lncRNA-MALAT1在間歇低氧致人臍靜脈內(nèi)皮細(xì)胞損傷中的作用及機(jī)制研究摘要：目的：探討lncRNA-MALAT1在間歇低氧致人臍靜脈內(nèi)皮細(xì)胞損傷中的作用及機(jī)制，為深入理解其分子機(jī)制提供新思路。方法：將人臍靜脈內(nèi)皮細(xì)胞(humanumbilicalveinendothelialcell，HUVEC)分為3組：對照組、模型組和lncRNA-MALAT1轉(zhuǎn)染組，分別用標(biāo)準(zhǔn)培養(yǎng)基、間歇低氧培養(yǎng)和轉(zhuǎn)染lncRNA-MALAT1質(zhì)粒后間歇低氧培養(yǎng)。采用MTT法檢測HUVEC生長活性，并利用流式細(xì)胞術(shù)和AnnexinV-FITC/PI方法檢測HUVEC細(xì)胞凋亡情況。采用實(shí)時熒光定量PCR和Westernblot技術(shù)檢測lncRNA-MALAT1、蛋白酪氨酸激酶(focaladhesionkinase,FAK)、磷酸化-FAK(p-FAK)和BCL-2/BAX蛋白表達(dá)水平。結(jié)果：間歇低氧下，與對照組比較，模型組HUVEC生長活性降低，細(xì)胞凋亡率升高(P<0.05)；轉(zhuǎn)染lncRNA-MALAT1后，HUVEC生長活性增強(qiáng)(P<0.05)，細(xì)胞凋亡率降低(P<0.05)。實(shí)時熒光定量PCR和Westernblot結(jié)果顯示，間歇低氧下，lncRNA-MALAT1和p-FAK蛋白的表達(dá)量升高(P<0.05)，細(xì)胞凋亡相關(guān)蛋白BAX表達(dá)升高，BCL-2表達(dá)降低(P<0.05)；而轉(zhuǎn)染lncRNA-MALAT1后，p-FAK蛋白表達(dá)量降低，BAX表達(dá)量降低，BCL-2表達(dá)量升高(P<0.05)。結(jié)論：lncRNA-MALAT1通過調(diào)控FAK的磷酸化水平，降低間歇低氧引起的HUVEC凋亡，并通過調(diào)節(jié)BCL-2/BAX蛋白表達(dá)水平參與了間歇低氧引起的HUVEC損傷，為開發(fā)治療間歇低氧引起的血管內(nèi)皮細(xì)胞損傷的新靶點(diǎn)提供新方法。

關(guān)鍵詞：lncRNA-MALAT1；磷酸化-FAK；BCL-2/BAX；HUVEC；間歇低氧

Abstract:Objectives:ToinvestigatetheroleandmechanismoflncRNA-MALAT1inintermittenthypoxia-inducedinjuryofhumanumbilicalveinendothelialcells(HUVEC),andprovidenewideasforfurtherunderstandingitsmolecularmechanism.Methods:HUVECsweredividedintothreegroups:controlgroup,modelgroupandlncRNA-MALAT1transfectiongroup,andwereculturedinstandardculturemedium,intermittenthypoxiaculture,andintermittenthypoxiacultureaftertransfectionwithlncRNA-MALAT1plasmid,respectively.MTTassaywasusedtodetectthegrowthactivityofHUVEC,andflowcytometryandAnnexinV-FITC/PIwereusedtodetecttheapoptosisofHUVEC.TheexpressionlevelsoflncRNA-MALAT1,focaladhesionkinase(FAK),phosphorylatedFAK(p-FAK),BCL-2/BAXproteinsweredetectedbyreal-timefluorescencequantitativePCRandWesternblot.Results:Underintermittenthypoxia,comparedwiththecontrolgroup,thegrowthactivityofHUVECinthemodelgroupdecreased,andtheapoptosisrateincreased(P<0.05);aftertransfectionwithlncRNA-MALAT1,thegrowthactivityofHUVECincreased(P<0.05),andtheapoptosisratedecreased(P<0.05).Real-timefluorescencequantitativePCRandWesternblotresultsshowedthatunderintermittenthypoxia,theexpressionlevelsoflncRNA-MALAT1andp-FAKproteinincreased,andtheexpressionofapoptosis-relatedproteinBAXincreasedandBCL-2decreased(P<0.05);whileaftertransfectionwithlncRNA-MALAT1,theexpressionofp-FAKproteindecreased,BAXexpressiondecreased,andBCL-2expressionincreased(P<0.05).Conclusion:lncRNA-MALAT1reducesHUVECapoptosiscausedbyintermittenthypoxiabyregulatingthephosphorylationlevelofFAK,andparticipatesinintermittenthypoxia-inducedHUVECinjurybyregulatingtheexpressionlevelsofBCL-2/BAXproteins,providingnewmethodsfordevelopingnewtargetsforthetreatmentofvascularendothelialcellsinjurycausedbyintermittenthypoxia.

Keywords:lncRNA-MALAT1;phosphorylatedFAK;BCL-2/BAX;HUVEC;intermittenthypoxiaIntermittenthypoxiaisacommonphenomenoninmanyclinicalconditions,includingobstructivesleepapneaandchronicobstructivepulmonarydisease.Thisconditioncanleadtovascularendothelialcellinjury,whichincreasestheriskofdevelopingcardiovasculardiseasessuchasatherosclerosisandhypertension.Therefore,understandingtheunderlyingmechanismsofvascularendothelialcellinjurycausedbyintermittenthypoxiaiscrucialfordevelopingeffectivetherapeuticstrategies.

Inthisstudy,wefocusedonlncRNA-MALAT1,whichhasbeenshowntobeinvolvedinvariousbiologicalprocesses,includingcellproliferationandapoptosis.OurresultsdemonstratedthatlncRNA-MALAT1reducedHUVECapoptosiscausedbyintermittenthypoxiabyregulatingthephosphorylationlevelofFAK.FAKisakeymoleculeinregulatingcelladhesion,migration,andproliferation.IthasbeenreportedthatthephosphorylationofFAKplaysacrucialroleinpromotingcellsurvivalunderstressconditions,suchashypoxia.

Furthermore,wefoundthatlncRNA-MALAT1participatedinintermittenthypoxia-inducedHUVECinjurybyregulatingtheexpressionlevelsofBCL-2/BAXproteins.BCL-2/BAXisawell-knownproteincomplexthatregulatestheapoptoticprocess.BCL-2isananti-apoptoticprotein,whileBAXisapro-apoptoticprotein.Thebalancebetweenthesetwoproteinsdeterminesthefateofthecells.OurresultsshowedthattheoverexpressionoflncRNA-MALAT1increasedtheexpressionofBCL-2anddecreasedtheexpressionofBAX,resultinginreducedHUVECapoptosis.

Overall,ourfindingssuggestthatlncRNA-MALAT1playsaprotectiveroleinintermittenthypoxia-inducedHUVECinjurybyregulatingthephosphorylationlevelofFAKandtheexpressionlevelsofBCL-2/BAXproteins.TargetinglncRNA-MALAT1mayprovideanoveltherapeuticstrategyforpreventingvascularendothelialcellinjurycausedbyintermittenthypoxia.However,furtherstudiesareneededtoelucidatetheexactmechanismoflncRNA-MALAT1inregulatingFAKphosphorylationandBCL-2/BAXexpressionFurtherstudiesarenecessarytodelineatethepossibledownstreameffectorsoflncRNA-MALAT1intheregulationofFAKphosphorylationandBCL-2/BAXexpression.ItwouldbeinterestingtoinvestigatethemolecularmechanismsbywhichlncRNA-MALAT1affectsthephosphorylationlevelofFAK.PreviousstudieshaveshownthatFAKactivationisregulatedbyvarioussignalingpathways,includingintegrinsignaling,growthfactorsignaling,andGprotein-coupledreceptorsignaling[1].ItispossiblethatlncRNA-MALAT1maymodulateoneormoreofthesepathwaystoregulateFAKphosphorylation.

Moreover,itremainsunclearhowlncRNA-MALAT1regulatestheexpressionlevelsofBCL-2/BAXproteins.BCL-2andBAXarekeyregulatorsofapoptosis,andtheirexpressionlevelsarefrequentlydysregulatedinvariousdiseases,includingcancerandcardiovasculardisease[2,3].ItispossiblethatlncRNA-MALAT1modulatestheexpressionofBCL-2/BAXbyeitherdirectlybindingtotheseproteinsorindirectlyregulatingtheexpressionoftheirupstreamregulators.

Inaddition,thepreciseroleoflncRNA-MALAT1inthepathogenesisofintermittenthypoxia-inducedvascularendothelialinjuryneedsfurtherinvestigation.WhileourfindingssuggestthatlncRNA-MALAT1playsaprotectiveroleinthisprocess,theexactmechanismsbywhichintermittenthypoxiainducesvascularendothelialdamageremainunclear.Previousstudieshaveimplicatedoxidativestress,inflammation,andmitochondrialdysfunctionasimportantcontributorstothepathologicaleffectsofintermittenthypoxiaonvascularendothelialcells[4,5].ItwouldbeinterestingtoinvestigatewhetherlncRNA-MALAT1modulatesthesepathwaystoprotectagainstintermittenthypoxia-inducedvascularendothelialinjury.

Inconclusion,ourstudysuggeststhatlncRNA-MALAT1playsanimportantroleinregulatingthephosphorylationlevelofFAKandtheexpressionlevelsofBCL-2/BAXproteinsinintermittenthypoxia-inducedHUVECinjury.TargetinglncRNA-MALAT1mayprovideapotentialtherapeuticstrategyforpreventingvascularendothelialcellinjurycausedbyintermittenthypoxia.However,furtherstudiesarenecessarytofullyelucidatethemechanismsunderlyingtheprotectiveeffectsoflncRNA-MALAT1anditspotentialasatherapeutictargetinthiscontextInconclusion,intermittenthypoxia-inducedvascularendothelialcellinjuryisacomplexprocessinvolvingmultiplesignalingpathwaysandmolecules.Understandingthemolecularmechanismsunderlyingthisprocessiscrucialtodevelopingeffectivetherapeuticstrategiestopreventortreatintermittenthypoxia-relateddiseases,suchassleepapnea,heartfailure,andstroke.

Inrecentyears,longnon-codingRNAshaveemergedasimportantregulatorsofgeneexpressionandcellularfunctionsinvariousphysiologicalandpathologicalconditions.Amongthem,lncRNA-MALAT1hasbeenshowntoplayacriticalroleinregulatingdiversecellularprocesses,includingproliferation,migration,apoptosis,andangiogenesis.

Inthisreview,wesummarizedthecurrentknowledgeontherolesoflncRNA-MALAT1invascularendothelialcellsanditspotentialimplicationsinintermittenthypoxia-inducedvascularendothelialcellinjury.WehighlightedtheevidencethatlncRNA-MALAT1exertsprotectiveeffectsonendothelialcellsviavariousmechanisms,includingregulatingtheproliferationandmigrationofcells,maintainingtheintegrityofendothelialbarrierfunction,andmodulatingapoptosis.

Furthermore,wediscussedthepotentialmolecularmechanismsunderlyingtheprotectiveeffectsoflncRNA-MALAT1inendothelialcells,includingtheregulationofFAKphosphorylationandBCL-2/BAXexpressionlevels.ThesefindingssuggestthatlncRNA-MALAT1isapromisingtherapeutictargetforpreventingortreatingintermittenthypoxia-inducedvascularendothelialcellinjuryandrelateddiseases.

However,furtherstudiesarenecessarytofullyelucidatethemechanismsunderlyingtheprotectiveeffectsoflncRNA-MALAT1anditspotentialasatherapeutictargetinthiscontext.Forexample,itwillbeimportanttodeterminethespecificdownstreamtargetsandsignalingpathwaysinvolvedinlncRNA-MALAT1-mediatedprotectiveeffectsinendothelialcells.Inaddition,the