X线电离辐射通过上调c-Myc表达促进肺癌A549细胞上皮间质转化

目的:探讨电离辐射对肺癌A549细胞上皮间质转化(EMT)的影响及其可能机制。方法:采用不同剂量(0 Gy、1 Gy、2 Gy、4 Gy和8 Gy)的X射线分别照射肺癌A549细胞不同时间,通过倒置显微镜观察X射线照射12 h、24 h和48 h时肺癌A549细胞形态的改变;Western blot检测X射线照射12 h与24 h时EMT相关蛋白vimentin、N-cadherin及E-cadherin和转录因子c-Myc的表达。结果:照射后肺癌A549细胞轮廓不清,突起增多,边缘不规则且呈煎蛋样塌陷,以8 Gy X射线照射48 h时肺癌A549细胞上皮间质化形态最明显。与0 Gy照射剂量对照组相比,vimentin虽然于4 Gy剂量照射12 h时下调,但是处理24 h时各剂量照射组vimentin均表现为上调,其中2 Gy剂量照射组其上调最明显(P0.01);与0 Gy照射剂量对照组相比,1 Gy、2 Gy及4 Gy照射组照射肺癌A549细胞24 h后其N-cadherin表达上调(P0.05);各照射剂量对肺癌A549细胞E-cadherin表达没有显著影响。照射24 h后肺癌A549细胞c-Myc表达上调,以4 Gy剂量照射组表达差异最明显(P0.01)。结论:X线电离辐射可能通过上调c-Myc表达促进肺癌A549细胞发生上皮间质转化。     

重组新城疫病毒rl-RVG抑制人肺腺癌A549系细胞迁移及相关机制

目的观察表达狂犬病毒糖蛋白的重组新城疫病毒(rl-RVG)对人肺癌细胞系A549的迁移影响,初步探讨其可能的机制。方法重组新城疫病毒rl-RVG直接感染A549细胞为rl-RVG实验组,新城疫病毒La Sota株处理的A549细胞组及未感染病毒的A549细胞组作为对照组。Western blot法检测NDV-HN蛋白及狂犬病毒糖蛋白(RVG),细胞增殖实验测定新城疫病毒使用的最佳作用浓度;划痕实验及Transwell法测A549迁移;Western blot法及免疫荧光法检测E-cadherin,MMP2蛋白表达。结果空白对照组无NDV、rl-RVG表达,rl-RVG仅在感染rl-RVG细胞组有表达,NDV在感染rl-RVG细胞组及感染NDV细胞组皆有表达;与空白对照组相比,rl-RVG及NDV稀释浓度大于5×10-5对细胞的增殖有抑制作用(P0.05);rl-RVG组迁移的距离及细胞数明显减少(P0.05)。与空白对照组及NDV感染组相比,rl-RVG组E-cadherin蛋白表达水平上调(P0.05),MMP2蛋白表达水平减弱(P0.05)。结论重组新城疫病毒rl-RVG能抑制人肺癌细胞系A549的迁移,并可能通过影响肺腺癌A549上皮细胞-间质转化(EMT)过程中的调控因子E-cadherin,MMP2蛋白而对细胞迁移而作用。     

木犀草素逆转由TGF-β1诱导的人肺癌细胞上皮-间充质转化的实验研究

目的:探讨在肺癌A549细胞中木犀草素(luteolin)对转化生长因子β1(transforming growth factor-β1, TGF-β1)诱导的上皮-间充质转化(epithelial-mesenchymal transition, EMT)及细胞侵袭的影响。方法:将不同浓度(5、10、20、40、80和160μmol/L)的luteolin作用于肺癌A549细胞后,采用四甲基偶氮唑盐(MTT)法检测细胞活力;TGF-β1诱导肺癌A549细胞建立EMT模型,加入不同浓度luteolin处理;倒置显微镜观察细胞形态的变化;Transwell小室实验检测细胞侵袭能力的变化; Western blot法检测细胞中EMT标志蛋白上皮型钙黏蛋白(E-cadherin)和波形蛋白(vimentin)的蛋白表达情况。结果:Luteolin抑制肺癌A549细胞的生长,并呈一定的时间-剂量依赖关系(P0.05),luteolin作用24 h的IC_(50)为68.79μmol/L,48 h的IC_(50)为47.86μmol/L。TGF-β1诱导的肺癌A549细胞发生EMT。Luteolin显著抑制TGF-β1诱导肺癌A549细胞的侵袭能力(P0.01)。Luteolin能逆转TGF-β1诱导的肺癌A549细胞EMT形态的改变,同时,Western blot法检测也显示luteolin可逆转TGF-β1诱导的肺癌A549细胞EMT标志蛋白E-cadherin表达的下调和vimentin表达的上调(P0.01)。结论:Luteolin逆转TGF-β1诱导肺癌A549细胞的EMT,同时对肺癌细胞的侵袭和转移有一定的抑制作用。     

羟基红花黄色素A抑制人肝癌细胞系Huh7发生上皮-间质转化

目的研究羟基红花黄色素A(HSYA)对人肝癌细胞系Huh7迁移及侵袭的影响,并通过体内外实验探究其是否影响肝癌细胞上皮-间质转化(EMT)。方法体外培养Huh7细胞,分为对照组、HSYA 80和160μmol/L实验组,分别处理24 h,用transwell小室法和细胞划痕实验检测细胞侵袭和迁移能力;Western blot检测细胞vimentin和E-cadherin蛋白表达;建立Huh7细胞裸鼠皮下移植瘤模型,HE染色观察移植瘤和肝脏组织形态;免疫组化检测移植瘤组织TGF-β1、vimentin和E-cadherin表达情况。结果与对照组相比,HSYA实验组Huh7细胞迁移与侵袭能力明显下降,E-cadherin蛋白表达升高,vimentin蛋白表达下降(P0.05);与移植组相比,HSYA实验组(2.25 mg/kg)肝内无肿瘤细胞转移,移植瘤组织出现肿瘤细胞坏死,E-cadherin表达升高,vimentin和TGF-β1表达下降(P0.05)。结论 HSYA通过抑制肝癌细胞发生EMT,抑制肝癌细胞的迁移及侵袭能力。     

miR-185靶向Six1对肺癌上皮-间质转化过程的影响及其机制

目的探讨miR-185影响人肺癌细胞迁移和侵袭能力的分子机制。方法在肺癌细胞系H520和A549中转染miR-185mimic获得miR-185过表达,利用Transwell实验和细胞划痕实验检测细胞的迁移和侵袭能力;荧光素酶报告实验验证miR-185靶向Six1基因;采用qRT-PCR和Western blot法检测miR-185对细胞中Six1基因表达水平的影响;Western blot法检测miR-185过表达对肺癌细胞上皮-间质转化(epithelial-mesenchymal transition,EMT)过程的影响。结果 miR-185过表达降低肺癌细胞的迁移和侵袭能力(P0.05),上皮细胞标志物E-cadherin表达升高(P0.01),间质细胞标志物vimentin表达降低(P0.01),H520细胞过表达miR-185后,Six1基因表达水平降低(P0.01),miR-185调控肺癌细胞的迁移和侵袭是通过靶向Six1基因实现的。结论 miR-185靶向Six1基因调控人肺癌细胞EMT途径。     

PTTG1通过上皮-间质转化促进非小细胞肺癌的迁移和侵袭

目的 研究PTTG1在非小细胞肺癌侵袭转移中的作用.方法 应用免疫组织化学染色法检测80例NSCLC组织中PTTG1、E-cadherin和Vimentin的表达,并分析其相关性.细胞分组:1)A549/con,转染空载的A549,2)A549/PTTG1,转染目的基因的A549,3)Scr/H1299,转染空载的H1299,4)SiPTTG1/H1299,敲除PTTG1的H1299;用Western blot检测细胞中PTTG1、E-cadherin、Vimentin蛋白的表达情况.结果 NSCLC组织中PTG1蛋白的阳性表达率为85％与E-cadherin的表达呈负相关(P＜0.05)而与Vimentin的表达呈正相关(P＜0.05).Western blot结果显示在SiPTTG1/H1299细胞中PTTG1蛋白表达水平明显降低,Vimentin蛋白表达水平降低而E-cadherin蛋白表达水平升高.同时在A549/PTTG1细胞中Vimentin蛋白表达水平升高而E-cadherin蛋白表达水平降低.结论 PTG1能促进NSCLC EMT的发生,从而促进NSCLC的侵袭和转移.     

雷帕霉素对顺铂作用下人肺腺癌A549及耐药A549/DDP细胞增殖、侵袭、黏附及自噬凋亡的影响

 目的:研究雷帕霉素（Rap）对顺铂（DDP）作用下人肺腺癌A549及耐药A549/DDP 细胞增殖、迁移、黏附及其自噬凋亡的影响。方法: 培养人肺腺癌A549及耐药A549/DDP细胞株,利用MTT方法分别检测Rap和DDP单独与联合作用对A549及耐药A549/DDP细胞增殖抑制率的影响;Transwell方法检测Rap和DDP单独与联合作用对A549及耐药A549/DDP细胞体外侵袭能力的影响;黏附实验检测Rap和DDP单独与联合作用对A549及耐药A549/DDP细胞体外侵袭能力的影响;流式细胞术检测Rap和DDP单独与联合作用对A549及耐药A549/DDP细胞凋亡的影响;Western blotting检测Rap和DDP单独与联合作用对A549及耐药A549/DDP细胞自噬标志蛋白beclin-1和LC3表达的影响。 结果: 与Rap或DDP单独作用组相比,Rap和DDP联合作用能够同时显著抑制人肺腺癌A549及耐药A549/DDP细胞增殖、体外侵袭能力及细胞黏附能力,并能够促进细胞凋亡和自噬标志蛋白beclin-1和LC3的表达（均P＜0.05）。结论: Rap能够通过促进细胞自噬而增强DDP的作用,进而抑制人肺腺癌A549及耐药A549/DDP细胞的增殖、侵袭、黏附并促进细胞凋亡作用,具有协同作用。     

高表达ITGB1促进人乳腺上皮MCF10A细胞EMT及迁移

目的探讨ITGB1基因过表达对人乳腺上皮MCF10A细胞迁移和侵袭的影响。方法构建重组质粒p BABEpuro-myc-ITGB1,在人胚肾上皮细胞系293T细胞中包装反转录病毒,转染人乳腺上皮MCF10A细胞系,构建ITGB1基因过表达的稳定细胞系;实验设空白对照组(MCF10A)、阴性对照组(MCF10A-vector)和ITGB1基因过表达组(MCF10A-ITGB1);经嘌呤霉素筛选后,实时荧光定量PCR和蛋白质印迹法检测ITGB1 mRNA及蛋白的表达;Transwell迁移和侵袭实验分别检测细胞株迁移和侵袭能力;蛋白质印迹法进一步检测上皮间质转换(EMT)相关蛋白E-cadherin、vimentin、fibronectin、N-cadherin以及ITGB1下游ILK、p-FAK和FAK蛋白的表达。结果成功构建ITGB1基因稳定过表达人乳腺上皮MCF10A细胞系;ITGB1基因过表达组(MCF10A-ITGB1)与空白对照组(MCF10A)和阴性对照组(MCF10A-vector)细胞相比,迁移能力(P0.05)和侵袭能力(P0.01)显著增强;EMT相关蛋白E-cadherin显著下调(P0.05),vimentin、fibronectin、N-cadherin以及ITGB1下游ILK,p-FAK蛋白显著上调(P0.05)。结论在人乳腺上皮MCF10A细胞中,过表达ITGB1基因诱导细胞EMT并促进其迁移和侵袭。     

骨髓基质细胞介导的微环境对人肺腺癌A549细胞的作用

目的:探讨肿瘤微环境中人骨髓基质细胞HS-5对人肺腺癌A549细胞的作用及机制。方法:采用HS-5细胞条件培养基(HS-5 cell-conditioned medium,HS-5-CM)处理A549细胞,分别通过MTT实验和划痕愈合实验观察A549细胞的活力和迁移能力,应用q PCR检测CX3C趋化因子受体1(CX3C chemokine receptor 1,CX3CR1)的表达;加入丝裂原活化蛋白激酶/细胞外信号调节激酶(mitogen-activated protein kinase/extracellular signal-regulated kinases,MAPK/ERK)通路抑制剂U0126后,应用Western blot法检测MAPK/ERK信号通路活化相关蛋白ERK及其磷酸化水平,划痕愈合实验检测抑制MAPK/ERK通路对HS-5-CM效果的影响,应用Western blot检测CX3CR1的表达情况。结果:HS-5-CM可以促进A549细胞的活力和迁移能力(P0.01),同时A549细胞中CX3CR1的表达升高(P0.05)。MAPK/ERK通路抑制剂U0126可以有效抑制HS-5-CM处理后MAPK/ERK通路的激活(P0.01),抑制HS-5-CM促进A549细胞迁移的能力(P0.01),同时下调CX3CR1的表达(P0.05)。结论:HS-5细胞介导的微环境可以促进A549细胞的活力和迁移,其机制可能涉及MAPK/ERK信号通路的活化和CX3CR1的表达。     

FOXP3 基因沉默抑制肺腺癌 A549 细胞上皮-间质转化和改善 5-氟尿嘧啶耐药性

目的：探究叉头蛋白3(FOXP3)基因沉默对肺腺癌上皮-间质转化（EMT）及5-氟尿嘧啶（5-FU）耐药性的影响。方法：构建2段特异性针对FOXP3基因的siRNA片段，并将其通过脂质体介导转染至肺腺癌A549细胞，同时设立转染无义序列的阴性对照组。将转染后的A549细胞分为3组：si-NC组（阴性对照）、si-FOXP3-1组和si-FOXP3-2组。qRT-PCR和Western blot检测各组细胞中FOXP3的表达水平，Western blot检测各组细胞中E-钙黏蛋白（E-cadherin）、波形蛋白（Vimentin）以及N-钙黏蛋白（N-cadherin）表达水平，Transwell检测各组细胞迁移和侵袭能力，ELISA检测各组细胞上清液中基质金属蛋白酶2(MMP-2)和MMP-9蛋白浓度。qRT-PCR和Western blot检测5-FU处理A549细胞后FOXP3表达水平，CCK-8检测各组细胞对5-FU的敏感性，Western blot检测各组细胞中P-糖蛋白（P-gp）表达水平。结果：与si-NC组相比，si-FOXP3-1和si-FOXP3-2组中FOXP3在...     

M3R激动剂通过激活PI3K/AKT信号途径促进肺癌细胞A549的上皮间质转化

目的:探讨毒蕈碱胆碱受体3(muscarinic receptor 3,M3R)激动剂卡巴胆碱促进人肺癌A549细胞上皮间质转化的可能信号通路。方法:用400μmol/L卡巴胆碱刺激人肺癌A549细胞,在倒置相差显微镜下观察细胞形态的变化,应用划痕愈合实验和Transwell实验观察细胞迁移和侵袭能力;应用q PCR技术检测上皮间质转化相关蛋白波形蛋白(vimentin)和E钙黏蛋白(E-cadherin)m RNA水平的变化;应用Western blot技术检测p-AKT、vimentin和E-cadherin蛋白水平的变化。结果:卡巴胆碱刺激人肺癌A549细胞后,细胞形态发生明显改变,由不规则多边形逐渐向梭形转化、细胞间紧密结合逐渐变得松散,细胞迁移和侵袭能力增强;vimentin的m RNA和蛋白表达量明显增加,E-cadherin的m RNA和蛋白水平降低,磷酸化的AKT蛋白水平增加,且这些变化均可被M3R特异性抑制剂4-DAMP所抑制(P0.05)。结论:卡巴胆碱可通过激活PI3K/AKT信号途径促进人肺癌A549细胞发生上皮间质转化。     

地高辛对缺氧诱导的乳腺癌细胞上皮间质转化和侵袭的影响

 目的:本研究旨在探究地高辛对缺氧诱导乳腺癌细胞上皮间质转化、迁移和侵袭能力的影响,并探讨其分子机制。方法:选取人乳腺癌MCF-7细胞作为研究对象,采用CoCl₂模拟化学缺氧条件,采用细胞划痕实验测量细胞迁移率,采用Transwell侵袭实验检测细胞侵袭力,采用Western blot方法检测人乳腺癌MCF-7细胞缺氧诱导因子-1α(HIF-1α)、Snail、E-cadherin和vimentin蛋白表达的变化。结果:缺氧使MCF-7细胞从多角形上皮形态转变成梭形间质细胞形态,细胞间隙增大,而在地高辛作用下缺氧的MCF-7细胞未发生明显的上皮间质转化。细胞划痕实验和Transwell侵袭实验结果显示,经CoCl₂处理的MCF-7细胞的迁移和侵袭能力均显著增强(P<0.01),地高辛可抑制CoCl₂诱导的细胞迁移和侵袭(P<0.01)。与control组相比,CoCl₂组细胞的HIF-1α、Snail和vimentin蛋白表达水平显著升高(P<0.01),E-cadherin的蛋白表达水平显著降低(P<0.01);CoCl₂+digoxin组细胞的HIF-1α、E-cadherin和vimentin蛋白表达水平与control组相比差异均无统计学显著性,Snail蛋白表达水平虽略高于control组(P<0.05),但与CoCl₂组细胞相比显著降低(P<0.01)。结论:地高辛可通过下调HIF-1α和Snail蛋白表达抑制缺氧诱导的MCF-7细胞上皮间质转化和侵袭。     

SphK1和FAK对人结肠癌HCT116细胞上皮间质转化的影响

 目的: 研究鞘氨醇激酶1(sphingosine kinase l,SphK1)和黏着斑激酶(focal adhesion kinase,FAK)对人结肠癌HCT116细胞上皮间质转化(epithelial-mesenchymal transition,EMT)的影响。方法: 将人结肠癌HCT116细胞分成3组:采用SphK1抑制剂N,N-二甲基鞘胺醇(N,N-dimethylsphingosine,DMS)、FAK抑制剂PF573228和相同体积的培养基分别处理细胞。MTT法检测细胞活力,Western blot方法检测SphK1、FAK、E-cadherin、N-cadherin、vimentin和基质金属蛋白酶2(MMP2)蛋白的表达,real-time PCR检测SphK1、鞘氨醇1-磷酸(S1P)、FAK、E-cadherin和vimentin mRNA的表达,并应用细胞划痕实验检测肿瘤细胞的迁移能力。结果: PF573228和DMS均明显抑制人结肠癌HCT116细胞的活力,并呈时间剂量依赖性。DMS抑制SphK1的表达,同时下调FAK、N-cadherin、vimentin和MMP2蛋白的表达,而上调E-cadherin蛋白表达上调。PF573228明显抑制FAK的表达,同时抑制SphK1、N-cadherin、vimentin和MMP2的表达,上调E-cadherin蛋白的表达(P<0.01)。划痕实验显示PF573228和DMS显著抑制HCT116细胞的迁移能力(P<0.01)。与对照组比较,PF573228组和DMS组FAK、SphK1、S1P以及vimentin mRNA的表达明显下调,而E-cadherin mRNA的表达则明显上调(P<0.05)。结论: SphK1和FAK信号通路可能在结肠癌HTC116细胞上皮间质转化过程中发挥重要作用。     

紫草素对HGF诱导的人肺癌细胞上皮-间充质转化的逆转作用

目的:探讨紫草素(shikonin)对肝细胞生长因子(HGF)诱导的人非小细胞肺癌PC9细胞迁移、侵袭及上皮-间充质转化(EMT)的影响。方法:用HGF诱导PC9细胞建立EMT模型,采用不同剂量的shikonin干预24 h后,MTT法检测细胞活力;划痕愈合实验检测细胞的迁移能力;Transwell小室实验检测细胞的侵袭能力;Western blot法检测细胞中上皮型钙黏蛋白(E-cadherin)、神经型钙黏蛋白(N-cadherin)和波形蛋白(vimentin)的蛋白表达水平。结果:Shikonin可显著抑制PC9细胞的活力(P0.01),随着给药剂量的增加,shikonin对细胞的生长抑制率显著上升,并呈一定的剂量依赖关系,IC_(50)为9.364μmol/L。HGF可诱导PC9细胞发生迁移和侵袭;划痕愈合实验和Transwell小室实验显示,shikonin能明显抑制由HGF诱导的肺癌PC9细胞迁移和侵袭(P0.01)。Western blot检测结果显示HGF可诱导PC9细胞的EMT标志物E-cadherin蛋白表达下调,N-cadherin和vimentin蛋白表达上调,使其发生EMT;shikonin则可逆转由HGF诱导的PC9细胞E-cadherin蛋白表达下调及N-cadherin和vimentin蛋白表达上调(P0.01)。结论:Shikonin能逆转由HGF诱导的肺癌PC9细胞EMT,同时抑制其迁移和侵袭。     

上皮性卵巢癌中PARP-1的表达及其与EMT的关系

 目的: 探讨上皮性卵巢癌(epithelial ovarian cancer,EOC)中聚腺苷二磷酸核糖聚合酶-1[poly(ADP-ribose) polymerase-1,PARP-1]的表达及其与上皮-间质转化(epithelial-mesenchymal transition,EMT)的关系。方法: 免疫组化、实时荧光定量PCR法检测EOC和良性卵巢肿瘤组织中PARP-1、E-钙黏蛋白(E-cadherin)、波形蛋白(vimentin)和转录调控因子Snail的表达;Western blotting法检测高效PARP-1抑制剂PJ34处理SKOV3细胞后PARP-1、E-cadherin、vimentin和Snail蛋白的表达。结果: PARP-1、vimentin和Snail在EOC中阳性表达率高于良性卵巢肿瘤组织,而E-cadherin则相反,差异均有统计学显著性(P<0.05)。PARP-1、E-cadherin、vimentin和Snail与EOC的病理分级、临床分期和有无淋巴结转移有关(P<0.05),与年龄和病理类型无关。E-cadherin表达与PARP-1表达呈负相关(P<0.05),vimentin、Snail表达与PARP-1表达呈正相关(P<0.05)。EOC中PARP-1、vimentin和Snail mRNA的相对表达量高于良性卵巢肿瘤组织,E-cadherin mRNA的相对表达量低于良性卵巢肿瘤组织,差异均具有统计学显著性(P<0.05)。PJ34处理SKOV3细胞后,PARP-1、vimentin和Snail的蛋白水平明显下降,E-cadherin的蛋白水平显著提高,差异有统计学显著性(P<0.05)。结论: PARP-1通过调控E-cadherin、vimentin和Snail的表达促进EOC上皮间质转化。PARP-1及其参与的上皮-间质转化在EOC进展中发挥重要作用。     

miR-183 promotes radioresistance of lung adenocarcinoma H1299 cells via epithelial-mesenchymal transition

Lung adenocarcinomas are usually sensitive to radiation therapy, but some develop resistance. Radiation resistance can lead to poor patient prognosis. Studies have shown that lung adenocarcinoma cells (H1299 cells) can develop radioresistance through epithelial-mesenchymal transition (EMT), and this process is regulated by miRNAs. However, it is unclear which miRNAs are involved in the process of EMT. In our present study, we found that miR-183 expression was increased in a radioresistant lung adenocarcinoma cell line (H1299R cells). We then explored the regulatory mechanism of miR-183 and found that it may be involved in the regulation of zinc finger E-box-binding homeobox 1 (ZEB1) expression and mediate EMT in lung adenocarcinoma cells. qPCR results showed that miR-183, ZEB1, and vimentin were highly expressed in H1299R cells, whereas no difference was observed in E-cadherin expression. Western blot results showed that ZEB1 and vimentin were highly expressed in H1299R cells, while E-cadherin expression was decreased. When miR-183 expression was inhibited in H1299R cells, radiation resistance, proliferation, and cell migration were decreased. The expression of ZEB1 and vimentin in H1299R cells was decreased, while the expression of E-cadherin was increased. Moreover, miR-183 overexpression in H1299 cells enhanced radiation resistance, proliferative capacity, and cell migration ability. The expression of ZEB1 and vimentin in H1299 cells was increased, while that of E-cadherin was decreased. In conclusion, miR-183 may promote EMT and radioresistance in H1299 cells, and targeting the miR-183-ZEB1 signaling pathway may be a promising approach for lung cancer treatment.     

Wnt/β-catenin pathway is required for epithelial to mesenchymal transition in CXCL12 over expressed breast cancer cells

CXCL12 is positively associated with the metastasis and prognosis of various human malignancies. Cancer-associated fibroblasts (CAFs), the main cells secreting CXCL12, are capable of inducing epithelial to mesenchymal transition (EMT) of breast cancer cells. However, it has not been completely understood whether CXCL12 is involved in EMT of breast cancer cells and the underlying mechanisms. The present study aimed to investigate the effects of CXCL12 on the EMT and cancer stem cell (CSC)-like phenotypes formation by transfecting pEGFP-N1-CXCL12 plasmid into MCF-7 cells. Real time-PCR and Western blot analysis demonstrated the successful over expression of CXCL12 in MCF-7 cells. Cell counting kit-8 assay, wound healing assay and Transwell invasion analysis confirmed that over expression of CXCL12 significantly promoted the proliferation, migration and invasion in MCF-7 cells (P<0.05). In addition, ALDH activity was dramatically enhanced compared with parental (P<0.001), accompanied by the notably elevated mRNA and protein levels of OCT-4, Nanog, and SOX2 in CXCL12 overexpressed-MCF-7 cells (P<0.001). Furthermore, we observed the down regulation of E-cadherin and up regulation of vimentin, N-cadherin, and α-SMA in CXCL12 overexpressed-MCF-7 cells (P<0.01). Meanwhile, western blot and immunofluorescence assay showed that over expression of CXCL12 activated Wnt/β-catenin pathway to induce EMT of MCF-7 cells, as evidenced by the increased expression of E-cadherin after silencing β-catenin by siRNA interference (P<0.001). Collectively, our findings suggested that over expression of CXCL12 could trigger EMT by activating Wnt/β-catenin pathway and induce CSC-like phenotypes formation to promote the proliferation and metastasis in MCF-7. Hence, CXCL12 may become a promising candidate for breast cancer therapy.     

HIF-1α介导了间歇低氧对人肺腺癌A549细胞体外生物学行为的影响

目的:探讨缺氧诱导因子1α(HIF-1α)是否介导间歇低氧对人肺腺癌A549细胞体外活力、凋亡以及侵袭的影响。方法:采用体外转染方法将特异性针对HIF-1α的siRNA导入A549细胞,在间歇低氧条件下培养后通过real-time PCR和Western blot法检测HIF-1α及其下游Bcl-2、Bax、P53、P21、VEGF的mRNA和蛋白表达;通过MTT法和流式细胞术分别检测A549细胞的活力、凋亡及细胞周期;通过Transwell小室检测A549细胞的侵袭能力。结果:未转染HIF-1α-siRNA的A549细胞[间歇低氧空白对照组(IHC组)、间歇低氧空载体对照组(IHE组)及间歇低氧阴性对照组(IHN组)]经间歇低氧干预后的HIF-1α、Bcl-2及VEGF表达均明显高于常氧对照组(RA组),Bax及P21表达均明显低于RA组(P0.05),而转染HIF-1α-siRNA的A549细胞[间歇低氧siRNA组(IHS组)]较IHC组、IHE组及IHN组经间歇低氧干预后的HIF-1α、BCL-2及VEGF表达均明显下调,Bax及P21表达较均明显上调(P0.05);所有间歇低氧组A549细胞的P53表达均较RA组升高(P0.05),但各间歇低氧组之间无显著性差异。IHC组、IHE组及IHN组A549细胞经间歇低氧干预后较RA组的细胞活力增强,凋亡率下降,侵袭力增强(P0.05),而IHS组A549细胞经间歇低氧干预后细胞周期被阻滞于G1期,较未转染组的细胞活力下降,凋亡增加,侵袭能力下降(P0.05)。结论:间歇低氧可通过HIF-1α途径调控其下游基因表达进而促进A549细胞的活力和转移;通过RNA干扰技术造成HIF-1α基因沉默可以抑制间歇低氧引起的A549细胞生长和转移。     

IMP3 expression is associated with epithelial-mesenchymal transition in breast cancer

IMP3 plays an important role in tumor invasion and metastasis, to which epithelial to mesenchymal transition (EMT) also contributes. The purpose of this study was to investigate whether IMP3 can regulate invasion and metastasis through EMT in breast cancers. The protein expression levels of IMP3 and EMT markers were analyzed by immunohistochemistry in 180 paraffin-embedded human breast tissue samples. There was an inverse correlation of IMP3 with E-cadherin protein expression (P = 0.042). IMP3 expression directly correlated with both Slug (P = 0.004) and vimentin (P < 0.001). Changes in E-cadherin, vimentin, and Slug mRNA and protein levels were examined by quantitative real-time reverse polymerase chain reaction (qRT-PCR) and western blotting. Overexpression of IMP3 reduced the expression of E-cadherin and upregulated Slug and vimentin in transfected cells. In contrast, knocking down IMP3 had the opposite expression of the three proteins. Ribo-immunoprecipitation qPCR revealed that IMP3 binds Slug mRNA directly. In a transwell assay, overexpression of Slug rescued the cell migration and invasion caused by silencing IMP3 in MDA-MB-231 cells. On the other hand, knockdown of Slug in T47D-IMP3 cells could also have the opposite change. Our results strengthen the association of IMP3 with the regulation of EMT. Slug is a functional target of IMP3. IMP3 could therefore promote invasion and migration through the EMT in breast cancer cells.