NFIC過表達(dá)載體的構(gòu)建及鑒定

王昭穎：NFIC過表達(dá)載體的構(gòu)建及鑒定 摘要 核因子I（NFI）家族在人體正常發(fā)育過程中發(fā)揮重要的作用且與人類的發(fā)育異常有關(guān)，它們通過對(duì)靶基因的轉(zhuǎn)錄控制發(fā)揮調(diào)節(jié)細(xì)胞的增殖和分化的功能。而核因子C（NFIC）作為家族的一員，參與多種細(xì)胞調(diào)控，隨著近年來NFIC的深入研究，NFIC在癌癥方面的作用日益清晰，NFIC蛋白或結(jié)合位點(diǎn)會(huì)影響信號(hào)通路，相關(guān)細(xì)胞的增殖與分化，乳腺癌等多種癌癥發(fā)育過程中是關(guān)鍵的調(diào)節(jié)因子。故通過探究癌細(xì)胞中NFIC過表達(dá)對(duì)相關(guān)基因和蛋白的影響，可以明確其致癌或腫瘤抑制潛力。為了探究NFIC的作用，本文做了以下探究。本文構(gòu)建NFIC真核過表達(dá)載體，觀察其轉(zhuǎn)染293T細(xì)胞后其mRNA及蛋白表達(dá)。根據(jù)NFIC基因CDS設(shè)計(jì)并合成一對(duì)只適用于該基因的引物。提取293T細(xì)胞總核糖核苷酸，反轉(zhuǎn)錄為cDNA，PCR擴(kuò)增基因片段。將其克隆到真核表達(dá)載體pcDNA3.1中，構(gòu)建重組載體。HindⅢ和XhoⅠ雙酶切并測(cè)序鑒定后，轉(zhuǎn)染至細(xì)胞。Real-timePCR和WesternBlot檢測(cè)NFIC的mRNA和蛋白表達(dá)水平。成功擴(kuò)增基因，并連接至表達(dá)載體中；NFICmRNA和蛋白表達(dá)量顯著增高。本實(shí)驗(yàn)成功構(gòu)建NFIC過表達(dá)載體，闡述NFIC對(duì)于腫瘤發(fā)生的影響。關(guān)鍵詞NFIC腫瘤細(xì)胞載體構(gòu)建293T細(xì)胞

ConstructionandidentificationofNFICoverexpressionvectorAbstractThenuclearfactorI(NFI)transcriptionfactorsplayimportantrolesduringnormaldevelopmentandhavebeenassociatedwithdevelopmentalabnormalitiesinhumans.Theyfunctionbyregulatingcellproliferationanddifferentiationviathetranscriptionalcontroloftheirtargetgenes.NFIC,asamemberoftheNFIfamily,isinvolvedinavarietyofcellularregulation.WiththerecentresearchonNFIC,theroleofNFICincancerhasbecomeincreasinglyclear,andNFICproteinsorbindingsitesaffectsignalingpathways.Itisakeyregulatorinosteoblastdifferentiation,proliferationanddifferentiationofrelatedcells,anddevelopmentofvariouscancerssuchasbreastcancer.Therefore,byexploringtheeffectsofNFICoverexpressiononrelatedgenesandproteinsincancercells,thepotentialofcarcinogenesisortumorsuppressioncanbeclarified.OnthebasisofexpoundingtheinfluenceofNFIfamilymemberNFIContumorigenesis,thisstudyfurtherconstructedanoverexpressionvectortoexplainthemechanismofactionofNFIConcancercells.ToconstructhumanNFICgeneoverexpressionvectorandidentifyitsexpressioninthecellsof293T.HumanNFICgenesequencewasobtainedbyPCRamplificationusing293TcellscDNAlibraryasatemplateandthenthegenesegmentwasintegratedintotheeukaryoticexpressionvectorpcDNA3.1,recombinant

plasmids

were

identified

using

HindIII

and

XhoI

restriction

enzymes

and

sequenced.

The

successfully

cloned

vector

was

then

transfected

into

cells

and

the

expression

of

NFIC

was

determined

by

RealTime-PCR

and

Western

blot.

The

human

NFIC

gene

was

successfully

amplified

and

ligated

into

the

vector；the

expression

of

NFIC

mRNA

and

protein

was

up-regulated

in

cells

after

transfection。NFICoverexpressionvectorissuccessfullyconstructed,whichcouldbeusedintothepoststudy.Keywords:NFICgeneTumorcellVectorconstruction293Tcells

目錄摘要 IAbstract II第一章緒論 11.1NFI家族 11.1.1簡(jiǎn)介 11.1.2作用 11.2NFIC簡(jiǎn)介 21.2.1NFIC對(duì)牙根發(fā)育的作用 21.2.2NFIC在骨細(xì)胞分化方面的作用 21.2.3NFIC對(duì)細(xì)胞增殖與凋亡的影響 21.2.4NFIC在癌癥中的作用 3第二章材料與方法 72.1主要試劑及器材 72.1.1材料 72.1.2儀器 72.1.3試劑 82.2方法 92.2.1引物設(shè)計(jì)合成 92.2.2目的片段獲取 92.2.3酶切連接 102.2.5轉(zhuǎn)化 112.2.6挑菌、搖菌、保菌 112.2.7質(zhì)粒小提 112.2.8酶切鑒定，測(cè)序 122.2.9質(zhì)粒大提 122.2.10293T細(xì)胞培養(yǎng) 122.2.11轉(zhuǎn)染 132.2.12NFICmRNA表達(dá)檢測(cè) 132.2.13NFIC蛋白檢測(cè) 13第三章 結(jié)果 143.1NCBI上查找NFIC（人源）基因及其CDS序列 153.2設(shè)計(jì)引物 163.3瓊脂糖凝膠電泳檢測(cè)PCR產(chǎn)物 163.4pcDNA3.1-NFIC重組質(zhì)粒的構(gòu)建 163.5雙酶切鑒定pcDNA3.1-NFIC 173.6目的基因測(cè)序 183.7Real-timePCR檢測(cè)目的基因mRNA 193.8Westernblot檢測(cè)目的蛋白表達(dá)量 19第四章討論 20第五章結(jié)論 21致謝 22參考文獻(xiàn) 23

第一章緒論1.1NFI家族1.1.1簡(jiǎn)介NFI家族ADDINEN.CITEADDINEN.CITE.DATA[1]是一類二聚體DNA結(jié)合蛋白，作為轉(zhuǎn)錄因子可以調(diào)控多種基因的表達(dá)。NFI家族在人類中包括NFIA，NFIB，NFIC和NFIX。具有特異結(jié)合位點(diǎn)，成員ADDINEN.CITE<EndNote><Cite><Author>M</Author><Year>1986</Year><RecNum>69</RecNum><DisplayText><styleface="superscript">[2]</style></DisplayText><record><rec-number>69</rec-number><foreign-keys><keyapp="EN"db-id="wt0zfwpza5r90uepsdw59s9y5fwzazftfffp"timestamp="1559912628">69</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>GronostajskiRM</author></authors></contributors><titles><title>AnalysisofnuclearfactorIbindingtoDNAusingdegenerateoligonucleotides.%JNucleicAcidsResearch</title></titles><volume>14</volume><number>22</number><keywords><keyword>BaseSequence</keyword><keyword>CCAAT-Enhancer-BindingProteins</keyword><keyword>CellNucleus</keyword><keyword>DNA</keyword><keyword>DNA-BindingProteins</keyword><keyword>HeLaCells</keyword><keyword>Humans</keyword><keyword>Kinetics</keyword><keyword>NFITranscriptionFactors</keyword><keyword>NuclearProteins</keyword><keyword>Oligodeoxyribonucleotides</keyword><keyword>ProteinBinding</keyword><keyword>Structure-ActivityRelationship</keyword><keyword>TranscriptionFactors</keyword><keyword>Y-Box-BindingProtein1</keyword></keywords><dates><year>1986</year></dates><isbn>0305-1048</isbn><urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>[2]含有一致序列TGG（N）6-7GCCAA的DNA緊密結(jié)合，形成同或異二聚體。1.1.2作用NFI在轉(zhuǎn)錄調(diào)控ADDINEN.CITE<EndNote><Cite><Author>M</Author><Year>2000</Year><RecNum>62</RecNum><DisplayText><styleface="superscript">[3]</style></DisplayText><record><rec-number>62</rec-number><foreign-keys><keyapp="EN"db-id="wt0zfwpza5r90uepsdw59s9y5fwzazftfffp"timestamp="1559833919">62</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>GronostajskiRM</author></authors></contributors><auth-address>DepartmentofCancerBiology,LernerResearchInstitute,ClevelandClinicFoundation,CaseWesternReserveUniversity,OH44195,USA.gronosr@</auth-address><titles><title>RolesoftheNFI/CTFgenefamilyintranscriptionanddevelopment.%JGene</title></titles><volume>249</volume><number>1-2</number><keywords><keyword>Animals</keyword><keyword>CCAAT-Enhancer-BindingProteins</keyword><keyword>DNA-BindingProteins</keyword><keyword>GeneExpressionRegulation</keyword><keyword>Developmental</keyword><keyword>Humans</keyword><keyword>MultigeneFamily</keyword><keyword>NFITranscriptionFactors</keyword><keyword>NuclearProteins</keyword><keyword>TranscriptionFactors</keyword><keyword>Transcription</keyword><keyword>Genetic</keyword><keyword>Y-Box-BindingProtein1</keyword></keywords><dates><year>2000</year></dates><isbn>0378-1119</isbn><urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>[3]特別是在發(fā)育過程中的轉(zhuǎn)錄調(diào)控有重要作用，且參與調(diào)控DNA復(fù)制和基因表達(dá)。所有的家族成員NFIA，NFIB，NFIC，NFIX，它們通過對(duì)靶基因的轉(zhuǎn)錄調(diào)控，調(diào)節(jié)細(xì)胞增殖和分化。通常調(diào)節(jié)細(xì)胞增殖，侵襲，分化等過程的轉(zhuǎn)錄因子在癌細(xì)胞發(fā)生時(shí)被破壞，要么直接通過突變，要么間接通過染色體易位。由這些轉(zhuǎn)錄因子調(diào)節(jié)的通路即可以和人體發(fā)育過程中的兒科腫瘤有關(guān)，也與由突變導(dǎo)致的成人癌癥有關(guān)，這種突變使成熟細(xì)胞回復(fù)突變?yōu)榭梢钥焖僭鲋车陌l(fā)育表型。NFI或CCAAT框結(jié)合轉(zhuǎn)錄因子（CTF）基因家族在刺激腺病毒DNA復(fù)制的啟動(dòng)方面發(fā)揮作用，在轉(zhuǎn)錄調(diào)控方面也發(fā)揮著重要作用，尤其是在發(fā)育過程中。最近，基于基因組分析和動(dòng)物模型的研究，NFI基因在多個(gè)器官系統(tǒng)的多種腫瘤中發(fā)現(xiàn)，包括CNS，乳腺和肺。驅(qū)動(dòng)造血功能，成骨細(xì)胞瘤，黑色素瘤都需要這些轉(zhuǎn)錄因子ADDINEN.CITEADDINEN.CITE.DATA[1]。通過基因敲除的方式發(fā)現(xiàn)，NFIA的丟失導(dǎo)致胼胝體的腦積水和發(fā)育不全ADDINEN.CITEADDINEN.CITE.DATA[4]，NFIA缺陷小鼠顯示出顯著的大腦表型，包括胼胝體的發(fā)育不全和中線神經(jīng)膠質(zhì)細(xì)胞群的畸形，所述神經(jīng)膠質(zhì)細(xì)胞群需要引導(dǎo)胼胝體軸突穿過發(fā)育中腦的中線；NFIB缺乏導(dǎo)致神經(jīng)缺陷和嚴(yán)重的肺發(fā)育不全ADDINEN.CITEADDINEN.CITE.DATA[5]，NFIB在肺成熟期間調(diào)節(jié)增殖和上皮分化，NFIB敲除動(dòng)物有嚴(yán)重的肺發(fā)育不全和發(fā)育缺陷；NFIX缺乏導(dǎo)致嚴(yán)重的腦畸形和骨骼缺陷ADDINEN.CITE<EndNote><Cite><Author>Katrin</Author><Year>2007</Year><RecNum>63</RecNum><DisplayText><styleface="superscript">[6]</style></DisplayText><record><rec-number>63</rec-number><foreign-keys><keyapp="EN"db-id="wt0zfwpza5r90uepsdw59s9y5fwzazftfffp"timestamp="1559835114">63</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>DrillerKatrin</author><author>PagenstecherAxel</author><author>UhlMarkus</author><author>OmranHeymut</author><author>BerlisAnsgar</author><author>GründerAlbert</author><author>SippelAlbrechtE</author></authors></contributors><auth-address>InstitutfürBiologieIII,Fakult?tfürBiologie,Albert-LudwigsUniversit?tFreiburg,Sch?nzlestrasse1,D-79104Freiburg,Germany.</auth-address><titles><title>NuclearfactorIXdeficiencycausesbrainmalformationandsevereskeletaldefects.%JMolecularandCellularBiology</title></titles><volume>27</volume><number>10</number><keywords><keyword>Animals</keyword><keyword>BiologicalMarkers</keyword><keyword>Brain</keyword><keyword>CollagenTypeII</keyword><keyword>CorpusCallosum</keyword><keyword>Femur</keyword><keyword>GeneTargeting</keyword><keyword>Humans</keyword><keyword>Hydrocephalus</keyword><keyword>Lectins</keyword><keyword>C-Type</keyword><keyword>Mice</keyword><keyword>Mice</keyword><keyword>Knockout</keyword><keyword>NFITranscriptionFactors</keyword><keyword>Osteocalcin</keyword><keyword>Osteogenesis</keyword><keyword>Phenotype</keyword><keyword>Spine</keyword></keywords><dates><year>2007</year></dates><isbn>0270-7306</isbn><urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>[6]，NFIX的缺失是出生后致死的，并導(dǎo)致腦積水和胼胝體體部分發(fā)育不全。此外，NFIX缺陷小鼠發(fā)生脊柱變形，這是由于椎體骨化延遲和椎間盤逐漸退化；而機(jī)體缺失NFIC則ADDINEN.CITE<EndNote><Cite><Author>Tae-Yeon</Author><Year>2009</Year><RecNum>64</RecNum><DisplayText><styleface="superscript">[7]</style></DisplayText><record><rec-number>64</rec-number><foreign-keys><keyapp="EN"db-id="wt0zfwpza5r90uepsdw59s9y5fwzazftfffp"timestamp="1559835734">64</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>LeeTae-Yeon</author><author>LeeDong-Seol</author><author>KimHyun-Man</author><author>KoJeaSeung</author><author>GronostajskiRichardM</author><author>ChoMoon-Il</author><author>SonHo-Hyun</author><author>ParkJoo-Cheol</author></authors></contributors><auth-address>DepartmentofConservativeDentistryandDentalResearchInstitute,CollegeofDentistry,SeoulNationalUniversity,28Yeon-GunDong,Jong-RoGu,Seoul110-749,Korea.</auth-address><titles><title>DisruptionofNficcausesdissociationofodontoblastsbyinterferingwiththeformationofintercellularjunctionsandaberrantodontoblastdifferentiation.%JJournalofHistochemistryandCytochemistry</title></titles><volume>57</volume><number>5</number><keywords><keyword>Animals</keyword><keyword>CellDifferentiation</keyword><keyword>Dentin</keyword><keyword>Immunohistochemistry</keyword><keyword>Incisor</keyword><keyword>IntercellularJunctions</keyword><keyword>Mice</keyword><keyword>Mice</keyword><keyword>Knockout</keyword><keyword>Molar</keyword><keyword>NFITranscriptionFactors</keyword><keyword>Odontoblasts</keyword><keyword>ReverseTranscriptasePolymeraseChainReaction</keyword><keyword>ToothRoot</keyword></keywords><dates><year>2009</year></dates><isbn>0022-1554</isbn><urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>[7]會(huì)影響成牙本質(zhì)細(xì)胞的生成及分化，NFIC基因的缺失會(huì)干擾NFIC基因形成細(xì)胞間連接，導(dǎo)致異常成牙本質(zhì)細(xì)胞分化和牙本質(zhì)形成異常。成牙本質(zhì)細(xì)胞的這些變化共同促成了NFIC缺陷小鼠中具有短根和異常根的臼齒的發(fā)育。1.2NFIC簡(jiǎn)介NFIC是一類特異性的轉(zhuǎn)錄因子，屬于NFI家族，NFIC蛋白或結(jié)合位點(diǎn)會(huì)影響信號(hào)通路，在骨細(xì)胞分化，相關(guān)細(xì)胞的增殖與分化，腦部腫瘤等多種癌癥發(fā)育過程中是關(guān)鍵的調(diào)節(jié)因子。1.2.1NFIC對(duì)牙根發(fā)育的作用NFIC缺陷小鼠形成ADDINEN.CITE<EndNote><Cite><Author>HJ</Author><Year>2007</Year><RecNum>71</RecNum><DisplayText><styleface="superscript">[8]</style></DisplayText><record><rec-number>71</rec-number><foreign-keys><keyapp="EN"db-id="wt0zfwpza5r90uepsdw59s9y5fwzazftfffp"timestamp="1559973113">71</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>KimHJ</author><author>ParkJC</author><author>ChoMI</author><author>GronostajskiRM</author><author>HerrY</author></authors></contributors><auth-address>DepartmentofOralHistologyandAnatomy,CollegeofDentistry,ChosunUniversity,Gwang-Ju,Korea.jcapark@chosun.ac.kr</auth-address><titles><title>Nficgenedisruptioninhibitsdifferentiationofodontoblastsresponsibleforrootformationandresultsinformationofshortandabnormalrootsinmice.%JJournalofPeriodontology</title></titles><pages>1795-1802</pages><volume>78</volume><number>9</number><keywords><keyword>PlantRoots</keyword><keyword>Odontoblasts</keyword><keyword>Laboratorymice</keyword><keyword>differentiation</keyword><keyword>植物根</keyword><keyword>成牙質(zhì)細(xì)胞</keyword></keywords><dates><year>2007</year></dates><isbn>0022-3492</isbn><urls><related-urls><url>/details/detail.do?_type=perio&id=c9588565eeae8cde82cc7ccd9ffe4bfd</url></related-urls></urls><remote-database-provider>北京萬方數(shù)據(jù)股份有限公司</remote-database-provider><language>eng</language></record></Cite></EndNote>[8]正常的Hertwig上皮根鞘（HERS），但嚴(yán)重破壞成牙本質(zhì)細(xì)胞分化，導(dǎo)致在根形成的早期階段形成異常成牙本質(zhì)細(xì)胞。它們變得分離并呈多邊形，失去了方向和極性，并且不表達(dá)牙本質(zhì)涎磷蛋白。NFIC的丟失不會(huì)干擾HERS的形成，但會(huì)導(dǎo)致成牙本質(zhì)細(xì)胞分化，牙骨質(zhì)減少。因此，NFIC是根成牙本質(zhì)細(xì)胞分化和根形成的關(guān)鍵調(diào)節(jié)因子。1.2.2NFIC在骨細(xì)胞分化方面的作用NFIC對(duì)于骨發(fā)育有重要的調(diào)控作用。研究發(fā)現(xiàn)，NFI家族在人類成骨細(xì)胞中都表達(dá)。相比而言，NFIC在正常的成骨細(xì)胞中表達(dá)量較高ADDINEN.CITE<EndNote><Cite><Author>劉蒼維</Author><Year>2018</Year><RecNum>53</RecNum><DisplayText><styleface="superscript">[9]</style></DisplayText><record><rec-number>53</rec-number><foreign-keys><keyapp="EN"db-id="wt0zfwpza5r90uepsdw59s9y5fwzazftfffp"timestamp="1559187163">53</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>劉蒼維</author><author>周怡君</author><author>閆廣興</author><author>史冊(cè)</author><author>張雪</author><author>胡月</author><author>郝新青</author><author>趙歡</author><author>孫宏晨</author></authors></contributors><auth-address>吉林大學(xué)口腔醫(yī)院病理科吉林省牙發(fā)育及頜骨重塑與再生重點(diǎn)實(shí)驗(yàn)室;</auth-address><titles><title>骨形態(tài)發(fā)生蛋白信號(hào)通路在牙根發(fā)育中的作用%J華西口腔醫(yī)學(xué)雜志</title></titles><pages>559-563</pages><volume>36</volume><number>05</number><keywords><keyword>骨形態(tài)發(fā)生蛋白</keyword><keyword>牙發(fā)生</keyword><keyword>上皮根鞘</keyword></keywords><dates><year>2018</year></dates><isbn>1000-1182</isbn><call-num>51-1169/R</call-num><urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>[9]，且與BMP-Smad通路相關(guān)因子的表達(dá)有關(guān)。魏澤寧等ADDINEN.CITE<EndNote><Cite><Author>魏澤寧</Author><Year>2016</Year><RecNum>45</RecNum><DisplayText><styleface="superscript">[10]</style></DisplayText><record><rec-number>45</rec-number><foreign-keys><keyapp="EN"db-id="wt0zfwpza5r90uepsdw59s9y5fwzazftfffp"timestamp="1557971292">45</key></foreign-keys><ref-typename="Thesis">32</ref-type><contributors><authors><author>魏澤寧</author></authors><tertiary-authors><author>李亞男,</author></tertiary-authors></contributors><titles><title>1型糖尿病大鼠Nfic與成骨成脂相關(guān)基因表達(dá)的探究及胰島素的干預(yù)作用</title></titles><keywords><keyword>鏈脲佐菌素</keyword><keyword>1型糖尿病模型</keyword><keyword>糖尿病</keyword><keyword>Nfic</keyword><keyword>成骨細(xì)胞</keyword><keyword>成骨相關(guān)基因</keyword><keyword>Runx2</keyword><keyword>ALP</keyword><keyword>OSX</keyword><keyword>IGF-1</keyword><keyword>COL2A1</keyword><keyword>OC</keyword><keyword>BMP-2</keyword><keyword>PPARγ</keyword><keyword>成脂分化</keyword><keyword>胰島素</keyword><keyword>大鼠</keyword><keyword>mRNA</keyword></keywords><dates><year>2016</year></dates><publisher>中國(guó)人民解放軍醫(yī)學(xué)院</publisher><work-type>碩士</work-type><urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>[10]的研究發(fā)現(xiàn)NFIC基因與Runx2等成骨相關(guān)基因表達(dá)量呈正相關(guān)。說明NFIC基因影響成骨。LeeADDINEN.CITEADDINEN.CITE.DATA[11]等研究支持NFIC在BMP-Smad成骨通路中的作用。其在Runx2和Osterix基因之間起到中介傳感器的作用。1.2.3NFIC對(duì)細(xì)胞增殖與凋亡的影響位點(diǎn)特異性ADDINEN.CITE<EndNote><Cite><Author>M</Author><Year>1986</Year><RecNum>69</RecNum><DisplayText><styleface="superscript">[2]</style></DisplayText><record><rec-number>69</rec-number><foreign-keys><keyapp="EN"db-id="wt0zfwpza5r90uepsdw59s9y5fwzazftfffp"timestamp="1559912628">69</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>GronostajskiRM</author></authors></contributors><titles><title>AnalysisofnuclearfactorIbindingtoDNAusingdegenerateoligonucleotides.%JNucleicAcidsResearch</title></titles><volume>14</volume><number>22</number><keywords><keyword>BaseSequence</keyword><keyword>CCAAT-Enhancer-BindingProteins</keyword><keyword>CellNucleus</keyword><keyword>DNA</keyword><keyword>DNA-BindingProteins</keyword><keyword>HeLaCells</keyword><keyword>Humans</keyword><keyword>Kinetics</keyword><keyword>NFITranscriptionFactors</keyword><keyword>NuclearProteins</keyword><keyword>Oligodeoxyribonucleotides</keyword><keyword>ProteinBinding</keyword><keyword>Structure-ActivityRelationship</keyword><keyword>TranscriptionFactors</keyword><keyword>Y-Box-BindingProtein1</keyword></keywords><dates><year>1986</year></dates><isbn>0305-1048</isbn><urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>[2]DNA結(jié)合蛋白在調(diào)節(jié)細(xì)胞凋亡中發(fā)揮重要作用，NFIC參與多種細(xì)胞增殖與凋亡的調(diào)控。以下簡(jiǎn)單介紹一下NFIC對(duì)于根尖乳頭干細(xì)胞，肝增殖過程中的作用。近來研究ADDINEN.CITEADDINEN.CITE.DATA[12]已確定NFIC對(duì)人干細(xì)胞來源的根尖牙乳頭(SCAPs)增殖的作用，JingZhang等人推測(cè)NFIC可能是促進(jìn)牙本質(zhì)/根再生的重要靶點(diǎn)。通過MTT，ALP活性檢測(cè)，siRNA轉(zhuǎn)染，蛋白質(zhì)印記等方法對(duì)SCAP進(jìn)行研究，發(fā)現(xiàn)過度表達(dá)NFIC增加SCAP中細(xì)胞增殖，礦化結(jié)節(jié)形成和堿性磷酸酶（ALP）活動(dòng)增加。NFIC被siRNA敲除會(huì)抑制SCAP的礦化能力，下調(diào)牙源性相關(guān)標(biāo)記物的表達(dá)。即SCAP中NFIC活性的上調(diào)可能促進(jìn)SCAP的骨/成牙本質(zhì)細(xì)胞分化。NFIC作為調(diào)控因子作用于肝再生時(shí)的肝細(xì)胞增殖是由SimoneEdelmann等人ADDINEN.CITEADDINEN.CITE.DATA[13]提出的，對(duì)小鼠NFIC的基因敲除發(fā)現(xiàn)了小鼠附器皮膚創(chuàng)傷愈合異常和生長(zhǎng)異常，表明了NFIC在成體再生過程中的控制作用，雖然NFIC在肝臟中高表達(dá)，但尚未建立起該轉(zhuǎn)錄因子肝細(xì)胞增殖方面的功能模型。為了確定NFIC是否在肝細(xì)胞增殖和肝再生中起重要作用，分別在NFIC基因敲除(Ko)和野生型(Wt)鼠中，觀察肝再生和細(xì)胞增殖途徑。結(jié)果表明NFIC在野生小鼠細(xì)胞再生開始時(shí)首先起促進(jìn)肝細(xì)胞增殖的作用，隨后NFIC的短暫下降-正如轉(zhuǎn)化生長(zhǎng)因子β1(TGF-1)的自我調(diào)節(jié)反饋回路所解釋的那樣，可能限制了肝細(xì)胞進(jìn)入第一波細(xì)胞分裂期的數(shù)量和（或）阻止有絲分裂的后期啟動(dòng)。進(jìn)一步的實(shí)驗(yàn)發(fā)現(xiàn)：NFIC的缺乏導(dǎo)致PAI-1的過度表達(dá)，從而使PAI-1的表達(dá)減弱。盡管細(xì)胞周期進(jìn)程有很大的下降，uPA通路導(dǎo)致肝細(xì)胞生長(zhǎng)因子（HGF）活性降低，Ko小鼠由于肥大，其肝臟的再生速度和肝細(xì)胞的反應(yīng)與Wt小鼠相當(dāng)。此外，我們還發(fā)現(xiàn)Ko小鼠在肝再生開始時(shí)，TGF-1的激活有延遲和降低的趨勢(shì)。在Wt小鼠中，NFIC可能與啟動(dòng)子協(xié)同作用。在再生過程的早期，uPA等對(duì)肝細(xì)胞增殖有促進(jìn)作用。在稍后階段，NFIC的表達(dá)被下調(diào)，這可能是由于與TGF-1形成負(fù)反饋循環(huán)從而自我調(diào)控的結(jié)果，可能有助于抑制有絲分裂期，從而介導(dǎo)肝細(xì)胞的瞬時(shí)增殖。總的來說，NFIC作為一個(gè)調(diào)控因子在肝再生過程中進(jìn)行肝細(xì)胞階段性增殖的調(diào)控。研究發(fā)現(xiàn)ADDINEN.CITE<EndNote><Cite><Author>Dong-Seol</Author><Year>2009</Year><RecNum>72</RecNum><DisplayText><styleface="superscript">[14]</style></DisplayText><record><rec-number>72</rec-number><foreign-keys><keyapp="EN"db-id="wt0zfwpza5r90uepsdw59s9y5fwzazftfffp"timestamp="1560058662">72</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>LeeDong-Seol</author><author>ParkJong-Tae</author><author>KimHyun-Man</author><author>KoJeaSeung</author><author>SonHo-Hyun</author><author>GronostajskiRichardM</author><author>ChoMoon-Il</author><author>ChoungPill-Hoon</author><author>ParkJoo-Cheol</author></authors></contributors><auth-address>DepartmentofOralHistology-DevelopmentalBiology,DentalResearchInstitute,SchoolofDentistry,SeoulNationalUniversity,Seoul110-749,Korea.</auth-address><titles><title>NuclearfactorI-Cisessentialforodontogeniccellproliferationandodontoblastdifferentiationduringtoothrootdevelopment.%JJBCPapersinPress</title></titles><volume>284</volume><number>25</number><keywords><keyword>AminoAcidSequence</keyword><keyword>Animals</keyword><keyword>BaseSequence</keyword><keyword>CellDifferentiation</keyword><keyword>CellProliferation</keyword><keyword>Cells</keyword><keyword>Cultured</keyword><keyword>DNAPrimers</keyword><keyword>Dentin</keyword><keyword>GeneExpressionRegulation</keyword><keyword>Developmental</keyword><keyword>Mice</keyword><keyword>Mice</keyword><keyword>Knockout</keyword><keyword>MolecularSequenceData</keyword><keyword>NFITranscriptionFactors</keyword><keyword>Odontoblasts</keyword><keyword>Odontogenesis</keyword><keyword>Protein-Serine-ThreonineKina...</keyword></keywords><dates><year>2009</year></dates><urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>[14]，NFIC缺陷的原漿細(xì)胞中p21和p16的表達(dá)增加，但D1和B1（細(xì)胞周期蛋白）的表達(dá)量減少，表明了缺乏NFIC活性引起的細(xì)胞生長(zhǎng)停滯。對(duì)NFIC缺陷小鼠中的牙髓和異常牙本質(zhì)的分析揭示了凋亡活性的增加。此外，NFIC缺陷的原漿細(xì)胞表現(xiàn)出caspase-8和casepase-3（屬于線粒體凋亡途徑）活化的增加，而幾乎未檢測(cè)到降解狀態(tài)的Bid。這些結(jié)果表明，NFIC的喪失抑制牙本質(zhì)細(xì)胞增殖和分化的，并誘導(dǎo)異常成牙本質(zhì)細(xì)胞的凋亡。NFIC轉(zhuǎn)錄因子在一些組織細(xì)胞中具有調(diào)控細(xì)胞增殖作用，通過調(diào)控一些牙源標(biāo)志物，骨鈣素等控制根尖牙乳頭細(xì)胞的增殖；在肝增殖方面，通過控制下游的PAI-1和TGF-1來調(diào)控肝細(xì)胞的增殖。1.2.4NFIC在癌癥中的作用隨著ADDINEN.CITEADDINEN.CITE.DATA[1]近年來NFIC的深入研究，NFIC在癌癥方面的作用日益清晰，多種癌癥中發(fā)現(xiàn)NFIC及其蛋白的存在，本文就腦部相關(guān)腫瘤及乳腺癌中NFIC的作用做簡(jiǎn)要介紹。(1)腦部相關(guān)腫瘤NFI家族也參與調(diào)控腦部相關(guān)腫瘤，與NFI家族作用最密切的是膠質(zhì)母細(xì)胞瘤（GBM）。膠質(zhì)母細(xì)胞瘤是一種高度侵襲性腦腫瘤。NFI已經(jīng)被證明ADDINEN.CITE<EndNote><Cite><Author>Miranda</Author><Year>2009</Year><RecNum>68</RecNum><DisplayText><styleface="superscript">[15]</style></DisplayText><record><rec-number>68</rec-number><foreign-keys><keyapp="EN"db-id="wt0zfwpza5r90uepsdw59s9y5fwzazftfffp"timestamp="1559874279">68</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>BrunMiranda</author><author>ColesJeffreyE</author><author>MoncktonElizabethA</author><author>GlubrechtDarrylD</author><author>BisgroveDwayne</author><author>GodboutRoseline</author></authors></contributors><auth-address>DepartmentofOncology,CrossCancerInstitute,UniversityofAlberta,Alberta,Canada.</auth-address><titles><title>NuclearfactorIregulatesbrainfattyacid-bindingproteinandglialfibrillaryacidicproteingeneexpressioninmalignantgliomacelllines.%JJMBOnline</title></titles><volume>391</volume><number>2</number><keywords><keyword>3'FlankingRegion</keyword><keyword>BaseSequence</keyword><keyword>BindingSites</keyword><keyword>CarrierProteins</keyword><keyword>CellLine</keyword><keyword>Tumor</keyword><keyword>GeneExpressionRegulation</keyword><keyword>Neoplastic</keyword><keyword>GeneKnockdownTechniques</keyword><keyword>GlialFibrillaryAcidicProtein</keyword><keyword>Glioma</keyword><keyword>Humans</keyword><keyword>MolecularSequenceData</keyword><keyword>Mutation</keyword><keyword>NFITranscriptionFactors</keyword><keyword>PromoterRegions</keyword><keyword>Genetic</keyword><keyword>TumorSup...</keyword></keywords><dates><year>2009</year></dates><urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>[15]可以調(diào)節(jié)星形膠質(zhì)細(xì)胞標(biāo)記物例如膠質(zhì)纖維酸性蛋白(GFAP)在正常腦和GBM細(xì)胞中的表達(dá)。MirandaBrun等人ADDINEN.CITEADDINEN.CITE.DATA[16]通過CHIP（染色體免疫共沉淀）來確定GBM細(xì)胞中額外的NFI靶標(biāo)。通過對(duì)芯片數(shù)據(jù)的分析，發(fā)現(xiàn)了約400個(gè)NFI靶基因，其中包括ADDINEN.CITE<EndNote><Cite><Author>Maddalena</Author><Year>2010</Year><RecNum>70</RecNum><DisplayText><styleface="superscript">[17]</style></DisplayText><record><rec-number>70</rec-number><foreign-keys><keyapp="EN"db-id="wt0zfwpza5r90uepsdw59s9y5fwzazftfffp"timestamp="1559915962">70</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>LinoMariaMaddalena</author><author>MerloAdrian</author><author>BoulayJean-Louis</author></authors></contributors><auth-address>DepartmentofBiomedicine,UniversityHospitalBasel,Basel,Switzerland.</auth-address><titles><title>Notchsignalinginglioblastoma:adevelopmentaldrugtarget?%JBMCMedicine</title></titles><volume>8</volume><keywords><keyword>Anoxia</keyword><keyword>CellDifferentiation</keyword><keyword>CellProliferation</keyword><keyword>Glioblastoma</keyword><keyword>Humans</keyword><keyword>Neovascularization</keyword><keyword>Pathologic</keyword><keyword>Receptors</keyword><keyword>Notch</keyword><keyword>SignalTransduction</keyword></keywords><dates><year>2010</year></dates><urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>[17]Notch信號(hào)的一個(gè)效應(yīng)因子HEY1，參與ADDINEN.CITE<EndNote><Cite><Author>Esther</Author><Year>2008</Year><RecNum>65</RecNum><DisplayText><styleface="superscript">[18]</style></DisplayText><record><rec-number>65</rec-number><foreign-keys><keyapp="EN"db-id="wt0zfwpza5r90uepsdw59s9y5fwzazftfffp"timestamp="1559836146">65</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>HullemanEsther</author><author>QuartoMicaela</author><author>VernellRichard</author><author>MasserdottiGiacomo</author><author>ColliElena</author><author>KrosJohanM</author><author>LeviDaniel</author><author>GaetaniPaolo</author><author>TuniciPatrizia</author><author>FinocchiaroGaetano</author><author>BaenaRiccardoRodriguezY</author><author>CapraMaria</author><author>HelinKristian</author></authors></contributors><auth-address>EuropeanInstituteofOncology,ViaRipamonti,Milan,Italy.</auth-address><titles><title>AroleforthetranscriptionfactorHEY1inglioblastoma.%JJournalofCellularandMolecularMedicine(Online)</title></titles><volume>13</volume><number>1</number><keywords><keyword>Animals</keyword><keyword>BasicHelix-Loop-HelixTranscriptionFactors</keyword><keyword>BrainNeoplasms</keyword><keyword>CellCycleProteins</keyword><keyword>CellLine</keyword><keyword>CellProliferation</keyword><keyword>DiseaseProgression</keyword><keyword>E2FTranscriptionFactors</keyword><keyword>Glioblastoma</keyword><keyword>Humans</keyword><keyword>Mice</keyword><keyword>Mice</keyword><keyword>InbredC57BL</keyword><keyword>RNAInterference</keyword><keyword>Receptors</keyword><keyword>Notch</keyword><keyword>SignalTransduction</keyword><keyword>TissueArrayAnalysis</keyword></keywords><dates><year>2008</year></dates><urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>[18]神經(jīng)干細(xì)胞的維持。所有NFI（NFIA，NFIB，NFIC和NFIX）都結(jié)合到位于HEY1轉(zhuǎn)錄位點(diǎn)上游1kb以內(nèi)的NFI識(shí)別位點(diǎn)。進(jìn)一步實(shí)驗(yàn)表明，NFI負(fù)調(diào)控HEY1的表達(dá)，GBM細(xì)胞中所有四種NFI均被敲除導(dǎo)致HEY1RNA水平升高。GBM細(xì)胞中HEY1的敲除降低了細(xì)胞增殖，細(xì)胞遷移增加，神經(jīng)球細(xì)胞形成減少。最后，我們發(fā)現(xiàn)HEY1水平升高與腦神經(jīng)干/祖細(xì)胞表達(dá)之間存在著普遍的相關(guān)性。HEY1基因敲除導(dǎo)致GFAP星形膠質(zhì)細(xì)胞分化標(biāo)志RNA水平升高?？偟膩碚f，HEY1是由NFI家族成員負(fù)調(diào)控的，與GBM細(xì)胞增殖增加，遷移減少和干細(xì)胞特性增加有關(guān)。NFI抑制HEY1表達(dá)和結(jié)合，CHIP和凝膠轉(zhuǎn)移實(shí)驗(yàn)共同顯示NFI與HEY1啟動(dòng)子中的三個(gè)不同區(qū)域，表明NFI家族在調(diào)節(jié)啟動(dòng)子表達(dá)方面的作用。因?yàn)镹FI家族包括四個(gè)家族成員，所以要找出哪個(gè)家族成員優(yōu)先綁定于HEY1轉(zhuǎn)錄起始位點(diǎn)上游的NFI識(shí)別位點(diǎn)，檢測(cè)了NFI水平的變化是否會(huì)影響內(nèi)源性HEY1mRNA的水平。用對(duì)照siRNA或特異性靶向NFI的siRNA單獨(dú)或聯(lián)合轉(zhuǎn)染U251細(xì)胞，基因敲除后，內(nèi)源性HEY1mRNA水平無明顯變化。然而，當(dāng)四個(gè)NFI全部耗盡時(shí)，觀察到HEY1mRNA水平增加了2.4倍，兩輪的NFIsiRNA轉(zhuǎn)染產(chǎn)生了更好的結(jié)果：增加了(4.6倍)的HEY1mRNA水平。這些數(shù)據(jù)表明NFI家族的多個(gè)成員參與了HEY1的調(diào)控。與對(duì)照siRNA相比，所有四個(gè)NFI的轉(zhuǎn)錄活性提高了5.6倍，NFIC基因敲除時(shí)HEY1的轉(zhuǎn)錄活性有最大的增加，這表明NFIC是抑制HEY1啟動(dòng)子激活的關(guān)鍵分子。GBM細(xì)胞中HEY1參與神經(jīng)干細(xì)胞的維持，而NFI家族對(duì)于此基因的表達(dá)起到負(fù)調(diào)控的作用，而在NFI的四個(gè)成員中，NFIC的敲除對(duì)于HEY1的轉(zhuǎn)錄活性影響最大，即NFI家族共同調(diào)控HEY1的表達(dá)，NFIC調(diào)控效應(yīng)最大。(2)乳腺癌Marie等研究ADDINEN.CITEADDINEN.CITE.DATA[19]了特定的同種型NF1C2結(jié)合到小鼠CEL（羧酸酯脂肪酶）基因啟動(dòng)子中的NF1結(jié)合位點(diǎn)。NF1C2（一種50kDa的磷蛋白）的DNA結(jié)合活性，在小鼠懷孕第13天增加，在乳腺退化時(shí)減少，這與CEL基因的表達(dá)一致。我們還發(fā)現(xiàn)NF1C2的結(jié)合增加了小鼠乳腺上皮細(xì)胞系HC11中CEL基因的表達(dá)，這種量降低至約15％。此外，通過顯示NF1C2與NF1結(jié)合位點(diǎn)的結(jié)合比NF1A1親和力更高，我們證明不同NF1家族成員可以以不同的親和力結(jié)合同一個(gè)位點(diǎn)。結(jié)果NF1C與小鼠CEL基因啟動(dòng)子的結(jié)合受到細(xì)胞分化階段的影響（早期我們對(duì)鼠乳腺啟動(dòng)子的研究），衍生細(xì)胞揭示了CEL基因啟動(dòng)子中的主要陽性元件與NF1家族的成員相互作用。先前已顯示上皮細(xì)胞中表達(dá)NF1C家族的因子，50kDa的NF1C蛋白與CEL基因啟動(dòng)子結(jié)合-顯示NF1蛋白從30到100kDa，這是由于存在許多同種型和不同種類的例如磷酸化和糖基化翻譯后修飾。懷孕期間NF1C2的結(jié)合增加不是在mRNA水平調(diào)節(jié)，NF1-C2與CEL基因啟動(dòng)子中NF1結(jié)合位點(diǎn)的結(jié)合與NF1C2蛋白的表達(dá)一致地增加和減少。NF1C2有助于乳腺中的CEL基因的組織特異性表達(dá)，我們之前已經(jīng)說明NF1在CEL基因調(diào)控中的重要性是乳腺特異性的，因?yàn)镹F1結(jié)合的影響在胰腺細(xì)胞中沒有檢測(cè)到。研究發(fā)現(xiàn)ADDINEN.CITEADDINEN.CITE.DATA[20-22]，EMT與癌細(xì)胞入侵和轉(zhuǎn)移有關(guān)，轉(zhuǎn)移是很多癌癥致死的主要原因，NFIC抑制乳腺癌細(xì)胞中的EMT，遷移和侵襲。在ER陰性乳腺癌細(xì)胞系中NFIC2的過表達(dá)抑制了細(xì)胞增殖和腫瘤增長(zhǎng)，有類似觀察結(jié)果報(bào)道NFIC的敲低導(dǎo)致增殖增加和CCND1致癌基因的過表達(dá)。NFIC2的已知功能是激活p53并參與懷孕期間乳蛋白基因表達(dá)的建立。NFIC2抑制EMT主要通過兩種途徑：①FoxF1是NFIC2的直接抑制靶標(biāo)。FoxF1誘導(dǎo)EMT和侵襲性并增強(qiáng)裸鼠的異種移植致瘤性，并且在入侵性的乳腺癌細(xì)胞中高度過表達(dá)。FoxF1的過度表達(dá)與間充質(zhì)表型，體外侵襲性增加和裸鼠中乳腺癌異種移植物生長(zhǎng)增強(qiáng)有關(guān)。②NFIC-KLF4-E-鈣粘蛋白途徑。GiselaNilsson等人ADDINEN.CITEADDINEN.CITE.DATA[21]使用Affyme組trix微陣列檢測(cè)小鼠乳腺上皮細(xì)胞NFIC2或FoxF1過表達(dá)時(shí)的細(xì)胞系轉(zhuǎn)錄的變化。為了闡明FoxF1過表達(dá)的影響和信號(hào)傳導(dǎo)，研究了經(jīng)RNAi和抑制劑處理后的體外侵襲能力以及轉(zhuǎn)錄和蛋白質(zhì)表達(dá)的變化。結(jié)果表明FoxF1以LOX依賴的方式增強(qiáng)入侵，并通過上調(diào)賴氨酰氧化酶來進(jìn)行細(xì)胞遷移，并抑制Smad2/3信號(hào)傳導(dǎo)促進(jìn)乳腺癌細(xì)胞遷移。JeanetteNilsson等ADDINEN.CITEADDINEN.CITE.DATA[22]認(rèn)為催乳素/核Janus激活的激酶2的兩個(gè)下游轉(zhuǎn)錄因子：NFIC2，叉頭F1對(duì)上皮到間質(zhì)的轉(zhuǎn)化（EMT），運(yùn)動(dòng)性，侵襲性這些過程有相反的影響。通過穩(wěn)定的變體或小的干擾RNA操縱NFIC2的水平表明NFIC2阻礙EMT，運(yùn)動(dòng)型，侵襲性和腫瘤生長(zhǎng)，發(fā)現(xiàn)了FoxF1是NFIC2的直接抑制靶標(biāo)及FoxF1在癌癥和上皮細(xì)胞中EMT調(diào)節(jié)作用的證據(jù)。FoxF1的過度表達(dá)與間充質(zhì)表型，體外侵襲性增加和裸鼠中乳腺癌異種移植物生長(zhǎng)增強(qiáng)有關(guān)。FoxF1的表達(dá)與乳腺癌細(xì)胞分離物間充質(zhì)表型之間的相關(guān)性加強(qiáng)了這些發(fā)現(xiàn)之間的相關(guān)性，與FoxF1促進(jìn)侵襲和轉(zhuǎn)移的解釋一致。我們先前描述了一個(gè)催乳素通過激活的核Janus激活的激酶2維持NF1-C2的新途徑。通過Jka2增加NFIC2活性的機(jī)制獨(dú)立于信號(hào)傳感器和轉(zhuǎn)錄激活器（Stat）途徑，依賴于Jak2和NFIC2之間的直接相互作用，并涉及NFIC2的酪氨酸磷酸化和蛋白酶體降解的保護(hù)作用。作者展示了NFIC2在腫瘤發(fā)展和乳腺癌細(xì)胞上皮-間質(zhì)轉(zhuǎn)化過程（EMT）中的新作用。結(jié)果發(fā)現(xiàn)：（1）與腫瘤細(xì)胞相比，正常腺細(xì)胞中NFIC2的水平更高，并且淋巴結(jié)轉(zhuǎn)移中幾乎不存在NFIC2蛋白；（2）乳腺癌細(xì)胞中有核因子NFIC的患者預(yù)后效果更好；（3）NFIC2的強(qiáng)制表達(dá)消除了裸鼠的致瘤性；（4）NFIC2是EMT的抑制因子；