miR-494在乳腺癌中的表达及其临床意义

目的探讨miR-494在乳腺癌组织和乳腺癌细胞系中的表达及其与乳腺癌临床分期、转移和预后的关系。方法实时荧光定量聚合酶链反应(qRT-PCR)检测5种乳腺癌细胞系MDA-MB-231、MDA-MB-453、HCC-1937、MDA-MB-468、MCF-7和乳腺上皮细胞系HBL-100中miR-494的表达水平;收集54对乳腺癌组织和癌旁组织,用qRT-PCR法检测其miR-494的表达水平,并分析其与乳腺癌临床分期、转移和预后的关系。结果 qRT-PCR检测结果显示,与正常乳腺上皮细胞HBL-100相比,MDA-MB-231、MDA-MB-453、HCC-1937、MDA-MB-468、MCF-7乳腺癌细胞系中miR-494的表达水平均明显降低(t分别为212.9、37.73、27.53、10.61、19.46,P均0.05)。此外,miR-494在乳腺癌组织中的表达水平亦明显低于癌旁组织(t=5.80,P0.01),且与临床分期(χ2=17.41,P0.05)、组织病理分级(χ2=5.33,P0.05)、C-erb B-2表达情况(χ2=9.83,P0.05)、Ki-67阳性细胞百分比(χ2=6.13,P0.05)有关。结论 miR-494在乳腺癌组织和乳腺癌细胞系中低表达,且与乳腺癌临床分期、转移和预后关系密切,可成为乳腺癌辅助诊断和预后判断的分子标志。     

印记基因SLC22A18的表达与乳腺癌侵袭能力的关系

目的:研究印记基因SLC22A18(solute carrier family 22,member 18)在乳腺癌中的表达情况及其与乳腺癌侵袭能力的关系。方法:采用Transwell方法评估2种不同恶性程度的乳腺癌细胞株MDA-MB-231(恶性程度高)和MCF-7(恶性程度低)的侵袭转移能力。分别采用实时荧光定量逆转录聚合酶链反应(RT-PCR)和蛋白质印迹法检测SLC22A18的mRNA和蛋白在这2种乳腺癌细胞株中的表达情况。结果:MDA-MB-231细胞株恶性程度高,穿过膜的细胞多,侵袭能力强;MCF-7细胞株恶性程度低,穿过膜的细胞少,侵袭能力弱;SLC22A18在MCF-7中的mRNA和蛋白表达水平高于MDA-MB-231;差异有统计学意义(P0.01)。结论:印记基因SLC22A18的表达与乳腺癌细胞的侵袭能力相关,该基因有望作为一个抑癌基因抑制乳腺癌的转移。     

表柔比星对乳腺癌细胞c-FLIP表达的影响

目的:探讨表柔比星对乳腺痛细胞中c-FLIP表达的影响.方法:以MCF-7及MDA-MB-231乳腺癌细胞株为对象分两组:处理组中加入2 mg/L的表柔比星分别作用24 h、48 h和72 h,对照组加入等量生理盐水.采用RT-PCR技术检测两组细胞株中c-FLIP的表达,漉式细胞仪检测细胞凋亡百分率.结果:MCF-7及MDA-MB-231乳腺癌细胞中有c-FLIP的表达;与对照组相比,表柔比星作用24 h,乳腺癌细胞中c-FLIP的表达明显下降,48 h时c-FLIP表达有所升高,72 h时表达最低.随着表柔比星作用时间延长,细胞凋亡率逐渐增加,72 h达到最高.结论:表柔比星通过抑制c-FLIP的表达促进乳腺癌细胞的凋亡.     

表没食子儿茶素没食子酸酯对乳腺癌细胞生长及迁移的影响

目的：探讨表没食子儿茶素没食子酸酯（EGCG）对乳腺癌细胞MCF-7生长的影响及对乳腺癌细胞MDA-MB-231迁移的影响。方法：MCF-7细胞培养贴壁之后,加入EGCG处理,2d后收集蛋白,采用Western Blot检测磷酸化p38丝裂原活化蛋白激酶（phospho-p38MAPK）的表达;同样处理后收集活细胞,用细胞计数法检测细胞的存活;取对数生长期的MDA-MB-231细胞,分至6孔板培养,使用EGCG处理后,采用细胞划线法探测乳腺癌细胞的迁移。结果：使用EGCG处理乳腺癌细胞后,phospho-p38MAPK的表达降低,EGCG处理乳腺癌细胞4d后其增殖率降低50%,迁移活性降低。结论：EGCG处理乳腺癌细胞能抑制肿瘤细胞的生长以及迁移,这与p38 MAPK信号通路相关。     

人三阴性乳腺癌细胞耐药株的建立及特性研究

目的建立人三阴性乳腺癌细胞株MDA-MB-231的多西紫杉醇(Doc)耐药模型(MDA-MB-231/Doc)和表阿霉素(Epi)耐药模型(MDA-MB-231/Epi),探讨其生物学特性。方法采用Doc和EPi低浓度逐步加量诱导法历时12个月分别建立MDA-MB-231/Doc和MDA-MB-231/Epi耐药细胞株。通过细胞形态学观察、MTT法和流式细胞术分析、比较其生物学特性,实时荧光定量PCR检测多药耐药基因(MDR1)mRNA表达,Western Blot法检测P糖蛋白(P-gp)、雌激素受体(ER)、孕激素受体(PR)和人表皮生长因子受体2(Her-2)的表达状况。结果所构建的MDA-MB-231/Doc和MDA-MB-231/Epi耐药株可分别在12nmol/L Doc和800nmol/L Epi中稳定生长,在相同的药物浓度下,耐药细胞株的生长增殖率明显高于亲代细胞,其耐药指数分别为亲代敏感细胞的8.32倍和64.93倍,且相互呈交叉耐药状态。与亲代细胞相比,两株耐药细胞处于G1期和G2期的细胞增加、处于S期的细胞减少,随撤药时间的延长,细胞的增殖速度加快。两株耐药株的MDR1基因表达水平增高,分别为亲代细胞的4.05倍和5.96倍,P-gp表达为阳性。与MCF-7细胞株相比,MDA-MB-231细胞株ER、PR、HER2表达阴性,是典型的三阴性乳腺癌细胞株。结论成功建立MDA-MB-231/Doc和MDA-MB-231/Epi的耐药细胞株,其生长及耐药性稳定。     

miR-126和miR-199对乳腺癌细胞周期进程的影响

目的探讨miR-126和miR-199在乳腺癌细胞中的表达对肿瘤细胞周期的影响。方法以qRT-PCR检测分析在乳腺癌细胞中miR-126和miR-199的表达情况;以miRNA mimics转染乳腺癌细胞株MDA-MB-231,检测miR-126和miR-199在表达增高的情况下对乳腺癌细胞周期的影响。结果miR-126和miR-199在乳腺癌细胞MDA-MB-231中的表达较MCF-7细胞株明显更低;转染miRNA mimics 48h后,乳腺癌细胞株MDA-MB-231中的miR-126和miR-199的表达显著上调,可促进细胞凋亡,阻滞乳腺癌细胞周期进程。结论提高miR-126和miR-199在乳腺癌细胞中的表达可抑制肿瘤细胞周期进程,发挥抗肿瘤效果。     

Stathmin表达水平与乳腺癌细胞侵袭能力相关性研究

目的 探讨乳腺癌细胞MDA-MB-231和MCF-7中Stathmin基因表达水平与细胞生长、黏附、侵袭等生物学行为之间的关系,为进一步研究乳腺癌转移机制奠定实验基础。方法 应用RT-PCR和Western Blot方法检测MDA-MB-231和MCF-7细胞中Stathmin基因的表达水平,同时利用细胞增殖试验、细胞黏附试验和细胞侵袭试验检测MDA-MB-231和MCF-7细胞的生长、黏附、侵袭能力,分析Stathmin表达与细胞的生长、黏附、侵袭能力之间的关系。结果 RT-PCR和Western Blot检测结果显示,Stathmin基因在MDA-MB-231和MCF-7细胞中表达均高于正常对照细胞(F=10.173,P<0.05),且MDA-MB-231细胞中的表达水平明显高于MCF-7细胞中的表达水平(t=4.562,P<0.05)。而MDA-MB-231细胞在生长、黏附和侵袭能力方面均强于MCF-7细胞(P<0.05)。结论 Stathmin表达水平高的乳腺癌细胞相应的生长、黏附、侵袭能力较强,Stathmin表达水平与细胞侵袭能力密切相关。     

Rab27A在4种不同人乳癌细胞株中的表达

目的检测Rab27A在4种人乳癌细胞中的定位、表达情况,初步研究Rab27A表达对乳癌细胞生物学特性的影响。方法采用RT-PCR技术,检测Rab27A mRNA在4种人乳癌细胞MCF-7、MDA-MB-231、MDA-MB-435和MDA-MB-435HM中的表达并进行半定量分析。结果Rab27A mRNA在人乳癌细胞MDA-MB-231、MDA-MB-435及MDA-MB-435HM中的表达水平分别是MCF-7中的2.1、3.4和6.9倍,差异有显著意义（F=17.74～136.23,P〈0.05、0.01）;Rab27A蛋白在人乳癌细胞MDA-MB-231、MDA-MB-435及MDA-MB-435HM中的表达分别是MCF-7中的2.8、4.9和9.2倍,差异有显著性（F=37.74～154.29,P〈0.05、0.01）。Rab27A蛋白弥散性分布于MCF-7、MDA-MB-231和MDA-MB-435细胞浆中,MDA-MB-231和MDA-MB-435中又可见Rab27A蛋白在核周凝聚。结论Rab27A表达可能与乳癌细胞侵袭转移能力有关。     

硫酸乙酰肝素在MDA-MB-231细胞增殖抑制中的作用

目的:探讨硫酸乙酰肝素(heparan sufate,HS)在抑制人乳腺癌MDA-MB-231细胞增殖过程中对葡萄糖调节蛋白78(glucose-regulated pr otein,GRP78)及相关凋亡蛋白表达的影响.方法:培养人乳腺癌上皮MCF-7细胞株,生长达汇合期及汇合后期时收集培养液,提取其中的HS链.采用噻唑蓝比色法检测HS对MDA-MB-231细胞增殖的抑制作用.采用流式细胞仪检测细胞凋亡情况.应用Westem blot法分别检测GRP78蛋白和凋亡相关蛋白Bcl-2、Bax的表达.结果:HS对MDA-MB-231细胞增殖有明显的抑制作用,且具有剂量和时间依赖性(P<0.05).HS可诱导MDA-MB-231细胞凋亡,并随着剂量的增大,细胞的凋亡率升高(P<0.05).HS抑制MDA-MB-231细胞GRP78蛋白的表达(P<0.05).HS可促进Bax蛋白表达(P<0.05),但抑制Bcl-2蛋白的表达(P<0.05).结论:HS通过减少MDA-MB-231细胞的GRF78、Bcl-2的表达,增加Bax表达来促进肿瘤细胞的凋亡,发挥其抗肿瘤作用.     

μ阿片受体通过调控Smad2抑制乳腺癌细胞迁移侵袭

目的:研究μ阿片受体(MOR)对乳腺癌细胞侵袭及迁移的影响及其机制。方法:Western blot(WB)及RT-q PCR检测乳腺癌细胞系MCF-7、BT-549、SKBR3、MDA-MB-231、BT-474、T47D中MOR的表达水平。在MDA-MB-231细胞中,慢病毒转染过表达MOR。用划痕实验、transwell实验以及WB实验来研究MOR表达量改变对乳腺癌MDA-MB-231细胞侵袭、迁移的影响及可能的机制。结果:WB及RT-q PCR结果示,MOR在MDA-MB-231中的表达量明显低于MCF-7、BT-549、SKBR3、BT-474、T47D。划痕实验及transwell实验示,过表达MOR后MDA-MB-231细胞愈合、迁移及侵袭能力明显减弱。WB结果示,与对照组相比,过表达组的Smad2、p Smad2、MMP9、MMP2、N-cadherin表达量下调。结论:与MCF-7等低转移潜能细胞相比,MOR在高转移潜能乳腺癌细胞系MDA-MB-231中表达量低。过表达MOR后,MDA-MB-231细胞迁移、侵袭能力减弱。WB结果提示MOR对MDA-MB-231细胞迁移侵袭的影响可能与Smad2及其下游蛋白的表达下调有关。     

人乳腺癌上皮细胞miR-217对DNMT1调控作用的研究

目的 验证在乳腺癌上皮细胞系中,miR-217通过直接作用于DNMT1 3′UTR调控DNMT1表达,为后期动物实验和临床试验提供实验基础.方法 利用瞬时转染技术,在对应细胞系中,过表达、抑制miR-217,Western blot检测各实验组中DNMT1蛋白表达,并用含有miR-217野生型及突变型识别位点的DNMT1 3′UTR载体质粒分别转染293T细胞,检测相对荧光素酶活性改变.结果 过表达miR-217,DNMT1表达下降;抑制miR-217,DNMT1表达增高;双荧光素酶检测实验,DNMT1-UTR-WT的相对荧光素酶活性显著低于DNMT1-UTR-MUT,差异有统计学意义（P〈0.05）.结论 在乳腺癌上皮细胞系中,miR-217对DNMT1表达具有调控作用,且miR-217通过直接作用于DNMT1 3′UTR调控DNMT1表达.     

TNF-α可诱导乳腺癌细胞凋亡

目的研究TNF-α对乳腺癌的影响。方法采用RT-PCR和WesternBlotting分析30例乳腺浸润性导管癌及癌旁正常乳腺组织,乳腺正常上皮细胞系及乳腺癌细胞系中TNF-α的表达情况;采用流式细胞术观察TNF-α对乳腺癌细胞凋亡的影响。结果RT-PCR和Western Blotting结果碌示,TNF-αmRNA和蛋白在乳腺癌组织中表达都明显低于配对的癌旁正常组织（P〈0．05）,在乳腺上皮细胞系中表达均高于乳腺癌三利一细胞系,流式细胞术检测结果显示与未处理组桐比．经TNF-α处理的MDA-MB-435S（16．7±0．31）和MCF7（18．6±0．42）细胞的凋亡率明显增加,差异均有统计学意义（P〈0．05）。结论TNF-α可促进乳腺癌细胞的凋亡,TNF-α可为乳腺癌的治疗提供新靶点。     

巨噬细胞迁移抑制因子受体CD74与乳腺癌转移及预后的相关性

目的：研究巨噬细胞迁移抑制因子受体CD74在乳腺癌细胞株MCF-7和MDAMB-231以及人乳腺癌组织中的表达及其与乳腺癌患者预后的关系。方法：应用荧光定量聚合酶链式反应（fluorescentquantitativepo[ymerasechainreaction,FQ-PCR）和蛋白质印迹（Westernblotting）方法分别检测不同转移潜能乳腺癌细胞株MCF-7和MDA—MB-231CD74的mRNA和蛋白表达量,并比较CD74在两者间的表达差异;应用免疫组织化学检测89例乳腺癌组织中CD74的表达情况,分析CD74与乳腺癌患者总生存率和无瘤生存率的关系。结果：高转移潜能乳腺癌细胞株MDA—MB-231中CD74表达显著高于低转移潜能细胞株MCF-7（P〈0．001）。CD74表达水平是乳腺癌总生存率（P=0．001）和无瘤生存率（P〈0．001）的独立预测因素。结论：CD74的表达水平与乳腺癌患者预后呈负相关。     

5,7,4⿲-Trihydroxy-6,8-diprenylisoflavone and lupalbigenin,active components of Derris scandens,induce cell death on breast cancer cell lines

BackgroundNatural products are a potential source for cancer chemotherapeutic development. This current study was performed to investigate the anti-tumor potential of 5,7,4⿲-trihydroxy-6,8-diprenylisoflavone (TD) and lupalbigenin (LB), plant flavonoids found in Derris scandens Benth (family: Leguminosae), in cancer and normal cell lines.MethodsThe human breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-468, the human colon cancer cell line SW-620, and the mouse fibroblast cell line L-929 were used to test their anti-cancer activity. Apoptotic cell levels were measured by staining with annexin-V and propidium iodide and Western blot analysis was performed to confirm the apoptotic mechanism.ResultsThe results revealed that TD and LB showed specific cytotoxicity against MDA-MB-231 and MCF-7 cells. To elucidate mode of cell death via cytotoxic activities, breast cancer cell lines were treated. TD and LB induced MDA-MB-231 and MCF-7 cells to apoptosis, with the highest number of apoptotic cells at 24 and 72 h, respectively. Furthermore, TD and LB inhibited cell cycle progression via up-regulation of p21. Both compounds stimulated apoptosis through down-regulation of bcl-2, up-regulation of bax and releasing of cytochrome C proteins.ConclusionsTD and LB have significant anti-cancer effects against human breast cancer cells via cell cycle arrest and the induction of apoptosis through mitochondria signaling pathways, and may be potential anti-cancer agents for the treatment of breast cancer.     

Benzyl isothiocyanate-induced apoptosis in human breast cancer cells is initiated by reactive oxygen species and regulated by Bax and Bak

Epidemiologic studies have revealed an inverse correlation between dietary intake of cruciferous vegetables and the risk of breast cancer. We now show that cruciferous vegetable constituent benzyl isothiocyanate (BITC) effectively suppresses growth of cultured human breast cancer cells (MDA-MB-231 and MCF-7) by causing G(2)-M phase cell cycle arrest and apoptosis induction. On the other hand, a normal mammary epithelial cell line (MCF-10A) is significantly more resistant to growth arrest and apoptosis by BITC compared with breast cancer cells. The BITC-mediated cell cycle arrest was associated with a decrease in levels of proteins involved in regulation of G(2)-M transition, including cyclin B1, cyclin-dependent kinase 1, and cell division cycle 25C. The BITC-induced apoptosis correlated with induction of proapoptotic proteins Bax (MCF-7) and Bak (MDA-MB-231 and MCF-7) and down-regulation of antiapoptotic proteins Bcl-2 and Bcl-xL (MDA-MB-231). The SV40-immortalized mouse embryonic fibroblasts derived from Bax and Bak double knockout mice were significantly more resistant to BITC-induced DNA fragmentation compared with wild-type mouse embryonic fibroblasts. The BITC treatment caused rapid disruption of the mitochondrial membrane potential, leading to cytosolic release of apoptogenic molecules, which was accompanied by formation of autophagosome-like structures as revealed by transmission electron microscopy. The BITC-mediated apoptosis was associated with generation of reactive oxygen species and cleavage of caspase-9, caspase-8, and caspase-3. Apoptosis induction by BITC was significantly attenuated in the presence of a combined superoxide dismutase and catalase mimetic EUK134 as well as caspase inhibitors. In conclusion, the present study reveals a complex signaling leading to growth arrest and apoptosis induction by BITC.     

Tamoxifen-resistant human breast cancer cell growth: inhibition by thioridazine, pimozide and the calmodulin antagonist, W-13.

Estrogen receptor (ER)-negative human breast cancer cell lines (MDA-MB-231 and MDA-MB-435) and ER-positive derivatives of the MCF-7 cell line selected for growth in the presence of antiestrogens (LY2 and RR) were used as in vitro models of tamoxifen-resistant human breast cancer in this study. The sensitivity of the tamoxifen-sensitive (MCF-7) and tamoxifen-resistant human breast cancer cell growth to two noncytotoxic neuroleptic drugs, pimozide and thioridazine, and the anticalmodulin agent, W-13, were compared. Inhibition of cell growth was measured as a decrease in cell number following a 72-h incubation with drug. Growth of the ER-negative cell lines MDA-MB-231 and MDA-MB-435 was inhibited by all three drugs. The average Ki values in these two lines were 6.3 and 3.8 microM for pimozide and 4.1 and 15 microM for thioridazine, respectively. Both ER-negative cell lines were more sensitive than MCF-7 cells to growth inhibition by W-13. MCF-7 cells selected for antiestrogen resistance were sensitive to growth inhibition by W-13 and thioridazine (LY2, average Ki = 10.4 microM; RR, average Ki = 5.2 microM). LY2 and RR cells were resistant to pimozide except when treated with estradiol (Ki = 4.6 and 7.9 microM, respectively). Pimozide, thioridazine and W-13 all exerted different effects on the distribution of human breast cancer cells within the cell cycle, suggesting that each drug may utilize a distinct pathway for inhibition of cell growth. We conclude that all three drugs are potential noncytotoxic alternatives to tamoxifen for the treatment of tamoxifen-resistant human breast cancer.     

Caprin-1 is a novel microRNA-223 target for regulating the proliferation and invasion of human breast cancer cells

MicroRNAs (miRNAs) are 21–22 nucleotides regulatory small non-coding RNAs that inhibit gene expression by binding to complementary sequences especially the 3’ untranslated region (3’UTR) of mRNA. One miRNA can target many messenger RNAs, leading to a complex metabolic network. Previous studies have shown that miRNA-223 regulates migration and invasion of tumor cells and targets cytoplasmic activation/proliferation-associated protein-1 (Caprin-1). In the present study, we detected the expression of miRNA-223 and Caprin-1 in MCF-7, T-47D and MDA-MB-231 cancer cell lines, and MCF-10A normal breast cell line, and analyzed the role of miRNA-223 in Caprin-1-induced proliferation and invasion of human breast cancer cells. We found that miRNA-223 expression levels are significantly lower in MCF-7, T-47D and MDA-MB-231 cancer cells than in MCF-10A normal breast cells, while Caprin-1 expression is higher in cancer cells than in normal breast cells. The most malignant cancer cell line MDA-MB-231 has the lowest expression of miR-223, but the highest expression of Caprin-1. Further, we found that miR-223 targets the 3’UTR of Caprin-1 miRNA and down-regulates the expression of Caprin-1. We also found that over-expression of Caprin-1 can promote the proliferation and the invasion of breast cancer cells, but miRNA-223 can inhibit the proliferation and the invasion. miRNA-223-induced inhibition can be reversed by ectopic over-expression of Caprin-1. These findings suggest that miR-223 may suppress the proliferation and invasion of cancer cells by directly targeting Caprin-1. Our study also indicates that expression levels of miR-223 and Caprin-1 can be used to predict the state of cancer in breast cancer patient.