Project description:Purpose: Identification of the miRNA signature carried by the exosomes released from embryonic hippocampal cells under ceramide treatment Outcome: We investigated the role of ceramide in amplifying the differentiation signal of HN9.10 cells. Treatment of HN9.10 cells with ceramide caused the release of exosomes carrying neutral sphingomyelinase and neutral ceramidase. The analysis of exosomal miRNAs showed that 38 miRNAs were differentially expressed in a statistically significant manner, with some overexpressed miRNAs regulating genes encoding for proteins involved in biological, homeostatic, biosynthetic and small molecule metabolic processes, embryo development and cell differentiation, all phenomena that could be relevant for HN9.10 cell differentiation.
Project description:MicroRNAs (miRNAs) are intrinsic regulators in the various cellular processes, and their abnormalities are considered to be involved in the onset of human disorders, including cancer. Circulating miRNA is focused as new cancer biomarker however it is regarded that circulating RNA are released not only from tumor but also by various pathways. Recently, exosomes, small membrane vesicles, have been a major interest in cancer research field, because of their unique biological properties. Exosomes are secreted from various cells and the components (Lipids, mRNAs, miRNAs and proteins) reflect origin of the cells secreting them. Identification of exosomal miRNAs from cancer cells is expected to provide useful biomarkers of cancer. To identify specific exosomal miRNAs as candidate biomarkers for colorectal cancer, we compared exosomal miRNA profiles of 5 colon cancer cell lines with that of normal colon-derived epithelial cells, and isolated a subset of miRNAs as commonly-secreted miRNAs from colon cancer cells Endogenously expression of microRNAs were analyzed by Agilent Human miRNA V3 Microarray (G4470C) using total RNAs of human colon-derived FHC cells and human colon cancer cell lines (HCT116 cells and SW480 cells) at two independent experiments. Exosomal microRNAs were analyzed by microRNA microarray using total RNAs of exosomes from conditioned media of FHC cells, HCT116 cells, and SW480 cells at three independent experiments.As negative control of exosomal microRNAs in conditioned media, FBS-exosomal microRNAs were analyzed at four independent experiments. Exosomes were prepared by step-wise ultra-centrifugation methods. RNA was prepared by Trizol or Trizol-LS reagent (Invitrogen) and RNeasy mini spin column (Qiagen).
Project description:RNA transfer via extracellular vesicles (EVs) influences cell phenotypes; however, lack of information regarding biogenesis of RNA-containing EVs has limited progress in the field. Here, we identify endoplasmic reticulum membrane contact sites (ER MCS) as platforms for generation of RNA-containing EVs. We identify a subpopulation of small EVs that is highly enriched in RNA and regulated by the ER MCS linker protein VAP-A. Functionally, VAP-A-regulated EVs are critical for miR-100 transfer between cells and in vivo tumor formation. Lipid analysis of VAP-A-knockdown EVs revealed reductions in the EV biogenesis lipid ceramide. Knockdown of the VAP-A-binding ceramide transfer protein CERT led to similar defects in EV RNA content. Imaging experiments revealed that VAP-A promotes lumenal filling of multivesicular bodies (MVBs). CERT localizes to MVBs, and the ceramide-generating enzyme neutral sphingomyelinase 2 colocalizes with VAP-A-positive ER. We propose that ceramide transfer via VAP-A-CERT linkages drives biogenesis of a select RNA-containing population.
Project description:Metastasis is a major cause of mortality, and remains a final frontier in the search for a cure for cancer. While there has been much research on the ‘seed’ (metastatic tumor cells) and the ‘soil’ (colonized host tissue), interactions between metastatic cancer cells and stromal endothelial cells, which occur at multiple stages during metastasis, are less well understood. Here we report a dynamic regulation of the endothelium by cancer cells through the formation of nanoscale intercellular membrane bridges, which act as physical conduits for intercellular communication in vitro and in vivo, including horizontal transfer of microRNAs (miRNA). The communication between the tumor cell and the endothelium upregulates markers associated with pathological endothelium, which is reversed by pharmacological inhibition of these nanoscale conduits. These results lead us to define the notion of “metastatic hijack”: cancer cell-induced transformation of healthy endothelium into pathological endothelium via horizontal communication through the nanoscale conduits. Pharmacological perturbation of these nanoscale membrane bridges decreases metastatic foci in syngeneic- and human xenograft-breast cancer models. Targeting the formation of these nanoscale membrane bridges may potentially emerge as a new therapeutic opportunity in the management of metastatic cancer. A miRNA microarray was used to evaluate the transport of endogenous microRNAs. The intercellular transfer-ve and intercellular transfer+ve samples were sorted from the same endothelial cell population with the only difference being the occurrence of intercellular transport. The heat map shows potential miRNA candidates for exogenous transfer on two independent biological replicates. These miRNA candidates were significantly up-regulated in the cells receiving transfer of intercellular contents. HUVECs that were not exposed to cancer cells were used as a baseline control.
Project description:MicroRNAs (miRNAs) are intrinsic regulators in the various cellular processes, and their abnormalities are considered to be involved in the onset of human disorders, including cancer. Circulating miRNA is focused as new cancer biomarker however it is regarded that circulating RNA are released not only from tumor but also by various pathways. Recently, exosomes, small membrane vesicles, have been a major interest in cancer research field, because of their unique biological properties. Exosomes are secreted from various cells and the components (Lipids, mRNAs, miRNAs and proteins) reflect origin of the cells secreting them. Identification of exosomal miRNAs from cancer cells is expected to provide useful biomarkers of cancer. To identify specific exosomal miRNAs as candidate biomarkers for colorectal cancer, we compared exosomal miRNA profiles of 5 colon cancer cell lines with that of normal colon-derived epithelial cells, and isolated a subset of miRNAs as commonly-secreted miRNAs from colon cancer cells Endogenously expression of microRNAs were analyzed by Agilent Human miRNA V3 Microarray (G4470C) using total RNA of three human colon cancer cell lines (HT-29 cells, SW48 cells, and RKO cells) at two independent experiments. Exosomal microRNAs were analyzed by microRNA microarray using total RNA of exosomes from conditioned media of three human colon cancer cell lines, HT-29 cells, SW48 cells, and RKO cells at three independent experiments. Exosomes were prepared by step-wise ultra-centrifugation methods. RNA was prepared by Trizol or Trizol-LS reagent (Invitrogen) and RNeasy mini spin column (Qiagen).
Project description:Identification of differentially expressed microRNAs in Colorectal Cancer Distant metastasis is the major determinant of patient outcome in colorectal cancer and microRNAs have emerged as an increasingly important class of molecules which can regulate several steps of the metastatic cascade. By systematically analysing the miR expression profiles of resected metastasis-, corresponding primary tumor- and normal tissues of colorectal cancer patients, we were able to delineate a miR-signature indicative of the metastatically critical microRNA landscape. 9 colorectal cancer patients were profiled comprising 5 patients with tissues from the primary tumor, normal mucosa, secondary metastasis and the background tissue in which the metastasis ocurred. In the remaining 4 patients, one of these four tissue entitities is missing. One patient had two synchronous primary tumors, one in the colon and the other in the rectum.
Project description:Abstract:
Accumulating experimental and clinical evidence suggest that the immune response to cancer is not exclusively anti-tumor. Indeed, the pro-tumor roles of the immune system - as suppliers of growth and pro-angiogenic factors or defenses against cytotoxic immune attacks, for example - have been long appreciated, but relatively few theoretical works have considered their effects. Inspired by the recently proposed "immune-mediated" theory of metastasis, we develop a mathematical model for tumor-immune interactions at two anatomically distant sites, which includes both anti- and pro-tumor immune effects, and the experimentally observed tumor-induced phenotypic plasticity of immune cells (tumor "education" of the immune cells). Upon confrontation of our model to experimental data, we use it to evaluate the implications of the immune-mediated theory of metastasis. We find that tumor education of immune cells may explain the relatively poor performance of immunotherapies, and that many metastatic phenomena, including metastatic blow-up, dormancy, and metastasis to sites of injury, can be explained by the immune-mediated theory of metastasis. Our results suggest that further work is warranted to fully elucidate the pro-tumor effects of the immune system in metastatic cancer.
Project description:Identification of differentially expressed microRNAs in Colorectal Cancer Distant metastasis is the major determinant of patient outcome in colorectal cancer and microRNAs have emerged as an increasingly important class of molecules which can regulate several steps of the metastatic cascade. By systematically analysing the miR expression profiles of resected metastasis-, corresponding primary tumor- and normal tissues of colorectal cancer patients, we were able to delineate a miR-signature indicative of the metastatically critical microRNA landscape.