Characterization of miRNA processing machinery in the embryonic chick lung

Lung development is a very complex process that relies on the interaction of several signaling pathways that are controlled by precise regulatory mechanisms. Recently, microRNAs (miRNAs), small non-coding regulatory RNAs, have emerged as new players involved in gene expression regulation controlling several biological processes, such as cellular differentiation, apoptosis and organogenesis, in both developmental and disease processes. Failure to correctly express some specific miRNAs or a component of their biosynthetic machinery during embryonic development is disastrous, resulting in severe abnormalities. Several miRNAs have already been identified as modulators of lung development. Regarding the spatial distribution of the processing machinery of miRNAs, only two of its members (dicer1 and argonaute) have been characterized. The present work characterizes the expression pattern of drosha, dgcr8, exportin-5 and dicer1 in early stages of the embryonic chick lung by whole mount in situ hybridization and cross-section analysis. Overall, these genes are co-expressed in dorsal and distal mesenchyme and also in growing epithelial regions. The expression pattern of miRNA processing machinery supports the previously recognized regulatory role of this mechanism in epithelial and mesenchymal morphogenesis.QRE

Down-Regulation of miR-92 in Breast Epithelial Cells and in Normal but Not Tumour Fibroblasts Contributes to Breast Carcinogenesis

Background MicroRNA (miR) expression is commonly dysregulated in many cancers, including breast. MiR–92 is one of six miRs encoded by the miR-17-92 cluster, one of the best-characterised oncogenic miR clusters. We examined expression of miR–92 in the breast epithelium and stroma during breast cancer progression. We also investigated the role of miR–92 in fibroblasts in vitro and showed that down-regulation in normal fibroblasts enhances the invasion of breast cancer epithelial cells. Methodology/Principal Findings We used laser microdissection (LMD) to isolate epithelial cells from matched normal, DCIS and invasive tissue from 9 breast cancer patients and analysed miR–92 expression by qRT-PCR. Expression of ERβ1, a direct miR–92 target, was concurrently analysed for each case by immunohistochemistry. LMD was also used to isolate matched normal (NFs) and cancer-associated fibroblasts (CAFs) from 14 further cases. Effects of miR–92 inhibition in fibroblasts on epithelial cell invasion in vitro was examined using a Matrigel™ assay. miR– 92 levels decreased in microdissected epithelial cells during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p<0.01) and being lowest in invasive breast tissue (p<0.01). This was accompanied by a shift in cell localisation of ERβ1 from nuclear expression in normal breast epithelium to increased cytoplasmic expression during progression to DCIS (p = 0.0078) and invasive breast cancer (p = 0.031). ERβ1 immunoreactivity was also seen in stromal fibroblasts in tissues. Where miR–92 expression was low in microdissected NFs this increased in matched CAFs; a trend also seen in cultured primary fibroblasts. Down-regulation of miR–92 levels in NFs but not CAFs enhanced invasion of both MCF–7 and MDA-MB–231 breast cancer epithelial cells. Conclusions miR–92 is gradually lost in breast epithelial cells during cancer progression correlating with a shift in ERβ1 immunoreactivity from nuclei to the cytoplasm. Our data support a functional role in fibroblasts where modification of miR–92 expression can influence the invasive capacity of breast cancer epithelial cells. However in silico analysis suggests that ERβ1 may not be the most important miR–92 target in breast cancer

Heavy vehicle state estimation and rollover risk evaluation using Kalman Filter and Sliding Mode Observer

Safety driving is due to the prevention of risks situation, one of the important risk is the rollover of a heavy vehicle. Preventing this accident requires the knowledge of the rollover coefficient which depends on the vehicle dynamic state and other vehicle parameters. Thus, we estimate the vehicle dynamic state using the Extended and Unscented Kalman Filter and the Sliding Mode Observer. Thereafter, we calculate the probability to have a rollover risk using the previous result and Monte-Carlo simulations

Elevated Mirc1/Mir17-92 cluster expression negatively regulates autophagy and CFTR (cystic fibrosis transmembrane conductance regulator) function in CF macrophages

Cystic fibrosis (CF) is a fatal, genetic disorder that critically affects the lungs and is directly caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR function. Macroautophagy/autophagy is a highly regulated biological process that provides energy during periods of stress and starvation. Autophagy clears pathogens and dysfunctional protein aggregates within macrophages. However, this process is impaired in CF patients and CF mice, as their macrophages exhibit limited autophagy activity. The study of microRNAs (Mirs), and other noncoding RNAs, continues to offer new therapeutic targets. The objective of this study was to elucidate the role of Mirs in dysregulated autophagy-related genes in CF macrophages, and then target them to restore this host-defense function and improve CFTR channel function. We identified the Mirc1/Mir17-92 cluster as a potential negative regulator of autophagy as CF macrophages exhibit decreased autophagy protein expression and increased cluster expression when compared to wild-type (WT) counterparts. The absence or reduced expression of the cluster increases autophagy protein expression, suggesting the canonical inverse relationship between Mirc1/Mir17-92 and autophagy gene expression. An in silico study for targets of Mirs that comprise the cluster suggested that the majority of the Mirs target autophagy mRNAs. Those targets were validated by luciferase assays. Notably, the ability of macrophages expressing mutant F508del CFTR to transport halide through their membranes is compromised and can be restored by downregulation of these inherently elevated Mirs, via restoration of autophagy. In vivo, downregulation of Mir17 and Mir20a partially restored autophagy expression and hence improved the clearance of Burkholderia cenocepacia. Thus, these data advance our understanding of mechanisms underlying the pathobiology of CF and provide a new therapeutic platform for restoring CFTR function and autophagy in patients with CF

A community of strangers:The dis-embedding of social ties

In this paper we explore two contrasting perspectives on individuals' participation in associations. On the one hand, some have considered participation the byproduct of pre-existing friendship ties--the more friends one already has in the association, the more likely he or she is to participate. On the other hand, some have considered participation to be driven by the association's capacity to form new identities--the more new friends one meets in the association, the more likely he or she is to participate. We use detailed temporal data from an online association to adjudicate between these two mechanisms and explore their interplay. Our results show a significant impact of new friendship ties on participation, compared to a negligible impact of pre-existing friends, defined here as ties to other members formed outside of the organization's context. We relate this finding to the sociological literature on participation and we explore its implications in the discussion

Abstract PR03: Macrophage phenotype drives tumor program via epigenetic machinery carried in secreted microvesicles

Abstract Macrophage phenotypes are reported to regulate tumor progression, angiogenesis, and metastasis in breast cancer by producing soluble factors modulating these programs. Macrophages also communicate via secreted microvesicles (MVs) which are taken-up by neighboring epithelium. MVs contain mRNAs coding the epigenetic regulating machinery, DNA methyltranferases (DNMTs) and histone deacetylases (HDACs), which augment or silence expression via promoter CpG island methylation. Tie2-expressing monocytes (TEMs) is a subset of monocytes reported to augment tumor angiogenesis and metastasis. Recently, we found that increased levels of colony stimulating factor-1 (CSF1) can expand the TEM population in circulation, enabling an influx into breast tumors. Interestingly, we also found that expansion of TEMs by hypoxia was regulated by HIF-1α; and not HIF-2α, but only once they enter the tumor proper. We hypothesized that MVs secreted from M1 and M2 macrophages or TEMs contain epigenetic regulatory machinery which regulate CpG island methylation and gene expression of tumor suppressor genes (TSGs) and genes driving epithelial-to-mesenchymal transition (EMT). We differentiated M1, M2, and TEMs in vitro from CD14+ monocytes isolated from peripheral blood. These cell populations were confirmed using flow cytometry for CD68 and CD80 for M1, CD163 for M2, and CD14/Tie2 for TEMs, as well as M1 (IL-6 and TNFα) and M2 (IL-10 and mannose receptor-1) gene expression profiles. After, we collected MVs using high speed centrifugation techniques characterized by flow cytometry and isolated their nucleic acid content. Using qRT-PCR, we found differential presence of mRNAs for DNMTs and HDACs between M1, M2, and TEM MVs. We cultured these MVs with MCF-10A normal mammary epithelial cells or BEAS-2B normal lung epithelial cells (target cells) for 24 hours and demonstrated MV uptake using Syto RNASelect (RNA) and DiIC16(3) (lipid membrane) and confocal microscopy. After, we isolated RNA and DNA from the target cells and analyzed DNMTs, HDACs, and EMT mRNA expression as well as methyl-specific PCR for CpG island methylation in the promoters of EMT genes. We found that MVs from M1 macrophages increased DNMTs mRNA expression compared to MVs produced from M2 and quiescent macrophages (M0) as well as untreated target cells. To the contrary, HDACs mRNA expression in these target cells cultured with M1-derived MVs was abrogated compared to target cells cultured with MVs from M2 and M0 macrophages and untreated target cells. As a result of the differential MV-carrying DNMTs and HDACs mRNA transferred to the target cells, we found significant differences in CpG island promoter methylation and resultant gene expression in a signature of EMT genes, including TWIST, WNT5A, VIM, FOXC2, KRT19, STAT3, SNAI1 BMP1, TGFb, DSP, AKT1, NUDT13, and ZEB1. The regulation of EMT and tumor suppressor gene promoter methylation and gene expression in MDA-MB-231 human breast cancer cells, as well as the disparate regulation of methylation and gene expression patterns on these target cells as well endothelial cells (HUVEC) by MVs collected from CD14+/Tie2+ TEMs is ongoing. Our current and ongoing work, we establish that M1 and M2 macrophages, and TEMs, secrete MVs containing distinct epigenetic profiles which are taken-up by target cells to regulate promoter methylation and gene expression of TSGs and genes driving EMT. This program of macrophage function may be important in the progression of solid tumors via inhibition of TSGs and activation of a signature of EMT genes in normal epithelial cells as well as to direct the endothelium to support tumor progression. This abstract is also presented as Poster A31. Citation Format: Duaa Dakhlallah, Ivory Patterson, Amy C. Gross, Randall Evans, Tim D. Eubank. Macrophage phenotype drives tumor program via epigenetic machinery carried in secreted microvesicles. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR03. doi:10.1158/1538-7445.CHTME14-PR03</jats:p