RESEARCHOpenAccessAnovelmechanismoflncRNAandmiRNAinteraction:CCAT2regulatesmiR-145expressionbysuppressingitsmaturationprocessincoloncancercellsYingjieYu1,3*,PratimaNangia-Makker1,2,3,LuluFarhana1,3andAdhipP.N.Majumdar1,2,3*AbstractBackground:AlthoughbothlongandmicroRNAsareemergingasimportantfunctionalcomponentsincolorectalcancer(CRC)progressionandmetastasis,themechanismoftheirinteractionremainspoorlyunderstood.CCAT2(Coloncancer-associatedtranscript-2),alongnoncodingRNA(lncRNA),hasbeenreportedtobeover-expressedinCRCandisfoundtopromotetumorgrowth.miRNAs,aclassofnaturallyoccurringshortRNAsnegativelycontroltheexpressionoftargetgenesbycleavingmRNAorthroughtranslationrepression.Recently,wereportedthatmiR-145andmiR-21cooperatetoregulatecoloncancerstemcell(CSC)proliferationanddifferentiation.ConsideringthatCCAT2ismainlylocatedinthenucleusandmiRNAmaturationprocessbeginsinthenucleus,wehypothesizethatCCAT2selectivelyblocksmiR-145maturationprocess,resultingindecreasedmaturemiR-145affectingcolonCSCproliferationanddifferentiation.Methods:ThelevelsofCCAT2weremanipulatedbytransfectionofCCAT2expressionplasmidorknockdownbysiRNAorbyCRISPR/Cas9.QuantitativeRT-PCRwasperformedtoexaminetheexpressionofCCAT2andpri-,pre-andmaturemiR-145/21.Fluorescenceinsituhybridization(FISH)wasusedtovisualizeCCAT2inthecells.Invitroprocessingofpri-miRNA-145wasperformedusingT7RNApolymeraseandrecombinanthumanDicer.Results:WehaveobservedthatmodulatedexpressionofCCAT2regulatestheexpressionofmiR-145incoloncancerHCT-116andHT-29cells.KnockoutofCCAT2increasesmiR-145andnegativelyregulatesmiR-21inHCT-116cells,impairsproliferationanddifferentiation.Incontrast,stableup-regulationofCCAT2decreasesmaturemiR-145andincreasestheexpressionofseveralCSCmarkersincoloncancercells.WehavealsoobservedthatCCAT2isenrichedinthenucleusandcorrelateswiththeexpressionofpre-miR-145butnotpre-miR-21inHCT-116cells.TheseresultsindicateCCAT2selectivelyblocksmiR-145maturationbyinhibitingpre-miR-145exporttocytoplasm.Further,werevealedthatCCAT2b...
