RASAL2 对胃癌细胞侵袭与上皮间质转化的影响*

目的:探讨RASAL2 表达改变对胃癌细胞侵袭与迁移能力的影响,以及对细胞上皮间质转化（epithelial-mesenchymal transition ,EMT ）的影响。方法:以胃癌细胞系SNU-1、SNU-16与AGS 为体外模型,通过RNA 干扰技术构建RASAL2 敲低细胞系,通过过表达慢病毒载体构建RASAL2 过表达细胞系。利用体外侵袭与迁移实验,划痕实验观察RASAL2 表达改变对两种胃癌细胞系侵袭与迁移能力的影响。同时利用免疫显微荧光技术与免疫印迹分析RASAL2 表达改变对胃癌细胞EMT 的影响。利用免疫印记分析RASAL2 对Ras-ERK通路的活化影响。结果:两种不同靶位的RASAL2-shRNA 均可以有效抑制RASAL2 在SNU-16与AGS 细胞中的表达。细胞体外侵袭与迁移能力在RASAL2 敲低后显著高于对照组。划痕实验也提示同样结果。上皮标志 E-cadherin表达在RASAL2 敲低后发生下调,同时间质标志Vimentin 发生上调。EMT 调控转录因子Snail 也发生上调。Ras-ERK 通路活化在RASAL2 敲低后显著上调。RASAL2 过表达抑制SNU-1 细胞迁移能力。结论:RASAL2 在胃癌中具有抑制细胞侵袭与迁移的能力。同时也是胃癌细胞EMT 的调控分子。RASAL2 发挥生物学功能至少是部分依赖Ras-ERK 通路的。      

CD4+CD25+调节性T 细胞对乳腺癌细胞上皮间质转化和ALDH1+干样细胞的影响*

目的:研究CD4+CD25+调节性T 细胞（regulatory T cells ,Tregs）对乳腺癌细胞上皮间质转化（epithelial-mesenchymal transition ,EMT ）、细胞迁移侵袭能力,及ALDH1+干样细胞比例的影响。方法:采用免疫磁珠法分离乳腺癌患者外周血中CD4+CD25+Tregs,CD4+CD25+Tregs与乳腺癌BT474、MCF-7 细胞系共培养（共培养组）,BT474、MCF-7 单独培养（对照组）。 检测共培养组和对照组乳腺癌细胞EMT 相关标志物表达的变化,及细胞迁移和侵袭能力的变化。此外,检测BT474 细胞中ALDH1+干样细胞、微球形成能力和自我更新能力的变化。结果:CD4+CD25+Tregs诱导BT474 和MCF-7 细胞间质性标志物表达增高,诱导MCF-7 细胞上皮性标志物E-cadherin 表达降低。CD4+CD25+Tregs诱导BT474 和MCF-7 细胞迁移和侵袭能力上调。共培养组BT474 细胞中ALDH1+干样细胞比例、微球体形成能力、自我更新能力较对照组增强。结论:CD4+CD25+Tregs可诱导乳腺癌细胞发生EMT ,增强细胞体外迁移和侵袭能力,同时促进ALDH1+干样细胞增加。      

缺氧微环境下HIF-1α对胰腺癌细胞上皮间质转化及侵袭迁移的影响

目的:探讨缺氧微环境下缺氧诱导因子-1α（hypoxia inducible factor-1α,HIF-1α）对胰腺癌细胞上皮间质转化（epithelial-mesenchymal transition,EMT）及侵袭迁移的影响。方法:首先比较缺氧和常氧培养的胰腺癌细胞形态和体外侵袭迁移能力,以及HIF-1α和EMT相关因子的表达水平。然后通过HIF-1α抑制剂YC-1和siRNA基因沉默分别从蛋白和mRNA水平抑制HIF-1α的表达,检测缺氧培养的胰腺癌细胞体外侵袭迁移能力,以及EMT相关基因的表达水平。结果:形态学和分子水平证实缺氧诱导人胰腺癌细胞株AsPC-1、Capan-2、Panc-1的EMT改变,增强其体外侵袭和迁移能力。缺氧上调HIF-1α、Snail、N-cadherin的表达,下调E-cadherin的表达。抑制HIF-1α的表达后,Snail和N-cadherin表达下调,E-cadherin表达上调,并且体外侵袭迁移能力降低。结论:HIF-1α介导EMT在缺氧促进胰腺癌细胞侵袭迁移中发挥重要作用。     

PAK5通过上皮-间质转化促进人乳腺癌细胞侵袭

目的:探讨p21活化激酶-5（p21-activated kinase 5,PAK5）蛋白对乳腺肿瘤细胞侵袭转移的作用机制。方法:观察PAK5过表达对乳腺癌细胞形态的影响,人乳腺肿瘤细胞MCF-7通过慢病毒感染稳定表达Flag-PAK5。然后提取感染细胞的总蛋白进行蛋白免疫印迹检测上皮-间质转化（EMT）标志物上皮-钙黏蛋白（E-cadherin）,纤维连接蛋白（Fibronectin）和波形蛋白（vimentin）的表达。最后通过Transwell实验检测过表达PAK5对乳腺癌细胞迁移和侵袭的影响。结果:过表达PAK5蛋白能够使细胞形态由鹅卵石样像梭形变化,下调E-cadherin蛋白,促进乳腺癌细胞迁移和侵袭。结论:PAK5可能参与调节乳腺肿瘤细胞发生上皮－间质转化,促进乳腺肿瘤侵袭转移的发生。     

阿克替苷通过调控ERK1/2信号通路抑制肝癌细胞上皮间质转化

目的探讨阿克替苷(ACT)通过调控ERK1/2通路抑制人肝癌HCCLM3细胞上皮间质转化(EMT)的作用及其机制。方法CCK-8法检测肝癌细胞的增殖能力;划痕实验和Transwell实验检测肝癌细胞侵袭及迁移能力;实时定量PCR和蛋白质印迹实验检测ACT对ERK1/2信号通路和上皮间质转化相关基因(E-cadherin、N-cadherin)mRNA和蛋白的表达。结果CCK-8法检测结果显示,ACT可降低肝癌HCCLM3细胞的活性,抑制肝癌细胞的增殖能力,并与药物浓度和时间有一定的相关性。划痕实验和Transwell实验结果显示,ACT能抑制肝癌HCCLM3细胞迁移和侵袭能力,并呈浓度依赖性。荧光定量PCR和蛋白质印迹实验表明,ACT可下调ERK1/2信号通路相关基因mRNA和蛋白的表达,上调EMT相关基因E-cadherin mRNA和蛋白的表达,下调N-cadherin mRNA和蛋白的表达。结论ACT可抑制人肝癌HCCLM3细胞EMT、侵袭及迁移,其作用机制与下调ERK1/2信号通路密切相关。     

MicroRNA-100调节mTOR表达对肝癌细胞侵袭转移及上皮间质转化的影响及机制研究

目的:探讨microRNA-100调节m TOR表达对肝癌细胞侵袭转移及上皮间质转化的影响并研究其可能的分子机制。方法:通过脂质体介导将microRNA-100模拟物及阴性对照转染Hep G2细胞,将细胞分为转染试剂对照组(mock)、阴性对照组(control)和microRNA-100 mimic(miRNA-100 mimic)组。采用realtime PCR检测转染后细胞中miRNA-100的表达水平,Western blot检测m TOR的表达变化,细胞划痕实验检测细胞的迁移能力,Tranwell检测细胞的侵袭能力,Western blot检测细胞中MMP-2、MMP-9、N-cadherin、Vimentin、α-SMA和E-cadherin的表达。结果:过表达miRNA-100通过抑制m TOR的表达抑制肝癌细胞的迁移及侵袭能力,并通过下调肝癌细胞中MMP-2、MMP-9、N-cadherin、Vimentin和α-SMA的表达,上调E-cadherin的表达,阻抑上皮细胞-间质转化(epithelial-mesenchymal transition,EMT)过程的发生。结论:MicroRNA-100可能通过降低m TOR的表达抑制肝癌细胞的转移侵袭能力,并抑制上皮间质转化过程的发生。     

乳腺癌上皮间质转化与耐药关系的研究进展

肿瘤上皮-间质转化(epithelial-mesenchymal transitions,EMT)是指肿瘤在各种因素作用下上皮细胞转变为具有高侵袭、转移能力间质表型的过程。EMT与细胞上皮表型标志物E-钙黏蛋白表达下调、间质表型标志物上调和相关基因表达改变有关。EMT的发生能够促进乳腺癌侵袭、转移和干细胞特性获得,诱导乳腺癌细胞对化疗、内分泌治疗和靶向药物治疗产生获得性耐药。同时研究发现,乳腺癌耐药细胞能够发生EMT,并获得干细胞表型。因此,针对乳腺癌细胞EMT的相关研究可以帮助人们在肿瘤抗耐药治疗中找到新的策略。本文旨在对EMT与乳腺癌发生获得性耐药之间关系的最新研究进展作一综述。     

SPON1促进胆管癌细胞迁移、侵袭和上皮间质转化

目的:探讨SPON1对胆管癌细胞迁移、侵袭能力和上皮间质转化的影响。方法:通过生物信息学和免疫组织化学分析SPON1的表达。将SPON1过表达质粒、SPON1敲低质粒和阴性对照质粒转入胆管癌细胞系(QBC939、HCCC-9810),通过划痕实验和Transwell实验检测细胞迁移和侵袭能力;免疫印迹检测SPON1对胆管癌细胞中上皮间质转化相关蛋白表达的影响。结果:生信分析结果和免疫组化结果均提示SPON1在胆管癌组织中高表达(P<0.05);过表达SPON1能够有效增强胆管癌细胞的迁移和侵袭能力(P<0.05);相反,沉默SPON1则抑制了胆管癌细胞的迁移和侵袭能力(P<0.05);SPON1通过上调Vimentin、下调E-cadherin促进胆管癌细胞上皮间质转化(P<0.05)。结论:SPON1在胆管癌中被上调,并促进胆管癌细胞迁移、侵袭和上皮间质转化。     

TGF-β上调Notch3诱导上皮间质转化在促进肺癌骨转移中的作用

目的:研究TGF-β上调Notch3表达诱导EMT在肺癌骨转移中的作用.方法:免疫荧光、蛋白印记和RT-PCR方法检测Notch3在TGF-β诱导下上皮、间质标记物以及上皮间质转化相关转录因子Twist、Snail和ZEB-1的表达变化;利用慢病毒包装Notch3正义表达载体和ZEB-1 siRNA载体分别上调Notch3和抑制ZEB-1的表达后,采用免疫荧光、蛋白印记方法检测抑制EMT转录因子ZEB-1的表达后,对上皮间质标记物的影响;进而通过体外迁移和侵袭试验观察抑制ZEB-1的表达后对Notch3高表达细胞系的迁移和侵袭能力的影响;最后体外ELISA法检测骨转移相关分子pTHrP和IL-6的表达,探讨Notch3通过诱导EMT在促进肺癌骨转移中的作用.结果:Notch3 siRNA抑制其表达后能够逆转TGF-β诱导的上皮标记物和间质标记物的表达变化,这一现象是通过影响EMT转录因子ZEB-1实现的.进而利用慢病毒包装系统,构建了Notch3过表达细胞株和ZEB-1 siRNA细胞株,免疫荧光和蛋白印记试验发现,Notch3过表达载体能够抑制上皮标记物的表达,上调间质标记物的表达,促进EMT的发生,而抑制ZEB-1的表达后能够逆转这一现象.最后,体外侵袭试验和ELISA试验发现,Notch3高表达能够增加肺癌细胞的侵袭能力以及pTHrP和IL-6的分泌,而抑制ZEB-1的表达后可明显降低其转移能力以及pTHrP和IL-6的分泌.结论:Notch3参与了TGF-β诱导肺癌细胞上皮间质转化过程,并在NSCLC骨转移中发挥作用.     

乳腺癌干细胞上皮-间质转化标志物表达变化及意义

目的 探讨乳腺癌干细胞上皮．间质转化标志物表达变化及其临床意义。方法采用无血清悬浮培养法,从MCF-7细胞培养乳腺癌微球体细胞,应用流式细胞仪检测微球体细胞中CD44和CD24的表达,采用Westernblot方法检测微球体细胞中E-钙黏素、N-钙黏素、纤维连接蛋白、波形蛋白的表达水平,体外穿膜实验检测肿瘤细胞的迁移、侵袭能力。结果乳腺微球体富集了CD44＋CD24-的乳腺癌干细胞,并能在含血清培养基中增殖分化。该微球体细胞的上皮标志物E-钙黏素表达水平下调,而间质标志物N-钙黏素、纤维连接蛋白、波形蛋白的表达水平上调,同时乳腺癌干细胞的迁移、侵袭能力显著增强。结论乳腺癌干细胞具有上皮-间质转化的特征,具有显著增强的迁移、侵袭能力。     

An in vivo model of epithelial to mesenchymal transition reveals a mitogenic switch

The epithelial to mesenchymal transition (EMT) is a process by which differentiated epithelial cells transition to a mesenchymal phenotype. EMT enables the escape of epithelial cells from the rigid structural constraints of the tissue architecture to a phenotype more amenable to cell migration and, therefore, invasion and metastasis. We characterized an in vivo model of EMT and discovered that marked changes in mitogenic signaling occurred during this process. DNA microarray analysis revealed that the expression of a number of genes varied significantly between post-EMT and pre-EMT breast cancer cells. Post-EMT cancer cells upregulated mRNA encoding c-Met and the PDGF and LPA receptors, and acquired increased responsiveness to HGF, PDGF, and LPA. This rendered the post-EMT cells responsive to the growth inhibitory effects of HGF, PDGF, and LPA receptor inhibitors/antagonists. Furthermore, post-EMT cells exhibited decreased basal Raf and Erk phosphorylation, and in comparison to pre-EMT cells, their proliferation was poorly inhibited by a MEK inhibitor. These studies suggest that therapies need to be designed to target both pre-EMT and post-EMT cancer cells and that signaling changes in post-EMT cells may allow them to take advantage of paracrine signaling from the stroma in vivo.     

IL-6通过活化STAT3/Notch信号促进癌相关成纤维细胞衰老及宫颈癌上皮细胞侵袭与放疗抵抗的机理研究

背景与目的：肿瘤微环境中衰老的癌相关成纤维细胞(cancer-associated fibroblasts,CAFs)介导上皮肿瘤的转移和放化疗抵抗,而CAFs分泌的炎性细胞因子IL-6可能促进宫颈癌的侵袭和放疗抵抗,但其作用机制并不清楚。该研究旨在探讨IL-6对CAFs衰老及宫颈癌上皮细胞侵袭与放疗抵抗的作用。方法：以宫颈癌CAFs、宫颈组织正常成纤维细胞(normal ifbroblasts,NFs)、宫颈癌细胞系HeLa、Siha和ME180为材料,采用IL-6、STAT3和Notch抑制剂处理不同细胞,用细胞染色、免疫荧光、蛋白[质]印迹法(Western blot)和流式细胞术等方法测定细胞衰老、STAT3、Notch信号、细胞侵袭能力和对放射线诱导的细胞凋亡变化。结果：CAFs条件培养基(conditioned medium,CM)或IL-6可以激活STAT3和Notch信号诱导细胞衰老或促进宫颈癌细胞的侵袭能力;CAFs与宫颈癌细胞混合培养能促进宫颈癌细胞的侵袭,但IL-6抗体、STAT3抑制剂S31-201或Notch抑制剂DAPT处理细胞则会抑制宫颈癌细胞的侵袭。IL-6/STAT3作为Notch信号的上游分子,可能主要通过自分泌或旁分泌的方式上调成纤维细胞或癌细胞中Notch信号关键配体Jagged-1而活化Notch信号,最后赋予宫颈癌细胞对放射线的抵抗作用。结论：CAFs在肿瘤微环境中可能通过IL-6/STAT3活化Notch信号诱导宫颈癌上皮细胞的侵袭和放疗抵抗,靶向STAT3/Notch信号相关分子有可能提高宫颈癌的放疗效果。     

Serum deprivation response inhibits breast cancer progression by blocking transforming growth factor‐β signaling

Serum deprivation response (SDPR), a key substrate for protein kinase C, play a critical role in inducing membrane curvature and participate in the formation of caveolae. However, the function of SDPR in cancer development and progression is still not clear. Here, we found that SDPR is downregulated in human breast cancer. Overexpression of SDPR suppresses cell proliferation and invasion in MDA‐MB‐231 cells, while depletion of SDPR promotes cell proliferation and invasion in MCF10A cells. Subsequently, SDPR depletion induces epithelial–mesenchymal transition (EMT)‐like phenotype. Finally, knockdown of SDPR activates transforming growth factor‐β (TGF‐β) signaling by upregulation of TGF‐β1 expression. In conclusion, our results showed that SDPR inhibits breast cancer progression by blocking TGF‐β signaling. Serum deprivation response suppresses cell proliferation and invasion in breast cancer cells. SDPR depletion induces epithelial–mesenchymal transition by activation of TGF‐β signaling.     

Eribulin mesilate suppresses experimental metastasis of breast cancer cells by reversing phenotype from epithelial–mesenchymal transition (EMT) to mesenchymal–epithelial transition (MET) states

Background:

Eribulin mesilate (eribulin), a non-taxane microtubule dynamics inhibitor, has shown trends towards greater overall survival (OS) compared with progression-free survival in late-stage metastatic breast cancer patients in the clinic. This finding suggests that eribulin may have additional, previously unrecognised antitumour mechanisms beyond its established antimitotic activity. To investigate this possibility, eribulin''s effects on the balance between epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) in human breast cancer cells were investigated.

Methods:

Triple negative breast cancer (TNBC) cells, which are oestrogen receptor (ER−)/progesterone receptor (PR−)/human epithelial growth receptor 2 (HER2−) and have a mesenchymal phenotype, were treated with eribulin for 7 days, followed by measurement of EMT-related gene and protein expression changes in the surviving cells by quantitative real-time PCR (qPCR) and immunoblot, respectively. In addition, proliferation, migration, and invasion assays were also conducted in eribulin-treated cells. To investigate the effects of eribulin on TGF-β/Smad signalling, the phosphorylation status of Smad proteins was analysed. In vivo, the EMT/MET status of TNBC xenografts in mice treated with eribulin was examined by qPCR, immunoblot, and immunohistochemical analysis. Finally, an experimental lung metastasis model was utilised to gauge the metastatic activity of eribulin-treated TNBC in the in vivo setting.

Results:

Treatment of TNBC cells with eribulin in vitro led to morphological changes consistent with transition from a mesenchymal to an epithelial phenotype. Expression analyses of EMT markers showed that eribulin treatment led to decreased expression of several mesenchymal marker genes, together with increased expression of several epithelial markers. In the TGF-β induced EMT model, eribulin treatment reversed EMT, coincident with inhibition of Smad2 and Smad3 phosphorylation. Consistent with these changes, TNBC cells treated with eribulin for 7 days showed decreased capacity for in vitro migration and invasiveness. In in vivo xenograft models, eribulin treatment reversed EMT and induced MET as assessed by qPCR, immunoblot, and immunohistochemical analyses of epithelial and mesenchymal marker proteins. Finally, surviving TNBC cells pretreated in vitro with eribulin for 7 days led to decreased numbers of lung metastasis when assessed in an in vivo experimental metastasis model.

Conclusions:

Eribulin exerted significant effects on EMT/MET-related pathway components in human breast cancer cells in vitro and in vivo, consistent with a phenotypic switch from mesenchymal to epithelial states, and corresponding to observed decreases in migration and invasiveness in vitro as well as experimental metastasis in vivo. These preclinical findings may provide a plausible scientific basis for clinical observations of prolonged OS by suppression of further spread of metastasis in breast cancer patients treated with eribulin.