Efficient adeno-associated virus-mediated gene expression in human placenta-derived mesenchymal cells

Mesenchymal cells from various sources are pluripotent and are attractive sources for cell transplantation. In this study, we analyzed recombinant adeno-associated virus (rAAV)-mediated gene expression in human placenta-derived mesenchymal cells (hPDMCs), which reside in placental villi. After transduction of AV-CAG-EGFP, a rAAV expressing enhanced green fluorescence protein (EGFP), hPDMCs showed much higher level of EGFP expression than human umbilical vein endothelial cells or rat aortic smooth muscle cells. The number of EGFP-positive hPDMCs infected by AV-CAG-EGFP alone did not increase significantly by coinfection of adenovirus, which enhanced expression level of the rAAV vector. Moreover, flow cytometric analysis showed discrete positive fraction of EGFP-expressing hPDMCs, which is about 15-20% of the cells infected with AV-CAG-EGFP. Therefore, some cell population in hPDMCs might be highly susceptible to rAAV-mediated gene transduction. In addition, stable EGFP expressions were observed in about 1% of hPDMCs infected with AV-CAG-EGFP at 4 weeks post-infection. Collectively, hPDMCs have characters favorable for rAAV-mediated gene expression.     

microRNA profiling in Epstein-Barr virus-associated B-cell lymphoma

The Epstein–Barr virus (EBV) is an oncogenic human Herpes virus found in ∼15% of diffuse large B-cell lymphoma (DLBCL). EBV encodes miRNAs and induces changes in the cellular miRNA profile of infected cells. MiRNAs are small, non-coding RNAs of ∼19–26 nt which suppress protein synthesis by inducing translational arrest or mRNA degradation. Here, we report a comprehensive miRNA-profiling study and show that hsa-miR-424, -223, -199a-3p, -199a-5p, -27b, -378, -26b, -23a, -23b were upregulated and hsa-miR-155, -20b, -221, -151-3p, -222, -29b/c, -106a were downregulated more than 2-fold due to EBV-infection of DLBCL. All known EBV miRNAs with the exception of the BHRF1 cluster as well as EBV-miR-BART15 and -20 were present. A computational analysis indicated potential targets such as c-MYB, LATS2, c-SKI and SIAH1. We show that c-MYB is targeted by miR-155 and miR-424, that the tumor suppressor SIAH1 is targeted by miR-424, and that c-SKI is potentially regulated by miR-155. Downregulation of SIAH1 protein in DLBCL was demonstrated by immunohistochemistry. The inhibition of SIAH1 is in line with the notion that EBV impedes various pro-apoptotic pathways during tumorigenesis. The down-modulation of the oncogenic c-MYB protein, although counter-intuitive, might be explained by its tight regulation in developmental processes.     

Altered microRNA expression in human heart disease.

MicroRNAs are recently discovered regulators of gene expression and are becoming increasingly recognized as important regulators of heart function. Genome-wide profiling of microRNAs in human heart failure has not been reported previously. We measured expression of 428 microRNAs in 67 human left ventricular samples belonging to control (n = 10), ischemic cardiomyopathy (ICM, n = 19), dilated cardiomyopathy (DCM, n = 25), or aortic stenosis (AS, n = 13) diagnostic groups. miRNA expression between disease and control groups was compared by ANOVA with Dunnett's post hoc test. We controlled for multiple testing by estimating the false discovery rate. Out of 428 microRNAs measured, 87 were confidently detected; 43 were differentially expressed in at least one disease group. In supervised clustering, microRNA expression profiles correctly grouped samples by their clinical diagnosis, indicating that microRNA expression profiles are distinct between diagnostic groups. This was further supported by class prediction approaches, in which the class (control, ICM, DCM, AS) predicted by a microRNA-based classifier matched the clinical diagnosis 69% of the time (P < 0.001). These data show that expression of many microRNAs is altered in heart disease and that different types of heart disease are associated with distinct changes in microRNA expression. These data will guide further studies of the contribution of microRNAs to heart disease pathogenesis.     

Ultra-deep sequencing reveals the microRNA expression pattern of the human stomach

Background

While microRNAs (miRNAs) play important roles in tissue differentiation and in maintaining basal physiology, little is known about the miRNA expression levels in stomach tissue. Alterations in the miRNA profile can lead to cell deregulation, which can induce neoplasia.

Methodology/Principal Findings

A small RNA library of stomach tissue was sequenced using high-throughput SOLiD sequencing technology. We obtained 261,274 quality reads with perfect matches to the human miRnome, and 42% of known miRNAs were identified. Digital Gene Expression profiling (DGE) was performed based on read abundance and showed that fifteen miRNAs were highly expressed in gastric tissue. Subsequently, the expression of these miRNAs was validated in 10 healthy individuals by RT-PCR showed a significant correlation of 83.97% (P<0.05). Six miRNAs showed a low variable pattern of expression (miR-29b, miR-29c, miR-19b, miR-31, miR-148a, miR-451) and could be considered part of the expression pattern of the healthy gastric tissue.

Conclusions/Significance

This study aimed to validate normal miRNA profiles of human gastric tissue to establish a reference profile for healthy individuals. Determining the regulatory processes acting in the stomach will be important in the fight against gastric cancer, which is the second-leading cause of cancer mortality worldwide.     

Real-time expression profiling of microRNA precursors in human cancer cell lines

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.     

HIV-1 Nef promotes cell proliferation and microRNA dysregulation in lung cells

Non-small cell lung cancer (NSCLC) represents about 85% of all lung cancer cases. Lung cancer is the most frequent non-AIDS-defining malignancies in HIV-infected patients. The mechanism of the increased risk for lung cancer in HIV-1 patients is poorly understood. HIV-1 Nef protein has been suggested to be one of the key players in HIV-related lung disease. In here, we showed the involvement of Nef protein in cell modifications such as fibroblasts (IMR-90) and normal (BEAS-2B) or cancerous (A549) epithelial cells. We demonstrated that Nef protein reprograms initial stages of lung cancer (e.g. changes in the metabolism, improved cell survival and invasion, increase the angiogenesis factor VEGF). Additionally, we showed that Nef is provoking a global decrease of mature miRNA and a decrease of DICER1 and AGO expression in lung cells. MiRNAs play a crucial role in cell signaling and homeostasis, functioning as oncogenes or tumor suppressors, and their dysregulation can contribute to the tumorigenic process. These results showed that HIV-1 Nef protein is directly involved in preventing cell death and contributes to tumor progression.     

Profiling microRNA expression with microarrays

The discovery of several types of small RNAs (sRNAs) has led to a steady increase in available RNA databases. Many of these sRNAs remain to be validated and functionally characterized. Recent advances in microRNA (miRNA)-expression profiling of different tissues, stages of development and physiological or pathological states are beginning to be explored using several technological approaches. In this review, these recent advances in miRNA microarray technology and their applications, particularly in basic research and clinical diagnosis, will be summarized and discussed. The methods for miRNA enrichment and probe design and labeling will also be discussed with an emphasis on evaluation of predicted miRNA sequences, analysis of miRNA expression and exploration of the potential roles of miRNA sequences in the regulation of stem cell differentiation and tissue- and time-specific profiling patterns of their target genes.     

Involvement of major histocompatibility complex class II antigen in Epstein-Barr virus-mediated B cell proliferation.

Five MHC class II monoclonal antibodies costimulated proliferation of cord blood leukocytes with Epstein-Barr virus. These agonistic antibodies were of different isotypes, but all of them were either specific for or cross-reacting with HLA-DR. The other MHC class II antibodies, including three that were specific for HLA-DQ and one that was specific for HLA-DP and also those that were specific for MHC class I or leukocyte common antigen, were not costimulatory. The agonistic effect of different MHC class II antibodies was additive, such that costimulation by different antibodies combined significantly exceeded that achieved by either of these antibodies alone. Spent culture media of B cell lines also costimulated B cell proliferation with the virus. Although MHC class II antibodies augmented the effects of suboptimal concentration of the conditioned media, their combined effects did not exceed the maximum costimulation achieved by either the antibodies or the spent culture media alone. These results raised the possibility that MHC class II antigen may contain distinct functional domains involved in the regulation of B cell progression.     

RAKE and LNA-ISH reveal microRNA expression and localization in archival human brain

microRNAs (miRNAs) are small (approximately 22 nucleotide) regulatory RNAs which play fundamental roles in many biological processes. Recent studies have shown that the expression of many miRNAs is altered in various human tumors and some miRNAs may function as oncogenes or tumor suppressor genes. However, with the exception of glioblastoma multiforme, the expression of miRNAs in brain tumors is unknown. Furthermore, methods to profile miRNAs from formalin-fixed, paraffin-embedded (FFPE) archival tissues or to study their cellular and subcellular localization in FFPE tissues have been lacking. Here we report the coordinated miRNA expression analysis from the tissue level to the subcellular level, using the RAKE (RNA-primed, array-based, Klenow Enzyme) miRNA microarray platform in conjunction with Locked Nucleic Acid (LNA)-based in situ hybridization (LNA-ISH) on archival FFPE human brains and oligodendroglial tumors. The ability to profile miRNAs from archival tissues at the tissue level, by RAKE microarrays, and at the cellular level by LNA-ISH, will accelerate studies of miRNAs in human diseases.