資源描述：
《短發(fā)夾RNA沉默S100A4基因表達(dá)對(duì)乳腺癌MCF-7細(xì)胞生長(zhǎng)和侵襲能力的影響》由會(huì)員上傳分享，免費(fèi)在線閱讀，更多相關(guān)內(nèi)容在行業(yè)資料-天天文庫(kù)。
1、腫瘤2009年8月第29卷第8期TumorVo1．29，No．8，August，2009·715·DOI：10．3781／j．issn．1000-7431．2009．08．003■I【sic0R．se神chl1．基0礎(chǔ)—磷憲短發(fā)夾RNA沉默S100A4基因表達(dá)對(duì)乳腺癌MCF-7細(xì)胞生長(zhǎng)和侵襲能力的影響吳愛(ài)國(guó)，馬雷，紀(jì)術(shù)峰，楊華峰(南方醫(yī)科大學(xué)附屬珠江醫(yī)院普外科，廣州510282)[摘要]目的：觀察靶向S100A4基因的短發(fā)夾RNA(shorthairpinRNA，shRNA)對(duì)乳腺癌MCF-7細(xì)胞生長(zhǎng)和侵襲的抑制作用。方法：構(gòu)建SIOOA4基
2、因特異性shRNA表達(dá)載體，經(jīng)過(guò)脂質(zhì)體介導(dǎo)將S100A4一shRNA表達(dá)載體轉(zhuǎn)染入MCF-7細(xì)胞。應(yīng)用實(shí)時(shí)熒光定量PCR(real—timefluorogenticquantitativePCR，RFQ—PCR)和Western印跡法檢測(cè)轉(zhuǎn)染后MCF-7細(xì)胞中S100A4的表達(dá)水平，運(yùn)用MTT法和FCM法檢測(cè)轉(zhuǎn)染后MCF-7細(xì)胞的增殖水平和凋亡率變化。轉(zhuǎn)染細(xì)胞經(jīng)G418篩選及克隆化培養(yǎng)后獲得穩(wěn)定轉(zhuǎn)染株，運(yùn)用Transwell小室法和劃痕實(shí)驗(yàn)檢測(cè)其侵襲力和遷移力的變化，裸鼠體內(nèi)成瘤實(shí)驗(yàn)檢測(cè)體內(nèi)細(xì)胞增殖情況。結(jié)果：經(jīng)測(cè)序鑒定證實(shí)成功構(gòu)建了S100
3、A4一shRNA表達(dá)載體。所構(gòu)建的S100A4一shRNA表達(dá)載體轉(zhuǎn)染MCF-7細(xì)胞48h后，能有效抑制S100A4的表達(dá)，并且顯著降低MCF一7細(xì)胞增殖水平，以及誘導(dǎo)細(xì)胞進(jìn)入晚期凋亡。穩(wěn)定轉(zhuǎn)染S100A4一shRNA表達(dá)載體的MCF-7細(xì)胞形態(tài)無(wú)顯著變化，但其侵襲力和遷移力明顯下降；裸鼠體內(nèi)成瘤實(shí)驗(yàn)也顯示實(shí)驗(yàn)組裸鼠移植瘤的體積和質(zhì)量明顯小于空白對(duì)照組和陰性對(duì)照組。結(jié)論：S100A4一shRNA表達(dá)載體能有效地抑制乳腺癌MCF-7細(xì)胞中S100A4的表達(dá)，從而降低細(xì)胞增殖水平以及細(xì)胞侵襲力和遷移力。[關(guān)鍵詞]乳腺腫瘤；S100蛋白質(zhì)類(lèi)；RNA
4、干擾；基因表達(dá)；細(xì)胞增殖；細(xì)胞運(yùn)動(dòng)；小鼠，裸[中圖分類(lèi)號(hào)]R737．9[文獻(xiàn)標(biāo)志碼]A[文章編號(hào)]1000-7431(2009)08-0715-06InhibitoryefectsofsilencingS100A4genebyshRNAonthegrowthandinvasivenessofbreastcancerMCF-7cellswUAi—guo，MALei，JIShu—feng，rANGHua—feng(DepartmentofGeneralSurgery，ZhujiangHospital，NanfangMedicalUni—versi
5、ty，Guangzhou510282，China)[ABSTRACT]Objective：TostudytheinhibitoryeffectsofshorthairpinRNA(shRNA)targetingS100A4geneonthegrowthandinvasivenessofbreastcancerMCF-7cells．Methods：TheS100A4specificshRNAexpressionvectorwasconstructedandconfirmedbysequencinganalysis．S100A4一shRNAexp
6、ressionvectorwastransfeetedintoMCF-7cellsvktlipofectamine2000．a(chǎn)ndtheexpressionlevelsofS100A4mRNAandproteinweredeterminedbyreal-timefluorogenticquantitativePCRandWesternblottingat48haftertrans-·fection．FlowcytometryandMTFassaywereperformedtoassesstheeffectoftheS100A4一shRNAon
7、theapoptosisandproliferationofMCF-7cells．ThecelllinewithstableexpressionofSIOOA4wasobtainedbyG418screeningandcolonyculture．InvasionandmigrationcapabilityofstablytransfectedMCF-7cellswasevaluatedbyusingTranswellchambermodelandwoundhealingassayinvitro．Thepro—liferationofstabl
8、ytransfeetedMCF-7cellswasevaluatedbytumorformationtestinnudemice．Results：S100A4一sh
