Integrated analysis of mutations,miRNA and mRNA expression in glioblastoma

Background  

Glioblastoma arises from complex interactions between a variety of genetic alterations and environmental perturbations. Little attention has been paid to understanding how genetic variations, altered gene expression and microRNA (miRNA) expression are integrated into networks which act together to alter regulation and finally lead to the emergence of complex phenotypes and glioblastoma.     

Integrated miRNA and mRNA expression profiling of mouse mammary tumor models identifies miRNA signatures associated with mammary tumor lineage

Background  

MicroRNAs (miRNAs) are small, non-coding, endogenous RNAs involved in regulating gene expression and protein translation. miRNA expression profiling of human breast cancers has identified miRNAs related to the clinical diversity of the disease and potentially provides novel diagnostic and prognostic tools for breast cancer therapy. In order to further understand the associations between oncogenic drivers and miRNA expression in sub-types of breast cancer, we performed miRNA expression profiling on mammary tumors from eight well-characterized genetically engineered mouse (GEM) models of human breast cancer, including MMTV-H-Ras, -Her2/neu, -c-Myc, -PymT, -Wnt1 and C3(1)/SV40 T/t-antigen transgenic mice, BRCA1 ^fl/fl ;p53 ^+/-;MMTV-cre knock-out mice and the p53 ^fl/fl ;MMTV-cre transplant model.     

Integrated analysis of miRNA and mRNA expression profiles in response to gut microbiota depletion in the abdomens of female Bactrocera dorsalis

Insect gut microbiota has been reported to participate in regulating host multiple biological processes including metabolism and reproduction. However, the corresponding molecular mechanisms remain largely unknown. Recent studies suggest that microRNAs (miRNAs) are involved in complex interactions between the gut microbiota and the host. Here, we used next-generation sequencing technology to characterize miRNA and mRNA expression profiles and construct the miRNA–gene regulatory network in response to gut microbiota depletion in the abdomens of female Bactrocera dorsalis. A total of 3016 differentially expressed genes (DEGs) and 18 differentially expressed miRNAs (DEMs) were identified. Based on the integrated analysis of miRNA and mRNA sequencing data, 229 negatively correlated miRNA–gene pairs were identified from the miRNA–mRNA network. Gene ontology enrichment analysis indicated that DEMs could target several genes involved in the metabolic process, oxidation–reduction process, oogenesis, and insulin signaling pathway. Finally, real-time quantitative polymerase chain reaction further verified the accuracy of RNA sequencing results. In conclusion, our study provides the profiles of miRNA and mRNA expressions under antibiotics treatment and provides an insight into the roles of miRNAs and their target genes in the interaction between the gut microbiota and its host.     

miRNA expression profile during osteogenic differentiation of human adipose-derived stem cells

Human adipose-derived stem cells (hADSC) are capable of differentiating into an osteogenic lineage. It is believed that microRNAs (miRNAs) play important roles in regulating this osteogenic differentiation of human adipose-derived cells, although its molecular mechanism remains unclear. We investigated the miRNA expression profile during osteogenic differentiation of hADSCs, and assessed the roles of involved miRNAs during the osteogenic differentiation. We obtained and cultured human adipose-derived stems cells from donors who underwent elective liposuction or other abdominal surgery at our institution. miRNA expression profiles pre- and post-osteogenic induction were obtained using microarray essay, and differently expressed miRNAs were verified using quantitative real-time polymerase chain reaction (qRT-PCR). The expression of osteogenic proteins was detected using an enzyme-linked immunosorbent assay. Putative targets of the miRNAs were predicted using online software MiRanda, TargetScan, and miRBase. Eight miRNAs were found differently expressed pre- and post-osteogenic induction, among which four miRNAs (miR-17, miR-20a, miR-20b, and miR-106a) were up-regulated and four miRNAs (miR-31, miR-125a-5p, miR-125b, and miR-193a) were down-regulated. qRT-PCR analysis further confirmed the results. Predicted target genes of the differentially expressed miRNAs based on the overlap from three public prediction algorithms: MiRanda, TargetScan, and miRBase Target have the known functions of regulating stem cell osteogenic differentiation, self-renewal, signal transduction, and cell cycle control. We identified a group of miRNAs that may play important roles in regulating hADSC cell differentiation toward an osteoblast lineage. Further study of these miRNAs may elucidate the mechanism of hADSC differentiation into adipose tissue, and thus provide basis for tissue engineering.     

Integrated miRNA and mRNA Expression Profiling in Inflamed Colon of Patients with Ulcerative Colitis

Background

Ulcerative colitis (UC) is associated with differential colonic expression of genes involved in immune response (e.g. IL8) and barrier integrity (e.g. cadherins). MicroRNAs (miRNAs) are regulators of gene expression and are involved in various immune-related diseases. In this study, we investigated (1) if miRNA expression in UC mucosa is altered and (2) if any of these changes correlate with mucosal mRNA expression. Integration of mRNA and miRNA expression profiling may allow the identification of functional links between dysregulated miRNAs and their target mRNA.

Methodology

Colonic mucosal biopsies were obtained from 17 UC (10 active and 7 inactive) patients and 10 normal controls. Total RNA was used to analyze miRNA and mRNA expression via Affymetrix miRNA 2.0 and Affymetrix Human Gene 1.0ST arrays, respectively. Both miRNA and gene expression profiles were integrated by correlation analysis to identify dysregulated miRNAs with their corresponding predicted target mRNA. Microarray data were validated with qRT-PCR. Regulation of IL8 and CDH11 expression by hsa-miR-200c-3p was determined by luciferase reporter assays.

Results

When comparing active UC patients vs. controls, 51 miRNAs and 1543 gene probe sets gave significantly different signals. In contrast, in inactive UC vs. controls, no significant miRNA expression differences were found while 155 gene probe sets had significantly different signals. We then identified potential target genes of the significantly dysregulated miRNAs and genes in active UC vs. controls and found a highly significant inverse correlation between hsa-miR-200c-3p and IL8, an inflammatory marker, and between hsa-miR-200c-3p and CDH11, a gene related to intestinal epithelial barrier function. We could demonstrate that hsa-miR-200c-3p directly regulates IL8 and CDH11 expression.

Conclusion

Differential expression of immune- and barrier-related genes in inflamed UC mucosa may be influenced by altered expression of miRNAs. Integrated analysis of miRNA and mRNA expression profiles revealed hsa-miR-200c-3p for use of miRNA mimics as therapeutics.     

The expression and functions of glycoconjugates in neural stem cells

The mammalian central nervous system is organized by a variety of cells such as neurons and glial cells. These cells are generated from a common progenitor, the neural stem cell (NSC). NSCs are defined as undifferentiated neural cells that are characterized by their high proliferative potential while retaining the capacity for self-renewal and multipotency. Glycoconjugates carrying carbohydrate antigens, including glycoproteins, glycolipids, and proteoglycans, are primarily localized on the plasma-membrane surface of cells and serve as excellent biomarkers at various stages of cellular differentiation. Moreover, they also play important functional roles in determining cell fate such as self-renewal, proliferation, and differentiation. In the present review, we discuss the expression pattern and possible functions of glycoconjugates and carbohydrate antigens in NSCs, with an emphasis on stage-specific embryonic antigen-1, human natural killer antigen-1, polysialic acid-neural cell-adhesion molecule, prominin-1, gp130, chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, cystatin C, galectin-1, glycolipids, and Notch.     

Epigenetic regulation of ganglioside expression in neural stem cells and neuronal cells

The structural diversity and localization of cell surface glycosphingolipids (GSLs), including gangliosides, in glycolipid-enriched microdomains (GEMs, also known as lipid rafts) render them ideally suited to play important roles in mediating intercellular recognition, interactions, adhesion, receptor function, and signaling. Gangliosides, sialic acid-containing GSLs, are most abundant in the nerve tissues. The quantity and expression pattern of gangliosides in brain change drastically throughout development and these changes are mainly regulated through stage-specific expression of glycosyltransferase genes. We previously demonstrated for the first time that efficient histone acetylation of the glycosyltransferase genes in mouse brain contributes to the developmental alteration of ganglioside expression. We further demonstrated that acetylation of histones H3 and H4 on the N-acetylgalactosaminyltransferase I (GalNAcT, GA2/GM2/GD2/GT2-synthase; B4galnt1) gene promoter resulted in recruitment of trans-activation factors. In addition, we showed that epigenetic activation of the GalNAcT gene was detected and accompanied by an apparent induction of neuronal differentiation of neural stem cells (NSCs) responding to an exogenous supplement of ganglioside GM1. Most recently, we found that nuclear GM1 binds with acetylated histones on the promoters of the GalNAcT as well as on the NeuroD1 genes in differentiated neurons. Here, we will introduce epigenetic regulation of ganglioside synthase genes in neural development and neuronal differentiation of NSCs.     

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.     

Quantitative analysis of gene expression in living adult neural stem cells by gene trapping

The potential of neural stem cells (NSCs) for the treatment of neurodegenerative diseases makes the identification and characterization of genes involved in neural stem cell responses therapeutically important. Although technologies exist for measuring gene expression in cells, they often provide only a representative expression profile specific to a stimulus and time. We developed a complementary technology based on a retroviral-vector gene-trap approach that uses beta-lactamase-induced disruption of fluorescence resonance energy transfer in the fluorophore CCF-2/AM. A library of 'tagged' adult rat NSCs was generated by transduction with gene-trap virus produced from a single-integrant packaging cell line that allowed us to quantitatively analyze dynamic gene expression changes in real time in living NSCs. Using this library we identified previously unknown genes regulated by oxidative stress, indomethacin and factors that induce differentiation, and show that one of the trapped genes, Sox6, is sufficient to induce astrocytic differentiation when overexpressed.     

Temporally regulated expression of Cre recombinase in neural stem cells

The use of mouse gene targeting to study molecules important in neural development is oftentimes impaired by early embryonic lethality. In order to address later roles for such molecules, specifically in neural stem cells, we generated transgenic mice that express both the tetracycline-inducible molecule rtTA-M2 and GFP under the control of the neural precursor specific form of nestin. Developmental analysis of these mice demonstrates that GFP expression is exclusive to the neural tube. Adult expression of GFP is seen only in known areas of adult neurogenesis, namely, the subventricular zone and the dentate gyrus. When crossed with a second transgenic mouse (TetOp-Cre) that expresses the Cre recombinase under the control of the tetracycline responsive promotor, we demonstrate temporal induction of Cre in bigenic animals exposed to doxycycline. We further demonstrate the feasibility of this approach by using the ROSA-26 reporter mouse to mediate recombination in neural precursor cells.     

Inducible regulation of GDNF expression in human neural stem cells

Glial cell derived neurotrophic factor (GDNF) holds promises for treating neurodegenerative diseases such as Parkinson’s disease. Human neural stem cells (hNSCs) have proved to be a suitable cell delivery vehicle for the safe and efficient introduction of GDNF into the brain. In this study, we used hNSCs-infected with a lentivirus encoding GDNF and the hygromycin resistance gene as such vehicles. A modified tetracycline operator 7 (tetO7) was inserted into a region upstream of the EF1-α promoter to drive GDNF expression. After hygromycin selection, hNSCs were infected with a lentivirus encoding a KRAB-tetracycline repressor fusion protein (TTS). TTS bound to tetO7 and suppressed the expression of GDNF in hNSCs. Upon administration of doxycycline (Dox) the TTS-tetO7 complex separated and the expression of GDNF resumed. The hNSCs infected with GDNF expressed the neural stem cell specific markers, nestin and sox2, and exhibited no significant change in proliferation rate. However, the rate of apoptosis in hNSCs expressing GDNF was lower compared with normal NSCs in response to actinomycin treatment. Furthermore, a higher percentage of Tuj-1 positive cells were obtained from GDNF-producing NSCs under conditions that induced differentiation compared to control NSCs. The inducible expression of GDNF in hNSCs may provide a system for the controllable delivery of GDNF in patients with neurodegenerative diseases.