gray matter from subjacent white matter in cerebral cortical studies, miRNA expression profiles were compared between gray matter and immediately contiguous white matter. The results suggest that for microarray studies, cellular composition is important, and dissecting white matter from gray matter improves the specificity of the results. Based on these data, recommendations for miRNA expression profiling in neural tissues, and considerations worthy of further study, are discussed.     

Systematic evaluation of microRNA processing patterns in tissues, cell lines, and tumors

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.     

Instability of miRNA and cDNAs derivatives in RNA preparations

Micro RNAs (miRNAs) are small RNA molecules, which function as important regulators of gene expression. We found that RNA preparation methods commonly utilized for miRNA expression studies yield highly unstable miRNAs. We studied the stability of four miRNAs belonging to different miRNAs families. A significant degradation of these molecules may be observed already three days after RNA isolation. Moreover, the respective cDNAs are highly unstable as well. Our findings indicate that instability of miRNAs and their cDNAs should be considered when designing miRNA expression studies.     

MicroRNAs in inflammatory lung disease - master regulators or target practice?

MicroRNAs (miRNAs) have emerged as a class of regulatory RNAs with immense significance in numerous biological processes. When aberrantly expressed miRNAs have been shown to play a role in the pathogenesis of several disease states. Extensive research has explored miRNA involvement in the development and fate of immune cells and in both the innate and adaptive immune responses whereby strong evidence links miRNA expression to signalling pathways and receptors with critical roles in the inflammatory response such as NF-κB and the toll-like receptors, respectively. Recent studies have revealed that unique miRNA expression profiles exist in inflammatory lung diseases such as cystic fibrosis, chronic obstructive pulmonary disease, asthma, idiopathic pulmonary fibrosis and lung cancer. Evaluation of the global expression of miRNAs provides a unique opportunity to identify important target gene sets regulating susceptibility and response to infection and treatment, and control of inflammation in chronic airway disorders. Over 800 human miRNAs have been discovered to date, however the biological function of the majority remains to be uncovered. Understanding the role that miRNAs play in the modulation of gene expression leading to sustained chronic pulmonary inflammation is important for the development of new therapies which focus on prevention of disease progression rather than symptom relief. Here we discuss the current understanding of miRNA involvement in innate immunity, specifically in LPS/TLR4 signalling and in the progression of the chronic inflammatory lung diseases cystic fibrosis, COPD and asthma. miRNA in lung cancer and IPF are also reviewed.     

Naive and primed murine pluripotent stem cells have distinct miRNA expression profiles

Over the last years, the microRNA (miRNA) pathway has emerged as a key component of the regulatory network of pluripotency. Although clearly distinct states of pluripotency have been described in vivo and ex vivo, differences in miRNA expression profiles associated with the developmental modulation of pluripotency have not been extensively studied so far. Here, we performed deep sequencing to profile miRNA expression in naive (embryonic stem cell [ESC]) and primed (epiblast stem cell [EpiSC]) pluripotent stem cells derived from mouse embryos of identical genetic background. We developed a graphical representation method allowing the rapid identification of miRNAs with an atypical profile including mirtrons, a small nucleolar RNA (snoRNA)-derived miRNA, and miRNAs whose biogenesis may differ between ESC and EpiSC. Comparison of mature miRNA profiles revealed that ESCs and EpiSCs exhibit very different miRNA signatures with one third of miRNAs being differentially expressed between the two cell types. Notably, differential expression of several clusters, including miR290-295, miR17-92, miR302/367, and a large repetitive cluster on chromosome 2, was observed. Our analysis also showed that differentiation priming of EpiSC compared to ESC is evidenced by changes in miRNA expression. These dynamic changes in miRNAs signature are likely to reflect both redundant and specific roles of miRNAs in the fine-tuning of pluripotency during development.     

Characterization of MicroRNA Expression Profiles and the Discovery of Novel MicroRNAs Involved in Cancer during Human Embryonic Development

MicroRNAs (miRNAs), approximately 22-nucleotide non-coding RNA molecules, regulate a variety of pivotal physiological or pathological processes, including embryonic development and tumorigenesis. To obtain comprehensive expression profiles of miRNAs in human embryos, we characterized miRNA expression in weeks 4-6 of human embryonic development using miRNA microarrays and identified 50 human-embryo-specific miRNAs (HES-miRNAs). Furthermore, we selected three non-conserved or primate-specific miRNAs, hsa-miR-638, -720, and -1280, and examined their expression levels in various normal and tumor tissues. The results show that expression of most miRNAs is extremely low during early human embryonic development. In addition, the expression of some non-conserved or primate-specific miRNAs is significantly different between tumor and the corresponding normal tissue samples, suggesting that the miRNAs are closely related to the pathological processes of various tumors. This study presents the first comprehensive overview of miRNA expression during human embryonic development and offers immediate evidence of the relationship between human early embryonic development and tumorigenesis.     

Integration of expressed sequence tag data flanking predicted RNA secondary structures facilitates novel non-coding RNA discovery

Many computational methods have been used to predict novel non-coding RNAs (ncRNAs), but none, to our knowledge, have explicitly investigated the impact of integrating existing cDNA-based Expressed Sequence Tag (EST) data that flank structural RNA predictions. To determine whether flanking EST data can assist in microRNA (miRNA) prediction, we identified genomic sites encoding putative miRNAs by combining functional RNA predictions with flanking ESTs data in a model consistent with miRNAs undergoing cleavage during maturation. In both human and mouse genomes, we observed that the inclusion of flanking ESTs adjacent to and not overlapping predicted miRNAs significantly improved the performance of various methods of miRNA prediction, including direct high-throughput sequencing of small RNA libraries. We analyzed the expression of hundreds of miRNAs predicted to be expressed during myogenic differentiation using a customized microarray and identified several known and predicted myogenic miRNA hairpins. Our results indicate that integrating ESTs flanking structural RNA predictions improves the quality of cleaved miRNA predictions and suggest that this strategy can be used to predict other non-coding RNAs undergoing cleavage during maturation.     

Solexa sequencing analysis of chicken pre-adipocyte microRNAs

MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in a variety of biological processes. Studies of miRNAs in mammals suggest that many are involved in lipid metabolism and adipocyte differentiation, but little is known about miRNA expression profiles during chicken adipogenesis. In this study, the Solexa sequencing approach was used to sequence a small RNA library prepared from Arbor Acres broiler pre-adipocytes, and more than 10? short sequence reads were obtained. From these, 159 known chicken miRNAs and 63 novel miRNAs were identified using a bioinformatics approach. Fifty-nine of these miRNA genes were further organized into 27 compact miRNA genomic clusters, and 34 new chicken mirtrons were also discovered, among which there were 27 mirtron candidates. These findings should serve as a foundation for future research on the functional roles of miRNAs in chicken adipocyte differentiation.     

Expression profiles of precursor and mature microRNAs under dehydration and high salinity shock in Populus euphratica

MicroRNAs (miRNAs) are small non-coding RNAs that play vital roles in plant abiotic stress responses via cleavage or translational inhibition of their target mRNAs. Populus euphratica is a typical stress-resistant sessile organism that grows in desert areas. Here, we identified sequences of 12 miRNA precursors from 11 families and 13 mature miRNAs from 12 families by PCR amplification in P. euphratica. To detect expression differences in mature miRNAs and their precursors under dehydration and high salinity shock in P. euphratica, we examined 14 miRNA precursors from 13 miRNA families and 17 mature miRNAs from 17 miRNA families using the SYBR Green RT–PCR assay. This is the first report of expression profiles for both precursor and mature miRNAs in P. euphratica. By profiling both the mature miRNAs and the precursors under abiotic stress shock, it was possible to identify miRNA whose processing is regulated during stress shock environments. A majority of the genes predicted to be targets for plant miRNAs are involved in development, stress resistance and metabolic processes. We have cloned and experimentally identified in vivo five of the predicted target genes and quantified the five target mRNAs from the same RNA sample simultaneously. Based on this study, we propose some regulatory pathways that illustrate the important role that miRNAs play in response to abiotic stress shock in P. euphratica.