Unknown,Transcriptomics,Genomics,Proteomics

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KRAS-Dependent Sorting of miRNAs and other small RNAs to secreted vesicles

ABSTRACT: Although long thought to act cell autonomously, mutant KRAS colorectal cancer (CRC) cells release protein-laden exosomes that can alter the tumor microenvironment. We have previously shown that mutant KRAS induces EGFR-ligand trafficking to exosomes and drastically alters exosomal protein contents, leading to activities that contribute to neoplastic growth. We have performed small library RNAseq analysis on cells and matched exosomes from isogenic CRC cell lines differing only in KRAS status to determine whether mutant KRAS regulates the composition of secreted small RNAs. Exosomal small RNA profiles were distinct from cellular profiles, with principle component analysis showing clusters of mutant KRAS cell-derived exosomes distinct from wild type KRAS cell-derived exosomes. Secreted RNA species encompassed several different classes of small RNAs, including ribosomal and tRNA fragments, as well as mature miRNA sequences. miR-10b, was selectively increased in wild type KRAS-derived exosomes, whereas miR-100 was selectively increased in mutant KRAS-derived exosomes. Ceramide inhibition resulted in accumulation of miR-100 in mutant KRAS cells, suggesting KRAS-dependent miRNA export. In Transwell cell culture experiments, mutant, but not wild type, KRAS donor cells conferred miR-100-mediated target repression in wild type KRAS recipient cells miRNAseq deep sequencing for both cell and exosome mirnas of Dks-8, DLD-1, and DKO-1 cell lines. The DKs-8 line contains a wild type KRAS alleles, the DLD-1 line contains both wild type and mutant (G13D) KRAS alleles, and the DKO-1 line contains only a mutant KRAS allele.

ORGANISM(S): Homo sapiens

SUBMITTER: Yongchao Dou

PROVIDER: E-GEOD-67004 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

KRAS-dependent sorting of miRNA to exosomes.

Cha Diana J DJ Franklin Jeffrey L JL Dou Yongchao Y Liu Qi Q Higginbotham James N JN Demory Beckler Michelle M Weaver Alissa M AM Vickers Kasey K Prasad Nirpesh N Levy Shawn S Zhang Bing B Coffey Robert J RJ Patton James G JG

eLife 20150701

Mutant KRAS colorectal cancer (CRC) cells release protein-laden exosomes that can alter the tumor microenvironment. To test whether exosomal RNAs also contribute to changes in gene expression in recipient cells, and whether mutant KRAS might regulate the composition of secreted microRNAs (miRNAs), we compared small RNAs of cells and matched exosomes from isogenic CRC cell lines differing only in KRAS status. We show that exosomal profiles are distinct from cellular profiles, and mutant exosomes ...[more]

PMID: 26132860

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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.

Dataset Information

KRAS-Dependent Sorting of miRNAs and other small RNAs to secreted vesicles

Publications

KRAS-dependent sorting of miRNA to exosomes.

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