Project description:Exosomes endogenous microRNAs (miRNAs) play critical roles in many biological processes.to obtain profiles of the miRNAs of exosomal and cell lysates in ovarian cancer cell lines.
Project description:We collected ovarian follicle fluids from 68 patients and assigned them to good group or bad group according to their oocyte quality. The exosomes were isolated and characterized. Exosomal microRNAs were extracted, the library was constructed and sequenced by Illumina hiseq platform. The exosomal microRNA expression was analyzed and profiled, the target genes were predicted, GO terms were enriched by GOSeq and KEGG pathway was analyzed using miranda.A total of 47 differential microRNAs was expressed significantly between good and bad group, of which 9 microRNAs were known microRNAs and 7 of them was upregulated in the bad group. In-silico analysis indicated that several of these exosomal microRNAs were involved in pathways implicated in oocyte quality.Our study suggests that exosomal microRNAs in ovarian follicle fluid are critical in maintaining the oocyte quality. Our study greatly improve our understanding of exosomal microRNAs in human ovarian follicular fluid, paving the way for further investigation on the microRNA functions in the ovarian microenvironment and the mechanism behind it.
Project description:To check the profile of exosomal and cellular miRNA in ovarian cancer cell lines, total RNA were extracted from exosomes and cells. Thirteen ovarian cancer cell lines (A2780, ES-2, CAOV3, SKOV3, OV-90, OAW42, MCAS, COV362, RMG-1, RMUG-S, KURAMOCHI, NIH-OVCAR3 and A2780cis) were investigated, and HOSE1, HOSE2 and HOSE3 (human ovarian surface epithelim cell lines) were used as control.
Project description:Over the last two decades, mounting evidence has demonstrated the aberrant expression of miRNAs in different human malignancies, including ovarian carcinomas. In this study, we found that miR-141 were secreted by ovarian cancer cells and transferred to the stromal cells by exosomal pathway. To clarify the mechanisms that miR-141 remodeling stromal fibroblast and reprogramming the relevant signals, miR-141 were overexpressed in WPMY-1 and their respective scrambled control were send for proteomic analysis.
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: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:Exososmes, potent intercellular communicators, are supposed to contribute to metastasis formation, which we confirmed for exosomes of the metastatic rat pancreatic adenocarcinoma line BSp73ASML that promote metastatic settlement in lymph nodes and lung of poorly metastatic BSp73ASML cells with a selective CD44v4-v7 (BSp73ASML-CD44vkd) knockdown. To define the molecular pathway(s), whereby exosomes contribute to premetastatic niche preparation, we profiled mRNA miRNA of BSp73ASMLwt and BSp73ASML-CD44vkd- exosomes and evaluated the impact on potential target cells. BSp73ASML exosomes are recovered in the draining lymph node after subcutaneous injection. In vitro, they preferentially bind and are taken-up by lymph node stroma cells (LnStr) and lung fibroblasts (LuFb) that were chosen as exosome targets. BSp73ASMLwt and BSp73ASML-CD44kd exosomes contain a restricted repertoire of mRNA and miRNA, hwere the lattter differe significantly between the two lines and even more pronounced, exosomes derived thereof with a not yet explored dominance of tumor-suppressor miRNA in ASML-CD44kd cells and exosomes. Both, exosomal mRNA and miRNA are recovered in target cells and exosome-uptake is accompanied by significant changes in gene expression. We didn't observe a correlation between exosomal mRNA and changes in target cell mRNA or proteins. Instead transferred miRNA significantly affected target cell mRNA translation as demonstrated for selected, most abundant ASML exosomal miRNA besides others, miR-494 known target MAL (myelin and lymphocytes protein)/cadherin17, and miR-542-3p which targets TRAF/cadherin17. Furthermore, MMP transcription suggested to accompany cadherin17 dwon-regulation was upregulated in miR-494 or miR542-3p transfected or exosome co-cultured LnStr. Taken together, tumor exosomes target in vivo non-transformed cells in premetastatic organs. Exosome uptake induced altered target celll gene expression is strongly promoted by exosomal miRNA where we demonstrate for the first time that exosomes/exosomal miRNA from a metastasizing tumor line can modulate stroma cells from premetastatic organs. Endothelial cells lines were treated with pancreatic adenocarcinoma (AS) derived exosomes or pancreatic adenocarcinoma derived exosomes expressing tetraspanin 8. Total RNA was isolated and used to perform the Agilent gene expression microarrays. In this assay a replicate of endothelial cell lines treated with ASTspan8 were also included. Moreover, total RNA from both base line expression of endothelial cells and rat endothelial fibroblasts were also used to perfrom gene expression microarrays. RNA isolated from Rat endothelial fibroblasts treated with the exosomes derived from rat pancreatic adenocarcinoma and exosomes derived from rat pancreatic adenocarcinoma expressing tetraspanin8 were individually used to perfrom gene expression microarrays. RNA isolated from exosomes derived from rat pancreatic adenocarcinoma cell lines expressing tetraspanin were used to peform gene expresiion to see the base line expression. Another replicate were also used. RNA isolated from base line or control of rat pancreatic adenocarcinoma wild type cells and also base line RNA isolated from rat pancreatic adenocarcinoma cells lines where CD44 was knock-down.
Project description:Background/Aim: We investigated alterations in the expression of serum exosomal miRNAs with the progression of liver fibrosis and evaluated their clinical applicability as biomarkers. Methods: This study prospectively enrolled 71 patients who underwent liver biopsy at an academic hospital in Korea. Exosomes were extracted from serum samples, followed by next-generation sequencing (NGS) of miRNAs and targeted real-time quantitative polymerase chain reaction. A model was derived to discriminate advanced fibrosis based on miRNA levels and the performance of this model was evaluated. Validation of the effect of miRNA on liver fibrosis in vitro was followed. Methods: This study prospectively enrolled 71 patients who underwent liver biopsy at an academic hospital in Korea. Exosomes were extracted from serum samples, followed by next-generation sequencing (NGS) of miRNAs and targeted real-time quantitative polymerase chain reaction. A model was derived to discriminate advanced fibrosis based on miRNA levels and the performance of this model was evaluated. Validation of the effect of miRNA on liver fibrosis in vitro was followed. Results: NGS data revealed that exosomal miR-660-5p, miR-125a-5p, and miR-122 expression were changed significantly with the progression of liver fibrosis, of which miR-122 exhibited high read counts enough to be used as a biomarker. The level of exosomal miR-122 decreased as the pathologic fibrosis grade progressed and patients with biopsy-proven advanced fibrosis had significantly lower levels of exosomal miR-122 (P < 0.001) than those without advanced fibrosis. Exosomal miR-122 exhibited a fair performance in discriminating advanced fibrosis especially in combination with fibrosis-4 score and transient elastography. In a subgroup of patients with a non-viral etiology of liver disease, the performance of exosomal miR-122 as a biomarker was greatly improved. Inhibition of miR-122 expression increased the proliferation of the human hepatic stellate cell line, LX-2, and upregulated the expression of various fibrosis related proteins. Conclusion: Exosomal miR-122 may serve as a useful non-invasive biomarker for liver fibrosis, especially in patients with non-viral etiologies of chronic liver disease.