Project description:We have identified several nucleotide motifs (caug, cgggag=S2) that promote exosome sorting of miRNA in different cell types including brown adipocytes. In order to identify which proteins might recognize and bind to these motifs, we have performed co-precipitations of proteins binding biotinylated forms of miRNAs containing the aforementioned motifs or none - using streptavidin beads incubated with brown adipocytes cell lysates. We have included two types of controls: negative poly-A control and a scramble miRNA.
Project description:Exosomal miRNAs secreted by cancer-associated fibroblasts (CAFs) play a critical role in facilitating head and neck cancer (HNC) progression. A few studies in other cancers have confirmed some CAF-specific miRNAs could be delivered to tumor cells via exosome, thus contributing to chemoresistance. To identify the specific miRNAs responsible for the capability of CAF-derived exosomes to promote cisplatin resistance in recipient HNC cells, a miRNA array was conducted to identify miRNAs involved in exosomes derived from CAFs and the paired normal fibroblasts (NFs).
Project description:We investigate the expression of miRNA in exosome of EBV-positive gastric carcinoma cells. The exosomes of EBV-positive and negative gastric carcinoma cells were separated by ultracentrifugation, the morphology of exosomes was identified by transmission electron microscopy, the exosome size was analyzed by Nanosight, and the expression of exosome membrane protein CD63 and CD81 was detected by western blot. High-throughput sequencing was used to detect miRNA expression profiles in gastric cancer cell lines and their exosomes. Under the ultra-microscopic electron microscope, the exosomes are seen as a typical translucent cup-like structure or a flat spherical structure. The nanoparticle tracking analyzer (Nanosight) showed that the exosomes were between 30 and 150 nm in diameter. Western blot(WB) assays showed that exosomes secreted by EBVaGC and EBVnGC cells expressed specific exosome membrane-associated proteins CD63 and CD81. High-throughput sequencing revealed that EBVaGC(SNU-719) and EBVnGC(AGS) and their secreted exosomes were highly expressed with certain human miRNAs, among which AGS-exo was highly expressed with hsa-miR-23b-3p, hsa-miR-320a-3p, and hsa-miR-4521. SNU-719-exo was highly expressed as hsa-miR-21-5p, hsa-miR-148a-3p and hsa-miR-7-5p. Nearly all EBV-related miRNAs (EBV-miRNA) were expressed in SNU-719 cells and their exosomes, among which EBV-miR-BART1-5p, EBV-miR-BART17-3p and EBV-miR-BART18-5p were the highest in SNU-719 cells, EBV-miR-BART1-5p, EBV-miR-BART18-5p and EBV-miR-BART17-3p were the highest in SNU-719-exo.