miR-100抑制乳腺癌细胞迁移的作用和机制

目的:探究miR-100对乳腺癌细胞株MDA-MB-231迁移能力的调节与机制.方法:Real time-PCR检测人正常乳腺上皮细胞MCF-10A和乳腺癌细胞株MDA-MB-231中miR-100的基础表达水平.应用脂质体法将 miR-100 mimic及阴性对照分别转染乳腺癌细胞株MDA-MB-231,通过real time-PCR检测转染后miR-100的表达水平,细胞划痕实验检测过表达miR-100对MDA-MB-231细胞迁移能力的影响,Western blot方法检测slug、snail和E-cadherin等EMT蛋白表达水平的变化.结果:miR-100在乳腺癌细胞株MDA-MB-231中的表达明显低于人正常乳腺上皮细胞MCF-10A.转染miR-100 mimic的乳腺癌细胞株MDA-MB-231的miR-100表达水平明显增高,细胞划痕实验显示过表达miR-100的MDA-MB-231细胞划痕愈合速度明显减慢.过表达miR-100的MDA-MB-231细胞E-cadherin蛋白表达水平明显增加,而slug和snail蛋白表达水平明显降低.结论:miR-100抑制乳腺癌细胞株MDA-MB-231的迁移能力与其上调E-cadherin,下调slug、snail蛋白表达,抑制EMT有关.     

乳腺癌MCF-7细胞通过TGF-β诱导的上皮-间质转化获得耐药北大核心CSCD

目的:通过研究高侵袭转移性乳腺癌细胞株MDA-MB-231和低侵袭转移性细胞株MCF-7中上皮-间质转化(epithelial-mesenchymal transition,EMT)和耐药的相关性,以探讨乳腺癌耐药的机制。方法:采用实时荧光定量PCR法检测MDA-MB-231和MCF-7细胞中EMT标志物E-钙黏蛋白(E-cadherin)、波形蛋白(vimentin)和纤黏蛋白(bronectin)以及转录因子Snail、Slug和锌指E-box同源结合框1(Zincnger E-box binding homeobox 1,ZEB1)mRNA的表达水平;划痕愈合实验和Transwell小室实验分别检测MDA-MB-231和MCF-7细胞的迁移和侵袭能力;CCK-8法检测MDA-MB-231和MCF-7细胞对化疗药物5-氟尿嘧啶、顺铂和紫杉醇的敏感性,并采用实时荧光定量PCR法检测耐药基因多重耐药相关蛋白1(multidrug resistance-associated protein 1,MDR1)和MDR相关蛋白1(MDR-associated protein,MRP1)mRNA的表达水平。用转化生长因子β(transforming growth factor-β,TGF-β)诱导MCF-7细胞发生EMT,或用靶向E-cadherin基因的E-cadherin-siRNA沉默EMT相关标志物E-cadherin的表达,再用CCK-8法检测MCF-7细胞对5-氟尿嘧啶敏感性的变化。结果:MDA-MB-231细胞中vimentin、fibronectin、Slug和ZEB1 mRNA的表达水平均高于MCF-7细胞(P值均<0.000 1),E-cadherin mRNA的表达水平明显低于MCF-7细胞(P=0.000 2),Snail mRNA的表达水平无明显差异。与MCF-7细胞相比,MDA-MB-231细胞的迁移和侵袭能力明显更强(P值均<0.000 1);5-氟尿嘧啶、顺铂和紫杉醇对MDAMB-231细胞的半数抑制浓度(half maximal inhibitory concentration,IC50)值明显高于MCF-7细胞(P值均<0.05);MDA-MB-231细胞中耐药相关基因MDR1和MRP1 mRNA表达水平明显更高(P值均<0.000 1)。TGF-β诱导MCF-7细胞发生EMT后,5-氟尿嘧啶对MCF-7细胞的IC50值明显上升(P<0.05);沉默E-cadherin表达后,MCF-7细胞对5-氟尿嘧啶的耐药性明显增强(P<0.05)。结论:乳腺癌中EMT和耐药存在相关性,诱导EMT发生可导致细胞耐药。     

MDM2对人乳腺癌细胞上皮间质转化的影响及其机制研究

目的 探讨鼠双微粒体2（MDM2）在上皮间质转化（EMT）过程中的作用及其分子机制。方法 采用Western blotting检测人乳腺癌MCF-7、MDA-MB-231和MDA-MB-435细胞株中MDM2及EMT相关标记分子（E-cadherin、N-cadherin及Vimentin）水平;分别于MCF-7细胞中瞬时转染MDM2空质粒pcmv、MDM2过表达质粒pcmv-MDM2,MDA-MB 231细胞中瞬时转染针对MDM2三个不同靶点的干扰质粒36h后观察细胞形态,并采用Western blotting和免疫荧光检测EMT相关标记分子及MDM2的表达情况;采用Western blotting及实时定量PCR检测在MCF-7、MDA-MB-231细胞过表达MDM2后对促EMT转录因子Snail1、Twist 蛋白和mRNA水平的影响。结果 MCF-7细胞过表达MDM2后形态由鹅卵石形变为纺锤形,E-cadherin蛋白随MDM2表达水平的上调表达降低;MDA-MB-231细胞敲低MDM2后形态由长梭形变为卵圆形,Vimentin、N-cadherin蛋白随MDM2表达水平的下调表达依次降低;MCF-7及MDA-MB-231细胞过表达MDM2后Snail1、Twist的蛋白及mRNA水平均升高。结论 在乳腺癌细胞株中MDM2可促进EMT发生,其可能通过上调转录因子Snail1和Twist表达来实现。     

CTEN在乳腺癌细胞上皮间质转化中的作用与机制研究

目的:探讨CTEN在乳腺癌细胞上皮间质转化中的作用及其分子机制。方法:采用定量PCR及Western blotting检测人乳腺癌MCF-7和MDA-MB-231细胞中CTEN的表达水平;然后用Lipofectamine 2000将CTEN高表达质粒pcmv-CTEN转染至乳腺癌MCF-7细胞,为高表达组,将对照质粒pcmv转染至MCF-7细胞,为对照组,用定量PCR检测两组细胞中CTEN、Snail和EMT标记分子mRNA水平的变化,用Western blotting检测CTEN、Snail和EMT标记分子蛋白水平的变化;应用Lipofectamine 2000将CTEN干扰质粒siCTEN转染至MDA-MB-231细胞,为干扰组,将对照质粒siNC转染至MDA-MB-231细胞,为对照组,分别用定量PCR和Western blotting检测两组细胞中CTEN、Snail和EMT标记分子mRNA及蛋白水平的变化;应用Lipofectamine 2000将Snail干扰质粒siSnail转染至MCF-7细胞,为干扰组,将对照质粒siNC转染至MCF-7细胞,为对照组,分别用定量PCR和Western blotting检测两组细胞中Snail mRNA及蛋白水平的表达情况,然后再将CTEN高表达质粒pcmv-CTEN转染入两组细胞,定量PCR检测CTEN和EMT标记分子mRNA水平的变化,Western blotting检测CTEN和EMT标记分子蛋白水平的变化。结果:CTEN在MDA-MB-231中的表达量高于MCF-7细胞;与对照组相比,高表达CTEN组的MCF-7细胞形态呈纺锥形,Snail表达升高,上皮标记分子E-cadherin表达下降,间质标记分子N-cadherin及Vimentin表达升高,促进EMT的发生;与对照组相比,敲低CTEN组的MDA-MB-231细胞形态呈鹅卵石形,Snail表达下降,上皮标记分子E-cadherin表达上升,间质标记分子N-cadherin及Vimentin表达下降,促进MET的发生;在MCF-7细胞中干扰Snail后再过表达CTEN,其促进EMT的作用明显减弱。结论:CTEN能够诱导乳腺癌细胞发生上皮间质转化,且其可能通过转录因子Snail发挥作用。     

人乳腺癌组织中miR-34a的表达及其在乳腺癌细胞系中的功能研究

目的 探讨miR-34a在人乳腺癌组织和细胞中的表达情况及对乳腺癌细胞系MDA-MB-231增殖、迁移侵袭和凋亡等生物学行为的影响,为研究乳腺癌组织中miR-34a的作用及深入了解乳腺癌发生发展的分子机制奠定理论基础。方法 通过实时荧光定量PCR（qRT-PCR）法检测miR-34a在20例人乳腺癌组织和癌旁正常组织中表达量的差异并比较其在人乳腺癌细胞系MDA-MB-231、MCF-7和正常乳腺上皮细胞MCF-10A中的表达差异;体外利用脂质体转染技术,转染miR-34a的模拟物（miR-34a mimic）和标记FAM（绿色荧光）的阴性对照(negative control ,miR-NC)进入MDA-MB-231细胞,研究miR-34a对细胞增殖活性、迁移和侵袭能力以及凋亡和周期分布的影响。结果 miR-34a在乳腺癌组织中的表达量较正常癌旁组织下调(P＜0.01);在MDA-MB-231、MCF-7和MCF-10A中的表达呈依次增高的趋势(P＜0.01);转染miR-34a mimic与转染miR-NC的MDA-MB-231相比,其增殖活力、迁移和侵袭能力均下降(P＜0.01),凋亡增加(P＜0.01),细胞周期被阻滞在G1/G0期（P＜0.01）。结论 miR-34a在乳腺癌组织和细胞系MDA-MB-231及MCF-7中的表达较正常组织和MCF-10A中都明显下调;miR-34a能够抑制肿瘤细胞MDA-MB-231的增殖、侵袭迁移,增加细胞凋亡率,使细胞周期阻滞在G0/G1;miR-34a可能起到抑癌作用,其表达水平与乳腺癌的发生发展密切相关。     

miR-26a通过调控E2F7、Myc抑制乳腺癌MDA-MB-231细胞的增殖和迁移能力

目的:研究miR-26a对乳腺癌MDA-MB-231细胞增殖和迁移能力的影响,并分析miR-26a 调控增殖与迁移的可能机制。方法:应用实时荧光定量PCR法(QPCR)检测乳腺癌细胞系和正常乳腺上皮细胞中miR-26a的表达水平,并检测三阴型乳腺癌组织及相应正常乳腺组织中miR-26a与E2F7 mRNA的表达水平。应用脂质体介导的方法,以miR-26a mimics与E2F7 siRNA瞬时转染MDA-MB-231细胞,实时荧光定量PCR法检测miR-26a表达水平,Western blot法检测E2F7、Myc蛋白的表达水平。MTT法检测MDA-MB-231细胞的增殖能力,划痕实验检测MDA-MB-231细胞迁移能力。结果:乳腺癌细胞中miR-26a的表达水平均低于正常乳腺细胞MCF-10A,且三阴型乳腺癌细胞表达水平降低最明显。三阴型乳腺癌组织中miR-26a相对于正常乳腺组织表达减低,而E2F7 mRNA表达则显著升高。miR-26a mimics转染后miR-26a表达水平显著升高,miR-26a过表达可抑制E2F7、Myc蛋白的表达;E2F7 siRNA转染后E2F7表达水平减低,Myc蛋白表达亦减低。MTT实验结果示miR-26a过表达可抑制MDA-MB-231细胞增殖,划痕实验示miR-26a过表达可抑制乳腺癌MDA-MB-231细胞迁移能力。结论:miR-26a可能通过抑制E2F7、Myc调控乳腺癌MDA-MB-231细胞的增殖与迁移能力。     

miRNA-34a抑制乳腺癌细胞MCF-7、MDA-MB-231的增殖与侵袭

目的:研究miRNA-34a(miR-34a)对乳腺癌细胞MCF-7、MDA-MB-231的生物调控作用。方法:采用定量PCR检测人乳腺上皮细胞MCF-10A,乳腺癌细胞株MCF-7、T47D、MDA-MB-231、MDA-MB-453、Hs578T中miR-34a的表达水平。通过miR-34a mimics分别上调MCF-7、MDA-MB-231细胞中miR-34a的表达水平,MTT和Transwell检测肿瘤细胞增殖能力、侵袭力等生物学行为的变化。结果:乳腺癌细胞MCF-7、T47D、MDA-MB-231、MDA-MB-453、Hs578T中miR-34a处于低表达水平。通过miR-34a mimics上调MCF-7、MDA-MB-231细胞中miR-34a的表达后,细胞的增殖能力被miR-34a抑制（P＜0.05）,miR-34a对细胞侵袭有显著抑制作用（P＜0.05）。结论:miR－34a在乳腺癌细胞MCF-7、T47D、MDA-MB-231、MDA-MB-453及Hs578T中低表达,miR-34a抑制乳腺癌细胞MCF-7、MDA-MB-231的细胞增殖和侵袭能力。     

沉默CD44表达对乳腺癌细胞MDA-MB-231增殖与侵袭的影响

目的:探索乳腺癌细胞MDA-MB-231及MCF-7中CD44分子的表达水平差异及沉默CD44对乳腺癌细胞MDA-MB-231增殖、侵袭和迁移的影响。方法:利用qRT-PCR及Western blot技术检测细胞中CD44基因表达水平;设计并合成CD44的siRNA片段(CD44-siRNA)转染乳腺癌细胞,利用qRT-PCR、Western blot技术检测细胞中CD44基因表达水平的变化;MTT检测MDA-MB-231细胞增殖;Transwell侵袭实验检测MDA-MB-231细胞的迁移与侵袭能力变化。结果:CD44在侵袭性乳腺癌细胞MDA-MB-231中的表达高于非侵袭性乳腺癌细胞MCF-7,CD44-siRNA下调了 MDA-MB-231细胞中CD44 mRNA与蛋白水平的表达,并抑制了细胞的增殖和侵袭转移能力。结论:CD44-siRNA能够下调CD44的表达,并有效抑制乳腺癌细胞MDA-MB-231的增殖及其侵袭迁移力。     

miR-21通过靶向PDCD4调控三阴性乳腺癌细胞的迁移和侵袭

目的： 研究miR-21在三阴性乳腺癌细胞MDA-MB-231中的表达，以及其是否通过调控PDCD4影响MDA-MB-231细胞的迁移和侵袭。方法： 采用实时定量PCR（qPCR）法检测MDA-MB-231细胞和正常乳腺细胞MCF-10A中miR-21和PDCD4 mRNA的表达。将MDA-MB-231细胞随机分为5组：空白对照组，转染miR-21模拟物组，模拟物对照组，转染miR-21抑制物组和抑制物对照组。采用Western blot法检测MDA-MB-231细胞PDCD4蛋白的表达；采用荧光素酶报告基因试剂盒检测转染不同载体后荧光强度的变化来判断miR-21的靶标；采用Transwell实验检测各组细胞的迁移和侵袭数目。结果： miR-21和PDCD4 mRNA在MDAMB-231细胞中的表达水平分别明显高于和低于MCF-10A细胞（P均 < 0.01）。过表达或抑制miR-21可调节PDCD4的表达水平。荧光素酶报告基因试剂盒检测结果显示miR-21可直接靶向调控PDCD4的表达。Transwell实验结果表明过表达miR-21表达能增强MDA-MB-231细胞的迁移和侵袭能力。结论： 在MDA-MB-231细胞中，miR-21通过靶向调控PDCD4表达影响细胞的迁移和侵袭。miR-21可能成为抑制三阴性乳腺癌迁移和侵袭的靶点。     

miR-125a增强多西他赛对乳腺癌生长的抑制作用

背景与目的:研究表明miR-125a通过下调Her-2或者Her-3的表达抑制乳腺癌细胞生长,可能是指导乳腺癌治疗的靶点.本研究旨在观察miR-125a是否具有增强多西他赛对乳腺癌细胞株和裸鼠荷瘤模型的生长抑制作用.方法:转染miR-125a联合不同浓度多西他赛处理MCF-7和MDA-MB-231乳腺癌细胞株和MDA-MB-231乳腺癌裸鼠模型,观察细胞株和裸鼠接种肿瘤的生长情况.结果:miR-125a与多西他赛可协同抑制MDA-MB-231及MCF-7乳腺癌细胞株、MDA-MB-231乳腺癌裸鼠模型肿瘤的增殖,单纯转染miR-125a也可抑制MDA-MB-231及MCF-7乳腺癌细胞株的增殖.结论:miR-125a与多西他赛有协同抗乳腺癌作用,可成为乳腺癌靶向治疗的潜在靶点.     

Hypoxia‐induced downregulation of miR‐30c promotes epithelial‐mesenchymal transition in human renal cell carcinoma

MicroRNAs (miRNAs), which negatively regulate protein expression by binding protein‐coding mRNAs, have been integrated into cancer development and progression as either oncogenes or tumor suppressor genes. miR‐30c was reported to be downregulated in several types of cancer. However, its role in human renal cell carcinoma (RCC) remains largely unknown. Here, we show that miR‐30c is significantly downregulated in human RCC tissues and cell lines. We found that miR‐30c downregulation could be induced by hypoxia in RCC cells in a hypoxia‐inducible factors (HIFs) dependent manner. Repression of miR‐30c through its inhibitor resulted in reduction of E‐cadherin production and promotion of epithelial‐mesenchymal transition (EMT), while overexpression of miR‐30c inhibited EMT in RCC cells. We identified Slug as a direct target of miR‐30c in RCC cells. Slug was upregulated in RCC tissues and its expression could be induced by hypoxia, which is consistent with downregulation of miR‐30c by hypoxia. Forced overexpression of Slug in 786‐O cells reduced E‐cadherin production, and promoted EMT as well as cell migration. Moreover, Slug overexpression abrogated the inhibitory role of miR‐30c in regulating EMT and cell migration, indicating miR‐30c regulates EMT through Slug in RCC cells. Our findings propose a model that hypoxia induces EMT in RCC cells through downregulation of miR‐30c, which leads to subsequent increase of Slug expression and repression of E‐cadherin production, and suggest a potential application of miR‐30c in RCC treatment.     

53BP1 suppresses epithelial–mesenchymal transition by downregulating ZEB1 through microRNA‐200b/429 in breast cancer

Epithelial–mesenchymal transition (EMT) is an important mechanism of cancer invasion and metastasis. Although p53 binding protein 1 (53BP1) has been implicated in several biological processes, its function in EMT of human cancers has not yet been reported. Here, we show that 53BP1 negatively regulated EMT by modulating ZEB1 through targeting microRNA (miR)‐200b and miR‐429. Furthermore, 53BP1 promoted ZEB1‐mediated upregulation of E‐cadherin and also inhibited the expressions of mesenchymal markers, leading to increased migration and invasion in MDA‐MB‐231 breast cancer cells. Consistently, in MCF‐7 breast cancer cells, low 53BP1 expression reduced E‐cadherin expression, resulting in increased migration and invasion. These effects were reversed by miR‐200b and miR‐429 inhibition or overexpression. Sections of tumor xenograft model showed increased ZEB1 expression and decreased E‐cadherin expression with the downregulation of 53BP1. In 18 clinical tissue samples, expression of 53BP1 was positively correlated with miR‐200b and mir‐429 and negatively correlated with ZEB1. It was also found that 53BP1 was associated with lymph node metastasis. Taken together, these results suggest that 53BP1 functioned as a tumor suppressor gene by its novel negative control of EMT through regulating the expression of miR‐200b/429 and their target gene ZEB1.     

整合素连接激酶在非小细胞肺癌中过度表达及促进肺癌细胞的侵袭和迁移

目的:探讨整合素连接激酶(ILK)在非小细胞肺癌(NSCLC)中的表达及在侵袭和迁移中的作用和相关分子机制。方法:免疫组化法检测ILK蛋白在NSCLC患者中的表达,细胞转染、siRNA干扰、细胞划痕试验、实时定量PCR、Westernblot方法探讨ILK在肺癌A549细胞中的表达及分子机制。结果:ILK蛋白在原发性NSCLC组织中过度表达30.6%(33/108)并且和TNM分期(P=0.001)、淋巴结转移(P=0.033)相关。ILK在A549细胞中过度表达并且通过下调E-cadherin,上调波形蛋白、纤维连接蛋白、Snail、Slug导致上皮-间质转化(EMT)。此外,NF-κB抑制剂BAY11-7028和小干扰靶RNA(siRNA)NF-p65可诱导E-cadher in的表达下调。结论:ILK在原发性NSCLC组织中高表达并与TNM分期和淋巴结转移相关,其促进肺癌细胞的侵袭和迁徙机制可能是经NF-κB信号通路诱导EMT所致。     

Retracted: Glabridin attenuates the migratory and invasive capacity of breast cancer cells by activating microRNA‐200c

Current treatments for breast cancer, a common malignancy in human females, are less than satisfactory because of high rates of metastasis. Glabridin (GLA), which acts through the FAK/ROS signaling pathway, has been used as an antioxidant and anti‐metastatic agent. However, little is known regarding the effect of microRNA (miRNA) on GLA's anti‐metastatic activity. The miRNA‐200 family, which is frequently expressed at low levels in triple negative breast cancers, inhibits metastasis by blocking the epithelial–mesenchymal transition. Here, we found that GLA attenuated the migratory and invasive capacity of breast cancer cells by activating miR‐200c. GLA induced the mesenchymal–epithelial transition in vitro and in vivo, as determined by increased expression of the epithelial marker, E‐cadherin, and decreased expression of the mesenchymal marker, vimentin. Overexpression of miR‐200c enhanced the expression of E‐cadherin and decreased the expression of vimentin. Furthermore, in MDA‐MB‐231 and BT‐549 breast cancer cells exposed to GLA, knockdown of miR‐200c blocked the GLA‐induced mesenchymal–epithelial transition and alleviated the GLA‐induced inhibition of migration and invasion. Thus, elevation of miR‐200c by GLA has considerable therapeutic potential for anti‐metastatic therapy for breast cancer patients.     

整合素连接激酶在非小细胞肺癌中过度表达及促进肺癌细胞的侵袭和迁移

目的:探讨整合素连接激酶（ILK）在非小细胞肺癌（NSCLC）中的表达及在侵袭和迁移中的作用和相关分子机制。方法:免疫组化法检测ILK蛋白在NSCLC患者中的表达,细胞转染、siRNA干扰、细胞划痕试验、实时定量PCR、Western blot方法探讨ILK在肺癌A549细胞中的表达及分子机制。结果:ILK蛋白在原发性NSCLC组织中过度表达30.6％（33/108）并且和TNM分期（P=0.001）、淋巴结转移（P=0.033）相关。ILK在A549细胞中过度表达并且通过下调E-cadherin,上调波形蛋白、纤维连接蛋白、Snail、Slug导致上皮-间质转化（EMT）。此外,NF-κB抑制剂BAY 11-7028和小干扰靶RNA（siRNA）NF-p65可诱导E-cadherin的表达下调。结论:ILK在原发性NSCLC组织中高表达并与TNM分期和淋巴结转移相关,其促进肺癌细胞的侵袭和迁徙机制可能是经NF-κB信号通路诱导EMT所致。     

ZIC1联合苦参碱对人乳腺癌 MDA - MB -231细胞增殖、迁移及凋亡的影响

目的：探讨 ZIC1联合苦参碱对人乳腺癌 MDA - MB -231细胞的增殖、迁移能力及凋亡的影响。方法：将慢病毒载体 rLV - Zic1- PGK - puro 稳定转染人乳腺癌 MDA - MB -231细胞株并得到稳定传代的细胞株,Western blot 法检测转染效果。以人乳腺癌 MDA - MB -231细胞及 ZIC1基因稳定转染的细胞为研究对象进行药物实验,MTT 法筛选出苦参碱的半数抑制浓度（IC50）后分组,划分 A 空载不加药组（阴性对照组）,B空载加药组,C 转染不加药组和 D 转染加药组。对四组细胞采用黏附试验检测细胞黏附（增殖）能力、划痕实验检测细胞迁移能力、流式细胞术检测细胞凋亡。结果：ZIC1或苦参碱单独作用均可抑制乳腺癌 MDA - MB-231细胞黏附（增殖）、迁移及增强其凋亡,差异较对照组有统计学意义（P <0.05）,且在抑制细胞迁移上存在时间依赖性。两者联合作用抗癌效果更明显,差异较其它三组有统计学意义（P <0.05）。结论：苦参碱联合 ZIC1可明显增强对人乳腺癌 MDA - MB -231细胞的抑癌效果。