The sequence of older 5 conventional miR21 (5-TAGCTTATCAGACTGATGTTGA-3), mature 5-miR21 super variant (5-TAGCTTATCAGACTGATGTTGACA-3) or mature 5-miR21 precursor variant (5-TAGCTTATCAGACTGATGTTGACTA-3) were used as the forward primer and the 3 universal reverse primer was provided by the QuantiMir RT kit. For cellular miR21 expression analysis, 5?ng of total RNA was first reverse transcribed using the TaqMan microRNA reverse transcription kit (Thermo Fisher Scientific) and miR21 (000397) and U6 (001973) snRNA-specific primers and probes, and then quantified using real-time PCR on a CFX96 Touch real-time PCR PF-04634817 detection system (Bio-Rad Laboratories Inc.). cancer apoptosis and confers chemoresistance by binding to its direct novel target, APAF1. These data suggest that the malignant phenotype of metastatic ovarian cancer cells can be altered by miR21 delivered by exosomes derived from neighbouring stromal cells in the omental tumour microenvironment, and that inhibiting the transfer of stromal-derived miR21 is an alternative modality in the treatment of metastatic and recurrent ovarian cancer. Approximately 22,000 new cases of epithelial ovarian cancer have been diagnosed in the United States in 2015 Rabbit polyclonal to AMHR2 (ref. 1). Over 16,000 deaths per year occurred, making this cancer the most lethal gynaecologic malignancy. Although cancer patients initially respond to platinum- and taxane-based PF-04634817 chemotherapy following surgery, most of them experience recurrence within 12C24 months and die of progressively chemotherapy-resistant disease. One critically important, yet often overlooked, component of tumour progression is the tumour microenvironment, which is primarily composed of fibroblasts, extracellular matrix proteins, endothelial cells and lymphocytic infiltrate. The tumour microenvironment has been shown to directly affect cell growth, migration and differentiation through secreted proteins, cellCcell interactions and matrix remodelling2. As it can promote the tumour initiation of normal epithelial cells and facilitate the progression of malignant cells, the tumour microenvironment presents a unique opportunity to discover ways to better diagnose, understand and treat cancer. Recent studies have shown that in addition to initiation via soluble mediators, cellCcell communication can be initiated via surface interactions between circulating exosomes and transmembrane molecules expressed by target cells3. The fusion of exosomes with target cell membranes facilitates the transfer of cell surface molecules and receptors from donor to recipient cells3. Furthermore, the endocytosis of exosomes by their target cells results in the intracellular release of vesicular contents, including messenger RNA, microRNA (miRNA), proteins and lipids4. Tumour exosomes have been shown to have angiogenic properties. For example, colorectal cancer exosomes transfer mRNAs, which promote endothelial cell proliferation and facilitate angiogenesis5, whereas glioblastoma-derived exosomes promote tubule formation by recipient endothelial cells6. Furthermore, tumour exosomes secrete factors that suppress natural killer cell activity and induce T-cell apoptosis7. In this way, the tumour cells themselves create a tumour-friendly’ environment that promotes cancer metastasis and progression. The transfer of miRNA by exosomes is particularly interesting, because miRNAs are more stable and can control the expression of multiple target genes in the recipient cells. In addition, miRNAs have been shown to regulate cell differentiation, proliferation and apoptosis, and contribute to the development of multiple tumour types8,9,10,11. Although the miRNA signatures of tumour-derived exosomes have been identified in multiple tumour types, including ovarian cancer12,13, exosomal miRNA signatures from cancer-associated stromal cells have not been investigated and the functional roles of these exosomal miRNAs in modulating the malignant phenotypes of recipient cancer cells have not been elucidated. In this study, we PF-04634817 use next-generation sequencing to identify differential miRNA signatures in exosomes isolated from ovarian cancer cells and ovarian cancer-associated fibroblasts (CAFs) and adipocytes (CAAs). We demonstrate that specific miRNAs are directly transferred, through exosomes, from CAFs and CAAs to ovarian cancer cells, and we identify the molecular mechanisms by which miRNAs modulate the malignant phenotypes in ovarian cancer cells. Results CAF and CAA exosomes have higher miR21 copy number miRNAs that transfer PF-04634817 between living cells that are involved in cellCcell communication are frequently encapsulated in exosomes, which facilitate their targeted exchange14. To identify miRNAs that are PF-04634817 transferred by exosomes secreted.