Unknown,Transcriptomics,Genomics,Proteomics

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Mesenchymal stem cell-derived exosomes stimulate cycling quiescence and early breast cancer dormancy in bone marrow

ABSTRACT: Transwell cultures were established in 6-well plates with 0.4 micron inserts. Equal amounts (6x10e4) of BCCs and MSCs were added to the outer and inner wells, respectively, in DMEM with 10% exosome-free FCS. After 24 h, the inner wells with BCCs were discarded. The MSCs were washed with PBS and then incubated with DMEM with 2% exosome-free FCS. At 48 h, exosomes were isolated from the MSC media. The exosomes from triplicate wells were added to duplicate naÃ¯ve BCCs at 2.5x105/well of 6-well plates. qPCR miRNA expression profiling. RNA from MSCs-Exosomes were analyzed as indicated in the summary. The PCR was run on the 7300 Real-Time PCR System using the following thermal-cycling parameters: 94 0C for 15 min, 40 cycles at 940 C for 10 sec, 550 C for 30s, 700 C for 30 sec followed by a melting curve analysis.

ORGANISM(S): Homo sapiens

SUBMITTER: Pranela Rameshwar

PROVIDER: E-GEOD-85341 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:It is widely accepted that long-term changes in synapse structure and function are mediated by rapid activity-dependent gene transcription and new protein synthesis. A growing amount of evidence suggests that the microRNA (miRNA) pathway plays an important role in coordinating these processes. Despite recent advances in this field, there remains a critical need to identify specific activity-regulated miRNAs as well as their key messenger RNA (mRNA) targets. To address these questions, we used the larval Drosophila melanogaster neuromuscular junction (NMJ) as a model synapse in which to identify novel miRNA-mediated mechanisms that control activity-dependent synaptic growth. First, we developed a screen to identify miRNAs differentially regulated in the larval CNS following spaced synaptic stimulation. Surprisingly, we identified five miRNAs (miRs-1, -8, -289, -314, and -958) that were significantly downregulated by activity. Neuronal misexpression of three miRNAs (miRs-8, -289, and -958) suppressed activity-dependent synaptic growth suggesting that these miRNAs control the translation of biologically relevant target mRNAs. Functional annotation cluster analysis revealed that putative targets of miRs-8 and -289 are significantly enriched in clusters involved in the control of neuronal processes including axon development, pathfinding, and growth. In support of this, miR-8 regulated the expression of a wingless 3M-bM-^@M-^YUTR (wg 3M-bM-^@M-^Y untranslated region) reporter in vitro. Wg is an important presynaptic regulatory protein required for activity-dependent axon terminal growth at the fly NMJ. In conclusion, our results are consistent with a model where key activity-regulated miRNAs are required to coordinate the expression of genes involved in activity-dependent synaptogenesis. Three technical replicates (each in raw data) of three biological replicates of wild-type (CantonS) larvae in two treatment groups: (1) 5x spaced high K; (2) or 0x mock stimulation.

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Mesenchymal stem cell-derived exosomes stimulate cycling quiescence and early breast cancer dormancy in bone marrow

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