CXCR4和Survivin启动子的克隆及其在乳腺癌细胞系的转录特异性分析

目的:评估CXCR4和Survivin启动子在乳腺癌细胞系中的特异转录活性.方法:采用PCR方法扩增CXCR4和Survivin启动子序列,将其分别克隆到舍有报告基因增强绿色荧光蛋白(EGFP)的质粒载体pEGFP-1和含有荧光素酶(LUC)的质粒载体pGL3-Basic,经脂质体分别转染乳腺癌细胞系(MCF-7MDA-MB-231和T-47D)和乳腺上皮细胞(HBL-100).荧光显微镜下观察EGFP的表达情况,并测定2个启动子在乳腺癌细胞中的荧光素酶活性.结果:CXCR4和Sur-vivin启动子在3种乳腺癌细胞中均表现特异的转录活性.CXCR4启动子在MDA-MB-231中的活性高于Survivin启动子(P=0.004);而在MCF-7细胞中活性低于Survivin启动子(P=0.006),两者在T-47D细胞中的启动子活性相当,P=0.615.结论:CXCR4和Survivin启动子在乳腺癌细胞中具有强的特异转录活性,且在不同乳腺癌细胞类型表现的转录特异性有一定差异.     

miR-139通过下调CXCR4/CXCL12生物轴抑制乳腺癌定向转移的分子机制研究

摘 要：［目的］ 检测miR-139在乳腺癌细胞系及组织中的表达,验证CXCR4是否为miR-139直接调控的靶基因,并探究miR-139是否可通过靶向抑制CXCR4/CXCL12生物轴从而抑制乳腺癌细胞的定向转移。［方法］ 实时荧光定量聚合酶链式反应（qRT-PCR）用于检测miR-139在34对转移性乳腺癌患者的原发灶、转移灶及癌旁组织中,在5株乳腺癌细胞系及1株正常乳腺上皮细胞中的表达。将包含miR-139的重组慢病毒质粒及其阴性对照空载体质粒vector分别以病毒/细胞数量=20的比例感染MDA-MB-231细胞,经2.0μg/ml嘌呤霉素筛选,成功构建稳定表达miR-139或vector的MDA-MB-231细胞,将MDA-MB-231vector和MDA-MB-231miR-139细胞分别经尾静脉注射到BALB/c裸鼠中,构建乳腺癌裸鼠转移模型,观察过表达miR-139对裸鼠体内转移能力的影响。TargetScan软件用于预测miR-139的靶基因,根据预测结果,将野生型或突变型CXCR4的3’-UTR区域克隆到荧光素酶报告基因的下游（CXCR4-wt或CXCR4-mut）并分别与miR-139 mimics或scramble共转染后检测荧光素酶的活性。qRT-PCR和Western blot分别检测转染miR-139 mimics和scramble后CXCR4、CXCL12的mRNA和蛋白表达。［结果］ miR-139在转移性乳腺癌原发灶和转移灶中的表达均低于癌旁组织,且转移灶中表达最低,miR-139在乳腺癌细胞系中的表达水平显著低于正常乳腺上皮细胞MCF-10A,且MDA-MB-231细胞中最低。尾静脉注射MDA-MB-231miR-139的裸鼠肺转移灶的个数均显著低于MDA-MB-231vector细胞。TargetScan软件预测CXCR4为miR-139直接调控的靶基因,CXCR4-wt+miR-139共转染组比CXCR4-wt+scramble共转染组的荧光素酶活性强度显著降低,CXCR4-mut+miR-139共转染组和CXCR4-wt+scramble共转染组间荧光素酶活性强度无显著性差异。转染miR-139后,CXCR4和CXCL12的mRNA和蛋白表达水平均显著下调,CXCR4/CXCL12生物轴下游的p-AKT和p-ERK蛋白表达水平也显著下调。［结论］ miR-139可通过靶向调控CXCR4/CXCL12生物轴而抑制乳腺癌细胞MDA-MB-231定向转移。     

RNA干扰CXCR4表达抑制乳腺癌MDA-MB-231细胞的增殖、黏附和迁移

目的：构建CXC趋化因子受体4（CXC chemokine receptor 4, CXCR4）RNA干扰真核表达载体,研究其对人乳腺癌细胞MDA-MB-231增殖、黏附及迁移能力的抑制作用。方法：构建针对CXCR4的带发夹结构的小RNA干扰序列,并连接到pGCsi-U6-Neo-GFP载体中,转染293T细胞,筛选出干扰效率最高的表达载体。脂质体法转染MDA-MB-231细胞。利用CCK8法、细胞-基质黏附实验和划痕修复实验检测shRNA干扰CXCR4表达对MDA-MB-231细胞增殖、黏附和迁移能力的影响。结果：成功构建CXCR4-shRNA重组质粒,并转染293T细胞,利用RT-PCR及Western blotting检测发现CXCR4沉默效率最高可达81.3%。CXCR4-shRNA转染能显著抑制MDA-MB-231细胞的增殖(P<0.05)以及细胞与细胞外基质的黏附(P<005)。CXCR4-shRNA转染组MDA-MB-231细胞的迁移距离明显低于对照质粒组和空白对照组(P<0.01)。结论：CXCR4-shRNA干扰载体能特异性抑制CXCR4的表达,从而抑制乳腺癌MDA-MB-231细胞的增殖、黏附及迁移。     

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.     

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-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。     

乳腺癌细胞体外摄取2-脱氧葡萄糖荧光类似物

目的 观察2-脱氧葡萄糖(2-DG)荧光类似物2-N[7-硝基苯-2-乙二酸,34羟氨基]-2-脱氧葡萄糖(2-NBDG)被高表达葡萄糖转运蛋白1(GLUT-1)的乳腺癌细胞靶向摄取的情况.方法 应用逆转录聚合酶链反应(RT-PCR)法和免疫组化法检测乳腺癌MDA-MB-231细胞GLUT-1 mRNA和蛋白的表达,采用Western blot法比较乳腺癌MDA-MB-231细胞和MCF-7细胞GLUT-1的蛋白表达量.应用2-NBDG孵育人乳腺癌MDA-MB-231细胞,采用荧光显微镜及流式细胞仪观察、分析对2-NBDG的摄取情况,比较MDA-MB-231和MCF-7细胞吸收2-NBDG量的差异.结果 RT-PCR和免疫组化检测结果显示,MDA-MB-231细胞高表达GLUT-1;Western blot检测结果进一步显示,MDA-MB-231细胞的GLUT-1表达(0.946 4±0.007)高于MCF-7(0.833±0.010).荧光成像及流式细胞仪分析结果显示,MDA-MB-231细胞能快速摄取2-NBDG,且加入50 mmol/L D-葡萄糖后,荧光强度降低了46.0%.2-NBDG孵育乳腺癌细胞20 min后,MDA-MB-231细胞荧光强度(25.10±0.57)明显高于MCF-7细胞(10.12±0.62).结论 2-NBDG能迅速被高表达GLUT-1的乳腺癌MDA-MB-231细胞靶向吸收.     

趋化因子受体CXCR4在乳腺癌中表达的研究

目的探讨趋化受体CXCR4在乳腺癌中的表达及其与肿瘤转移的关系,以及脂多糖(LPS)对其表达的影响.方法采用流式细胞仪和RT-PCR法检测23例乳腺癌患者的癌组织、癌旁组织、正常组织以及MDA-MB-231细胞中CXCR4在蛋白质和mRNA水平的表达情况,以及LPS对MDA-MB-231细胞 CXCR4表达的影响;用Transwell板检测经LPS作用前后MDA-MB-231对趋化因子SDF-1趋化活性的影响.结果乳腺癌组织MDA-MB-231细胞的趋化因子受体CXCR4在蛋白质和mRNA水平的表达均显著高于癌旁组织和正常组织(P<0.05),并且CXCR4的表达与乳腺癌的转移密切相关;脂多糖(LPS)能下调CXCR4的表达,经LPS作用后乳腺癌细胞趋化活性降低.结论趋化因子受体在乳腺癌的转移中起重要作用,下调乳腺癌细胞CXCR4的表达水平,可减少或抑制其转移.     

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。     

微小RNA-148a抑制乳腺癌细胞MDA-MB-231迁移的作用机制

目的:研究微小RNA-148a(miR-148a)在乳腺癌细胞株MDA-MB-231中的表达,探讨上调miR-148a的表达对其迁移的影响及机制.方法:采用实时荧光定量PCR(qRT-PCR)检测正常乳腺上皮细胞MCF-10A和乳腺癌细胞株MDA-MB-231中miR-148a的表达水平.应用脂质体法将miR-148a mimic及阴性对照分别转染MDA-MB-231细胞,qRT-PCR检测转染效率;细胞划痕实验检测转染前后MDA-MB-231细胞迁移能力的变化;Western blot方法检测上皮间质转化(EMT)相关蛋白Slug、Snail和E-cadher-in表达水平的变化.结果:与MCF-10A细胞相比,MDA-MB-231细胞中miR-148a的表达水平明显降低(P<0.05);与阴性对照组相比,miR-148a转染组中miR-148a的表达水平显著升高(P<0.05);过表达miR-148a后,MDA-MB-231细胞迁移能力明显下降,Slug和Snail蛋白表达明显降低,E-cadherin蛋白表达明显增加.结论:miR-148a可通过调控Slug、Snail及E-cadherin蛋白的表达抑制EMT,进而抑制乳腺癌细胞的迁移能力.     

Negative regulation of chemokine receptor CXCR4 by tumor suppressor p53 in breast cancer cells: implications of p53 mutation or isoform expression on breast cancer cell invasion

Chemokine receptor CXCR4 and its ligand CXCL12 are suggested to be involved in migration, invasion and metastasis of breast cancer cells. Mutation of the tumor suppressor gene p53 in breast cancer is associated with metastasis and aggressive clinical phenotype. In this report, we demonstrate that wild type but not the dominant-negative mutant (V143A) or cancer-specific mutants (R175H or R280K) of p53 repress CXCR4 expression. Recently described cancer-specific p53 isoform, Delta133p53, also failed to repress CXCR4 promoter activity. Short-interfering RNA-mediated depletion of p53 increased endogenous CXCR4 expression in MCF-7 breast cancer cells that contain wild-type p53. Basal CXCR4 promoter activity in HCT116 colon carcinoma cells deleted of p53 [HCT116(p53KO)] was 10-fold higher compared to that in parental HCT116 cells with functional wild-type p53. Deletion analysis of CXCR4 promoter identified a seven-base pair p53-repressor element homologous to cyclic AMP/AP-1 response (CRE/AP-1) element. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed binding of ATF-1 and cJun to the CRE/AP-1 element. The p53 rescue drug PRIMA-1 reduced CXCR4 mRNA and cell surface expression in MDA-MB-231 cells, which express R280K mutant p53. CP-31398, another p53 rescue drug, similarly reduced cell surface levels of CXCR4. PRIMA-1-mediated decrease in CXCR4 expression correlated with reduced invasion of MDA-MB-231 cells through matrigel. These results suggest a mechanism for elevated CXCR4 expression and metastasis of breast cancers with p53 mutations or isoform expression. We propose that p53 rescue drugs either alone or in combination with chemotherapeutic drugs may be effective in reducing CXCR4-mediated metastasis.     

Inhibition of estrogen-dependent breast cell responses with phenylacetate

The aromatic fatty acid phenylacetate (PA) and its analogs have come under intense investigation due to their ability to cause the growth arrest of a variety of neoplasia, including human breast cancer. We have determined that PA and its halide derivative 4-chlorophenylacetate (4-CPA) showed marked antiproliferative activity on 3 of 6 human breast cancer cell lines tested. Interestingly, the 3 cell lines that were growth inhibited by PA and 4-CPA were estrogen receptor (ER) positive (T47-D, MCF-7 and ZR-75-1) whereas those that were little affected by these compounds were ER-negative (MDA-MB-157, MDA-MB-231 and SK-Br-3). Dose response studies indicated that 4-CPA inhibited the growth of the sensitive (ER+) cell lines with a potency 3-4 times that of PA. These findings suggest that there is "cross-talk" between the PA and estrogen signaling pathways such that PA can directly inhibit estrogen-dependent events. This hypothesis was directly tested in vitro using ER+ MCF-7 cells that were stably transfected with a luciferase reporter construct driven by the full length (1745 bp) cyclin D1 promoter (MCF-7-D1). Our experiments with MCF-7-D1 cells indicated that PA and 4-CPA inhibited basal and estrogen-induced reporter gene activity by up to 90%, resulting in almost complete elimination of estrogen-dependent cyclin D1 gene activation. Using a reporter gene construct (ERE(V)-tk-Luc) containing a canonical estrogen response element that was transiently transfected into MCF-7 and MDA-MB-231 cells, we have also demonstrated inhibition of promoter activity by PA and 4-CPA that was directly mediated by blockage of activity through the ERE. Taken together, these findings indicate that PA analogs possess potent antiestrogen properties that may, at least partly, account for their antiproliferative effects on ER+ breast cancer cells. The data suggests a novel mechanism of action that might bypass some of the limitations of conventional antiestrogen therapy.     

FGFB binding sites in cancers of the human breast]

We investigated binding characteristics of bFGF in membranes prepared from 4 human breast cancer cell lines (MCF-7, T-47D, BT-20 and MDA-MB-231) and 38 primary breast cancer biopsies. Results of competitive binding experiments were analysed using the "Ligand" program to determine binding site concentrations and affinities. bFGF mitogenic activity was also measured by [3H]-thymidine incorporation into DNA of breast cancer cell lines. The presence of high-affinity binding sites was demonstrated in each cell type (Kd: 0.5 nM). The presence of these high-affinity binding sites was confirmed by saturation experiments. A second class of low-affinity binding sites was detected in the 2 hormono-independent cells (BT-20: Kd = 2.9 nM; MDA-MB-231: Kd = 2.7 nM). bFGF stimulated the proliferation of MCF-7, 7-47D, BT-20 and not of MDA-MB-231 cell lines. In breast cancer biopsies, binding sites were detectable in 36/38 cases; high-affinity binding sites (Kd < 1 nM) were present in 19/39 cases and low-affinity binding sites (Kd > 2 nM) were present in 29/36 cases (the 2 classes of binding sites were present in 12 biopsies). No relation between FGF binding sites and node involvement nature or grade of tumor was evidenced. Negative correlations (Spearman test) were found between total bFGF binding site concentrations and estradiol receptor concentrations (P = 0.05) or progesterone receptor concentrations (P = 0.009). The demonstrations of 1), bFGF specific binding sites in breast cancer membranes; and 2) bFGF growth stimulation of some breast cancer cell lines, indicate that this factor could be involved in the growth of most breast cancers, and could act (among other factors) directly on the growth of cancer cells.