miR-7 and miR-218 epigenetically control tumor suppressor genes RASSF1A and Claudin-6 by targeting HoxB3 in breast cancer

Many microRNAs have been implicated as key regulators of cellular growth and differentiation and have been found to dysregulate proliferation in human tumors, including breast cancer. Cancer-linked microRNAs also alter the epigenetic landscape by way of DNA methylation and post-translational modifications of histones. Aberrations in Hox gene expression are important for oncogene or tumor suppressor during abnormal development and malignancy. Although recent studies suggest that HoxB3 is critical in breast cancer, the putative role(s) of microRNAs impinging on HoxB3 is not yet fully understood. In this study, we found that the expression levels of miR-7 and miR-218 were strongly and reversely associated with HoxB3 expression. Stable overexpression of miR-7 and miR-218 was accompanied by reactivation of tumor suppressor genes including RASSF1A and Claudin-6 by means of epigenetic switches in DNA methylation and histone modification, giving rise to inhibition of the cell cycle and clone formation of breast cancer cells. The current study provides a novel link between overexpression of collinear Hox genes and multiple microRNAs in human breast malignancy.     

Ago2 Immunoprecipitation Identifies Predicted MicroRNAs in Human Embryonic Stem Cells and Neural Precursors

Background

MicroRNAs are required for maintenance of pluripotency as well as differentiation, but since more microRNAs have been computationally predicted in genome than have been found, there are likely to be undiscovered microRNAs expressed early in stem cell differentiation.

Methodology/Principal Findings

SOLiD ultra-deep sequencing identified >10⁷ unique small RNAs from human embryonic stem cells (hESC) and neural-restricted precursors that were fit to a model of microRNA biogenesis to computationally predict 818 new microRNA genes. These predicted genomic loci are associated with chromatin patterns of modified histones that are predictive of regulated gene expression. 146 of the predicted microRNAs were enriched in Ago2-containing complexes along with 609 known microRNAs, demonstrating association with a functional RISC complex. This Ago2 IP-selected subset was consistently expressed in four independent hESC lines and exhibited complex patterns of regulation over development similar to previously-known microRNAs, including pluripotency-specific expression in both hESC and iPS cells. More than 30% of the Ago2 IP-enriched predicted microRNAs are new members of existing families since they share seed sequences with known microRNAs.

Conclusions/Significance

Extending the classic definition of microRNAs, this large number of new microRNA genes, the majority of which are less conserved than their canonical counterparts, likely represent evolutionarily recent regulators of early differentiation. The enrichment in Ago2 containing complexes, the presence of chromatin marks indicative of regulated gene expression, and differential expression over development all support the identification of 146 new microRNAs active during early hESC differentiation.     

Nuclear receptors and microRNAs: Who regulates the regulators in neural stem cells?

In this mini-review, we focus on regulatory loops between nuclear receptors and microRNAs, an emerging class of small RNA regulators of gene expression. Evidence supporting interactions between microRNAs and nuclear receptors in the regulation of gene expression networks is discussed in relation to its possible role in neural stem cell self renewal and differentiation. Furthermore, we discuss possible disturbances of the regulatory loops between microRNAs and nuclear receptors in human neurodegenerative disease. Finally, we discuss the possible use of nuclear receptors as pharmacological entry points to regulate neural stem cell self-renewal and differentiation.     

Expression and DNA sequence analysis of a human embryonic skeletal muscle myosin heavy chain gene.

Vertebrate myosin heavy chains (MHC) are represented by multiple genes that are expressed in a spatially and temporally distinct pattern during development. In order to obtain molecular probes for developmentally regulated human MHC isoforms, we used monoclonal antibodies to screen an expression cDNA library constructed from primary human myotube cultures. A 3.4 kb cDNA was isolated that encodes one of the first MHCs to be transcribed in human skeletal muscle development. A portion of the corresponding gene encoding this isoform has also been isolated. Expression of this embryonic MHC is a hallmark of muscle regeneration after birth and is a characteristic marker of human muscular dystrophies. During normal human development, expression is restricted to the embryonic period of development prior to birth. In primary human muscle cell cultures, devoid of other cell types, mRNA accumulation begins as myotubes form, reaches a peak 2 days later and declines to undetectable levels within 10 days. The expression of the protein encoded by the embryonic skeletal MHC gene follows a similar time course, lagging behind the mRNA by approximately two days. Thus, expression of the human embryonic gene is efficiently induced and then repressed in cultured muscle cells, as it is in muscle tissue. The study of the regulation of a human MHC isoform with a central role in muscle development and in muscle regeneration in disease states is therefore amendable to analysis at a molecular level.     

MicroRNA regulation of stem cell fate

MicroRNAs modulate target gene expression and are essential for normal development, but how does this pathway impact cell fate decisions? In this issue of Cell Stem Cell, Ivey et al. (2008) find that muscle-specific microRNAs repress nonmuscle genes to direct embryonic stem cell differentiation to mesoderm and muscle.     

Egr2::Cre Mediated Conditional Ablation of Dicer Disrupts Histogenesis of Mammalian Central Auditory Nuclei

Histogenesis of the auditory system requires extensive molecular orchestration. Recently, Dicer1, an essential gene for generation of microRNAs, and miR-96 were shown to be important for development of the peripheral auditory system. Here, we investigated their role for the formation of the auditory brainstem. Egr2::Cre-mediated early embryonic ablation of Dicer1 caused severe disruption of auditory brainstem structures. In adult animals, the volume of the cochlear nucleus complex (CNC) was reduced by 73.5%. This decrease is in part attributed to the lack of the microneuronal shell. In contrast, fusiform cells, which similar to the granular cells of the microneural shell are derived from Egr2 positive cells, were still present. The volume reduction of the CNC was already present at birth (67.2% decrease). The superior olivary complex was also drastically affected in these mice. Nissl staining as well as Vglut1 and Calbindin 1 immunolabeling revealed that principal SOC nuclei such as the medial nucleus of the trapezoid body and the lateral superior olive were absent. Only choline acetyltransferase positive neurons of the olivocochlear bundle were observed as a densely packed cell group in the ventrolateral area of the SOC. Mid-embryonic ablation of Dicer1 in the ventral cochlear nucleus by Atoh7::Cre-mediated recombination resulted in normal formation of the cochlear nucleus complex, indicating an early embryonic requirement of Dicer1. Quantitative RT-PCR analysis of miR-96 demonstrated low expression in the embryonic brainstem and up-regulation thereafter, suggesting that other microRNAs are required for proper histogenesis of the auditory brainstem. Together our data identify a critical role of Dicer activity during embryonic development of the auditory brainstem.     

Identification and Validation of Human Papillomavirus Encoded microRNAs

We report here identification and validation of the first papillomavirus encoded microRNAs expressed in human cervical lesions and cell lines. We established small RNA libraries from ten human papillomavirus associated cervical lesions including cancer and two human papillomavirus harboring cell lines. These libraries were sequenced using SOLiD 4 technology. We used the sequencing data to predict putative viral microRNAs and discovered nine putative papillomavirus encoded microRNAs. Validation was performed for five candidates, four of which were successfully validated by qPCR from cervical tissue samples and cell lines: two were encoded by HPV 16, one by HPV 38 and one by HPV 68. The expression of HPV 16 microRNAs was further confirmed by in situ hybridization, and colocalization with p16INK4A was established. Prediction of cellular target genes of HPV 16 encoded microRNAs suggests that they may play a role in cell cycle, immune functions, cell adhesion and migration, development, and cancer. Two putative viral target sites for the two validated HPV 16 miRNAs were mapped to the E5 gene, one in the E1 gene, two in the L1 gene and one in the LCR region. This is the first report to show that papillomaviruses encode their own microRNA species. Importantly, microRNAs were found in libraries established from human cervical disease and carcinoma cell lines, and their expression was confirmed in additional tissue samples. To our knowledge, this is also the first paper to use in situ hybridization to show the expression of a viral microRNA in human tissue.     

Functions of Noncoding RNAs in Neural Development and Neurological Diseases

The development of the central nervous system (CNS) relies on precisely orchestrated gene expression regulation. Dysregulation of both genetic and environmental factors can affect proper CNS development and results in neurological diseases. Recent studies have shown that similar to protein coding genes, noncoding RNA molecules have a significant impact on normal CNS development and on causes and progression of human neurological disorders. In this review, we have highlighted discoveries of functions of noncoding RNAs, in particular microRNAs and long noncoding RNAs, in neural development and neurological diseases. Emerging evidence has shown that microRNAs play an essential role in many aspects of neural development, such as proliferation of neural stem cells and progenitors, neuronal differentiation, maturation, and synaptogenesis. Misregulation of microRNAs is associated with some mental disorders and neurodegeneration diseases. In addition, long noncoding RNAs are found to play a role in neural development by regulating the expression of protein coding genes. Therefore, examining noncoding RNA-mediated gene regulations has revealed novel mechanisms of neural development and provided new insights into the etiology of human neurological diseases.     

MicroRNA in cell differentiation and development

The regulation of gene expression by microRNAs (miRNAs) is a recently discovered pattern of gene regulation in animals and plants. MiRNAs have been implicated in various aspects of animal development and cell differentiation, such as early embryonic development, neuronal development, muscle development, and lymphocyte development, by the analysis of genetic deletions of individual miRNAs in mammals. These studies show that miRNAs are key regulators in animal development and are potential causes of human diseases. Here we review some recent discoveries about the functions of miRNAs in cell differentiation and development. Supported by National Key Basic Research and Development Program of China (Grant No. 2005CB724602) and Knowledge Innovation Project of the Chinese Academy of Sciences (Grant Nos. KSCX2-YW-R-096, KSCX1-YW-R-64)     

Novel Pancreatic Endocrine Maturation Pathways Identified by Genomic Profiling and Causal Reasoning

We have used a previously unavailable model of pancreatic development, derived in vitro from human embryonic stem cells, to capture a time-course of gene, miRNA and histone modification levels in pancreatic endocrine cells. We investigated whether it is possible to better understand, and hence control, the biological pathways leading to pancreatic endocrine formation by analysing this information and combining it with the available scientific literature to generate models using a casual reasoning approach. We show that the embryonic stem cell differentiation protocol is highly reproducible in producing endocrine precursor cells and generates cells that recapitulate many aspects of human embryonic pancreas development, including maturation into functional endocrine cells when transplanted into recipient animals. The availability of whole genome gene and miRNA expression data from the early stages of human pancreatic development will be of great benefit to those in the fields of developmental biology and diabetes research. Our causal reasoning algorithm suggested the involvement of novel gene networks, such as NEUROG3/E2F1/KDM5B and SOCS3/STAT3/IL-6, in endocrine cell development We experimentally investigated the role of the top-ranked prediction by showing that addition of exogenous IL-6 could affect the expression of the endocrine progenitor genes NEUROG3 and NKX2.2.     

Prediction of Bacterial microRNAs and possible targets in human cell transcriptome

Recent studies have examined gene transfer from bacteria to humans that would result in vertical inheritance. Bacterial DNA appears to integrate into the human somatic genome through an RNA intermediate, and such integrations are detected more frequently in tumors than normal samples and in RNA than DNA samples. Also, vertebrate viruses encode products that interfere with the RNA silencing machinery, suggesting that RNA silencing may indeed be important for antiviral responses in vertebrates. RNA silencing in response to virus infection could be due to microRNAs encoded by either the virus or the host. We hypothesized that bacterial expression of RNA molecules with secondary structures is potentially able to generate miRNA molecules that can interact with the human host mRNA during bacterial infection. To test this hypothesis, we developed a pipelinebased bioinformatics approach to identify putative micro-RNAs derived from bacterial RNAs that may have the potential to regulate gene expression of the human host cell. Our results suggest that 68 bacterial RNAs predicted from 37 different bacterial genomes have predicted secondary structures potentially able to generate putative microRNAs that may interact with messenger RNAs of genes involved in 47 different human diseases. As an example, we examined the effect of transfecting three putative microRNAs into human embryonic kidney 293 (HEK293) cells. The results show that the bacterially derived microRNA sequence can significantly regulate the expression of the respective target human gene. We suggest that the study of these predicted microRNAs may yield important clues as to how the human host cell processes involved in human diseases like cancer, diabetes, rheumatoid arthritis, and others may respond to a particular bacterial environment.     

MicroRNA let-7b targets important cell cycle molecules in malignant melanoma cells and interferes with anchorage-independent growth

A microRNA expression screen was performed analyzing 157 different microRNAs in laser-microdissected tissues from benign melanocytic nevi (n = 10) and primary malignant melanomas (n = 10), using quantitative real-time PCR. Differential expression was found for 72 microRNAs. Members of the let-7 family of microRNAs were significantly downregulated in primary melanomas as compared with benign nevi, suggestive for a possible role of these molecules as tumor suppressors in malignant melanoma. Interestingly, similar findings had been described for lung and colon cancer. Overexpression of let-7b in melanoma cells in vitro downregulated the expression of cyclins D1, D3, and A, and cyclin-dependent kinase (Cdk) 4, all of which had been described to play a role in melanoma development. The effect of let-7b on protein expression was due to targeting of 3'-untranslated regions (3'UTRs) of individual mRNAs, as exemplified by reporter gene analyses for cyclin D1. In line with its downmodulating effects on cell cycle regulators, let-7b inhibited cell cycle progression and anchorage-independent growth of melanoma cells. Taken together, these findings not only point to new regulatory mechanisms of early melanoma development, but also may open avenues for future targeted therapies of this tumor.