1、湯文濤miR29a促進(jìn)結(jié)直腸癌遠(yuǎn)處轉(zhuǎn)移的機(jī)制研究中文摘要第一部分miR29a促進(jìn)結(jié)直腸癌遠(yuǎn)處轉(zhuǎn)移的研究目的和意義：遠(yuǎn)處轉(zhuǎn)移是結(jié)直腸癌(CRC)患者死亡的主要原因。腫瘤遠(yuǎn)處轉(zhuǎn)移是一個(gè)復(fù)雜的多步驟過程，包括原發(fā)腫瘤的生長(zhǎng)，血管生成，癌細(xì)胞侵襲，滲入血管或淋巴管，外滲到遠(yuǎn)處的器官以及轉(zhuǎn)移瘤的定居和生長(zhǎng)。大量研究證實(shí)miRNA在CRC遠(yuǎn)處轉(zhuǎn)移過程中起重要作用。本實(shí)驗(yàn)分析miR一29a在CRC患者臨床標(biāo)本中的表達(dá)及miR29a對(duì)CRC細(xì)胞惡性表型

2、的影響。評(píng)估m(xù)iR一29a在CRC患者診斷或預(yù)后判斷的應(yīng)用價(jià)值。研究方法：運(yùn)用熒光定量逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(qRTPCR)檢測(cè)68例CRC患者腫瘤組織中miR一29a的表達(dá)，并分析miR一29a表達(dá)水平與臨床病理特征的關(guān)系。然后將miR一29amimics和antmiR一29a分別轉(zhuǎn)染CRC細(xì)胞系LoVo和HCT116。運(yùn)用transwell小室分析miR一29a對(duì)CRC細(xì)胞侵襲的影響，運(yùn)用蛋白質(zhì)印跡(Westernblot)檢測(cè)基質(zhì)金屬

3、蛋白酶2和9(MMP2／9)蛋白的表達(dá)。最后，利用慢病毒技術(shù)構(gòu)建miR29a穩(wěn)定轉(zhuǎn)染的LoVo細(xì)胞株，建立裸鼠CRC肝轉(zhuǎn)移模型，體內(nèi)水平分析miR29a對(duì)CRC遠(yuǎn)處轉(zhuǎn)移的影響。研究結(jié)果：qRTPCR結(jié)果顯示miR29a表達(dá)水平與CRC肝轉(zhuǎn)移正相關(guān)，而與其它臨床病理特征均無顯著顯著相關(guān)性。細(xì)胞侵襲實(shí)驗(yàn)顯示miR29amimics促進(jìn)LoVo和HCT116細(xì)胞的侵襲，而anti—miR29a抑制LoVo和HCT116細(xì)胞侵襲。Western

4、blot結(jié)果顯示miR一29amimics促進(jìn)MMP2和MMP9蛋白的表達(dá)，而antmiR一29a抑制MMP2和MMP9表達(dá)。裸鼠CRC肝轉(zhuǎn)移模型顯示miR一29a促進(jìn)CRC體內(nèi)遠(yuǎn)處轉(zhuǎn)移。結(jié)論：miR。29a表達(dá)水平與CRC遠(yuǎn)處轉(zhuǎn)移正相關(guān)。miR一29a促進(jìn)CRC細(xì)胞侵襲和遠(yuǎn)處轉(zhuǎn)移，是CRC潛在的癌基因。湯文濤miR29a促進(jìn)結(jié)直腸癌遠(yuǎn)處轉(zhuǎn)移的機(jī)制研究3PARTIAbstractTheroleofmiR29aondistantmetas

5、tasisofcolorectalcancerobjectiveDistancemetastasisisamajorcauseforthedeathofcolorectalcancer(CRC)patientsThereisincreasingevidenceofmiRNAsinvolvementinCRCmetastasisInthisstudyweanalyzedtheexpressionlevelofmiR一29ainCRCtis

6、suesandtheinfluenceofmiR_29aonmalignantphenotypeofCRCinordertoevaluatetheapplicantvalueofmiR一29aforCRCdiagnosisandprognosisMethodsQuantitativereversetranscriptionpolymerasechainreaction(qRTPCR)wasusedtodetecttheexpressio

7、nlevelofmiR一29ain68casesofCRCtissues，andtheassociationofmiR一29aexpressionlevelandclinical—pathologicalfeatureswasanalyzedThenmiR29amimicsandantmiR一29awastransfectedintoCRCcelllines，LoVoandHCT116respectivelyTranswellchamp

8、erswereusedtoanalyzetheinfluenceofmiR一29aonCRCcellinvasion，andwestemblotwascarriedouttodetecttheexpressionofMatrixmetallopeptidase2／9(MMP2／9)LoVocellsectopicallyexpressingmiR29aconductedbylentivirusinfectionwereinjectedi

9、ntotheCecalwallofnudemicetoconstructCRClivermetastasismodelandanalyzetheinfluenceofmiR29aonCRCmetastasisinvivesultsTheresultsofqRTPCRrevealedthattheexpressionlevelofmiR一29awaspositivecorrectedwithCRClivermetastasis，while

10、therewasnosignificantassociationbetweenmiR29aandotherclinicalpathologicalfeaturesTranswellassaydemonstratedthatmiR一29apromotedcellinvasionbothinLoVoandHCT116cells，whileanti—miR一29ainhibitedcellinvasionWesternblotrevealed