Long non-coding RNAs with low expression levels in cells are enriched in secreted exosomes.

AbstractLong non-coding RNAs (lncRNAs) are involved in regulating chromatin modifications, gene transcription, mRNAtranslation,andproteinfunction.WerecentlyreportedahighvariationinthebasalexpressionlevelsofapaneloflncRNAsinHeLa and MCF-7 cells and their differential response to DNA damage induction. Here, we hypothesized that lncRNAmoleculeswithdifferentcellularexpressionmayhaveadifferentialabundanceinsecretedexosomes,andtheirexosomelevelswould reflect cellular response to DNA damage. MALAT1, HOTAIR, lincRNA-p21, GAS5, TUG1, CCND1-ncRNA inexosomes secreted from cultured cells were characterized. A different expression pattern of lncRNAs in exosomes was seencomparedtocells.RNAmoleculeswithrelativelowexpressionlevels(lincRNA-p21,HOTAIR,ncRNA-CCND1)werehighlyenrichedinexosomes.TUG1andGAS5levelsweremoderatelyelevatedinexosomes,whereasMALAT1—whichwasthemostabundantmoleculeincells—waspresentatlevelscomparabletoitscellularlevels.lincRNA-p21andncRNA-CCND1werethemainmolecules;exosomelevelsofthembestreflectthechangeoftheircellularlevelsuponexposureofthecellstobleomycin-induced DNA damage. In conclusion, we provide evidence that lncRNAs have a differential abundance in exosomes,indicating a selective loading.Keywords: long noncoding RNAs; exosomes; HeLa cells; MCF-7 cellsIntroductionMore than 90% of the human genome is transcribed intoRNA, whereas only about 2% is translated into proteins(Bertone et al., 2004; Wilusz et al., 2009). Most of thetranscripts serve as functional non-coding RNAs (ncRNAs),including microRNAs and long non-coding RNAs(lncRNAs).lncRNAsaredefinedasaclassofRNAmoleculesthat do not fall into any known class of small and structuralRNAs, and possess regulatory roles (Spizzo et al., 2012). Thehuman genome presumably contains about 9,000 lncRNAloci that potentially encode approximately 15,000 transcripts(Harrowetal.,2012).ProductionoflncRNAsissimilartothatof protein-coding genes with respect to splicing and post-transcriptional processing. They are roughly classified asintergenic,intragenic/intronic,andantisenselncRNAsbasedontheirpositionrelativetotheprotein-codinggenes(Merceret al., 2009). Biological activities of lncRNAs are still beingelucidated,butresearchshowsthattheyhaveatissue-specificexpression pattern (Derrien et al., 2012) and are involved inregulating chromatin modification, gene transcription,mRNA translation, and protein function (Wilusz et al.,2009; Mercer et al., 2009; Wang and Chang, 2011).Extracellular vesicles are important mediators of intercel-lular communication. The pathophysiological significanceofextracellularvesiclesisbeginningtobedefinedindiseases,including cancer (El Andaloussi et al., 2013). Exosomes aresmall membranous vesicles secreted from several types ofcells, ranging in size from 30 to 100nm in diameter, andarisingfromendosomalcompartmentscalledmultivesicularbodies (MVBs) (Heijnen et al., 1999; Denzer et al., 2000).During the biogenesis of exosomes, the membrane of theMVBs invaginates to form intraluminal vesicles (ILVs)(Denzer et al., 2000). Secretion of exosomes into theextracellularcompartmentisduetofusionoftheMVBswiththeplasmamembrane,whichcanoccurspontaneouslyorbeinduced by cellular machinery. Depending on the originat-ing cell and the secretion conditions, exosomes are involvedin diverse processes (Thery et al., 2002; Simons andRaposo, 2009). The discovery that exosomes contain