RESUMO
The regulation and functional roles of secreted coding and long noncoding RNAs (lncRNAs; >200 nt) are largely unknown. We previously showed that mutant KRAS colorectal cancer (CRC) cells release extracellular vesicles (EVs) containing distinct proteomes, microRNAs (miRNAs), and circular RNAs. Here, we comprehensively identify diverse classes of CRC extracellular long RNAs secreted in EVs and demonstrate differential export of specific RNAs. Distinct noncoding RNAs, including antisense transcripts and transcripts derived from pseudogenes, are enriched in EVs compared to cellular profiles. We detected strong enrichment of Rab13 in mutant KRAS EVs and demonstrate functional delivery of Rab13 mRNA to recipient cells. To assay functional transfer of lncRNAs, we implemented a CRISPR/Cas9-based RNA-tracking system to monitor delivery to recipient cells. We show that gRNAs containing export signals from secreted RNAs can be transferred from donor to recipient cells. Our data support the existence of cellular mechanisms to selectively export diverse classes of RNA.
Assuntos
Comunicação Celular , Neoplasias Colorretais/metabolismo , Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , Mutação , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Transporte Biológico , Movimento Celular , Neoplasias Colorretais/genética , Exossomos/genética , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Células Tumorais Cultivadas , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Recent studies have shown that circular RNAs (circRNAs) are abundant, widely expressed in mammals, and can display cell-type specific expression. However, how production of circRNAs is regulated and their precise biological function remains largely unknown. To study how circRNAs might be regulated during colorectal cancer progression, we used three isogenic colon cancer cell lines that differ only in KRAS mutation status. Cellular RNAs from the parental DLD-1 cells that contain both wild-type and G13D mutant KRAS alleles and isogenically-matched derivative cell lines, DKO-1 (mutant KRAS allele only) and DKs-8 (wild-type KRAS allele only) were analyzed using RNA-Seq. We developed a bioinformatics pipeline to identify and evaluate circRNA candidates from RNA-Seq data. Hundreds of high-quality circRNA candidates were identified in each cell line. Remarkably, circRNAs were significantly down-regulated at a global level in DLD-1 and DKO-1 cells compared to DKs-8 cells, indicating a widespread effect of mutant KRAS on circRNA abundance. This finding was confirmed in two independent colon cancer cell lines HCT116 (KRAS mutant) and HKe3 (KRAS WT). In all three cell lines, circRNAs were also found in secreted extracellular-vesicles, and circRNAs were more abundant in exosomes than cells. Our results suggest that circRNAs may serve as promising cancer biomarkers.
Assuntos
Neoplasias do Colo/genética , Exossomos/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA/genética , RNA CircularRESUMO
Secretion of RNAs in extracellular vesicles is a newly recognized form of intercellular communication. A potential regulatory protein for microRNA (miRNA) secretion is the critical RNA-induced silencing complex (RISC) component Argonaute 2 (Ago2). Here, we use isogenic colon cancer cell lines to show that overactivity of KRAS due to mutation inhibits localization of Ago2 to multivesicular endosomes (MVEs) and decreases Ago2 secretion in exosomes. Mechanistically, inhibition of mitogen-activated protein kinase kinases (MEKs) I and II, but not Akt, reverses the effect of the activating KRAS mutation and leads to increased Ago2-MVE association and increased exosomal secretion of Ago2. Analysis of cells expressing mutant Ago2 constructs revealed that phosphorylation of Ago2 on serine 387 prevents Ago2-MVE interactions and reduces Ago2 secretion into exosomes. Furthermore, regulation of Ago2 exosomal sorting controls the levels of three candidate miRNAs in exosomes. These data identify a key regulatory signaling event that controls Ago2 secretion in exosomes.
Assuntos
Proteínas Argonautas/metabolismo , Exossomos/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Humanos , MicroRNAs/metabolismo , Corpos Multivesiculares/metabolismo , Proteínas Mutantes/metabolismo , Fosforilação , Fosfosserina/metabolismo , Transporte Proteico , Frações Subcelulares/metabolismoRESUMO
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 cluster separately from wild-type KRAS exosomes. miR-10b was selectively increased in wild-type exosomes, while miR-100 was increased in mutant exosomes. Neutral sphingomyelinase inhibition caused accumulation of miR-100 only in mutant cells, suggesting KRAS-dependent miRNA export. In Transwell co-culture experiments, mutant donor cells conferred miR-100-mediated target repression in wild-type-recipient cells. These findings suggest that extracellular miRNAs can function in target cells and uncover a potential new mode of action for mutant KRAS in CRC.