miR-31-5p 通过调控TNS1 抑制乳腺癌细胞生物学行为及放疗抵抗的分子机制

目的：探讨miR-31-5p/张力蛋白1 基因（tension protein 1,TNS1）分子轴对乳腺癌细胞生物学行为的影响及其放疗抵抗的分子机制。方法：收集2017 年7 月至2017 年12 月南阳市中心医院肿瘤放疗科收治的、经手术切除的21 例乳腺癌患者癌及癌旁组织标本,以及乳腺癌细胞系MCF-7、MDA-MB-231 和SKBR-3,采用qPCR法检测癌组织和癌细胞系中miR-31-5p 的表达水平。通过6 MV-X射线照射MCF-7 细胞,构建放疗抵抗细胞株MCF-7R。随后,采用克隆形成实验、Transwell 小室法和AnnexinV/PI 染色流式细胞术检测过表达/敲降miR-31-5p 对MCF-7 和MCF-7R细胞放疗敏感性的影响;用双荧光素酶报告基因验证miR-31-5p 与TNS1 的靶向关系。结果：乳腺癌组织、细胞系和MCF-7R细胞中miR-31-5p 表达水平显著低于癌旁组织、人正常乳腺上皮细胞MCF-10A和MCF-7 细胞（均P<0.01）。过表达miR-31-5p 显著抑制MCF-7R细胞的侵袭并促进细胞凋亡（均P<0.01）,沉默miR-31-5p 在MCF-7 细胞中结果相反。双荧光素酶报告基因法证实TNS1 是miR-31-5p 靶基因。过表达miR-31-5p 通过靶向下调TNS1 显著抑制MCF-7R细胞侵袭能力并促进细胞凋亡（均P<0.01）,沉默miR-31-5p 通过上调TNS1 显著促进MCF-7 细胞侵袭和抑制细胞凋亡（均P<0.01）,从而上调MCF-7R对放射治疗的敏感性。结论：miR-31-5p/TNS1 分子轴与乳腺癌放疗抵抗存在调控关系,且过表达miR-31-5p 可逆转MCF-7R细胞对放疗抵抗作用。     

蛋白质精氨酸甲基转移酶2在乳腺癌细胞中的表达及其对SKBR-3细胞生长特性的影响

目的:探讨蛋白质精氨酸甲基转移酶2(protein arginine methyltransferase 2,PRMT2)基因在多种乳腺癌细胞株中的表达情况,以及外源性PRMT2基因过表达对乳腺癌SKBR-3细胞生长特性的影响.方法:采用real-time RT-PCR法检测PRMT2基因在不同乳腺癌细胞株中的表达,并建立稳定表达pcDNA3.1/NT-GFP-PRMT2的SKBR-3细胞株;激光共聚焦显微镜下观察外源性PRMT2蛋白在细胞中的定位,检测在雌激素和雌激素受体(estrogen receptor,ER)拮抗剂4-OHT作用下过表达PRMT2基因对SKBR-3细胞增殖的影响.结果:PRMT2基因在ERα阳性乳腺癌细胞株中表达水平明显高于ERα阴性乳腺癌细胞株;在转染PRMT2基因的SKBR-3细胞株中,雌激素反应元件-荧光素酶报告基因(estrogen response elements-luciferase reporter,ERE-luc)的转录活性明显升高.在无处理因子情况下,外源基因PRMT2在SKBR-3细胞中的表达对细胞的形态及生长速度无明显影响.与未转染及转染GFP空载体的SKBR-3细胞相比,稳定转染PRMT2基因的SKBR-3细胞对雌激素的敏感性明显下降,但其对4-OHT处理的敏感性降低无明显差异.结论:PRMT2基因表达的多少及部位与乳腺癌细胞中ERα的表达密切相关.外源性PRMT2基因在乳腺癌SKBR-3细胞中稳定表达使细胞对雌激素的敏感性降低,但不增加细胞对ER拮抗剂4-OHT的耐药性.     

LncRNA NBAT1通过调控miR-3664-5p/Caspase 9分子轴对人乳腺癌细胞增殖的影响

目的 探究lncRNA NBAT1通过调控miR-3664-5p/Caspase 9分子轴对人乳腺癌细胞增殖的影响。方法 通过在线生物软件分析TCGA(The Cancer Genome Atlas)数据库比较lncRNA NBAT1在乳腺癌和正常乳腺组织中的表达量。RT-qPCR方法比较正常细胞株MCF-10A和乳腺癌细胞株MCF-7、MDA-MB-231及MDA-MB-453中lncRNA NBAT1的表达水平。生物信息学预测lncRNA NBAT1、miR-3664-5p、Caspase 9三者之间关系,并进一步通过荧光素酶报告系统进行验证;lncRNA NBAT1过表达载体转染乳腺癌MCF-7细胞株,RT-qPCR检测转染后lncRNA NBAT1的表达,qPCR检测miR-3664-5的表达,Western blot检测Caspase 9的蛋白表达水平;CCK-8和克隆形成实验检测细胞的增殖能力;同时将lncRNA NBAT1过表达载体和mimics miR-3664-5p转染MCF-7细胞,观察mimics miR-3664-5p对lncRNA NBAT1抑制细胞增殖能力产生的缓解效应。结果 TCGA数据库分析发现,lncRNA NBAT1在乳腺癌组织中的表达量显著低于正常组织(P＜0.001);qPCR检测发现lncRNA NBAT1在正常细胞株MCF-10A细胞中表达量最高(P＜0.001),MDA-MB-231和MDA-MB-453细胞中表达量比MCF-10A细胞低(P＜0.001),MCF-7细胞表达量最低(P＜0.001);通过生物信息学预测lncRNA NBAT1可以吸附miR-3664-5p,Caspase 9是miR-3664-5p的靶基因,并进一步通过荧光素酶报告系统进行验证;lncRNA NBAT1过表达载体转染乳腺癌细胞株MCF-7,qRT-PCR检测发现pc-NBAT1组比pc-NC组LncRNA NBAT1的表达增高(P＜0.001),miR-3664-5的表达降低(P＜0.001),Western blot检测发现pc-NBAT1组比pc-NC组Caspase 9的表达增高(P＜0.001)。CCK-8和克隆形成实验发现pc-NBAT1组比pc-NC组细胞的增殖能力降低(P＜0.001);同时将lncRNA NBAT1过表达载体和mimics miR-3664-5p转染MCF-7细胞,发现mimics miR-3664-5p能够对lncRNA NBAT1抑制细胞增殖能力产生缓解效应(P＜0.001)。结论 lncRNA NBAT1通过调控miR-3664-5p/Caspase 9分子轴抑制乳腺癌细胞的增殖,为乳腺癌治疗提供新的潜在靶点。     

高与低表达RSK4基因人乳腺癌细胞株的筛选及鉴定

目的:探讨核糖体S6激酶4(RSK4)抑癌基因在人乳腺癌细胞株MDA-MB-231、T47D、Bcap37和MCF-7中的表达,并筛选出高和低表达RSK4基因的乳腺癌细胞。方法:逆转录聚合酶链反应(RT-PCR)和蛋白质印迹法从mRNA和蛋白水平检测RSK4在MDA-MB-231、T47D、Bcap-37和MCF-7 4种乳腺癌细胞株中的表达。结果:人乳腺癌细胞株MDA-MB-231、T47D、Bcap37和MCF-7中均有RSK4基因表达,其中,MCF-7细胞RSK4表达水平最高,为表达量最低的MDA-MB-231细胞的2.9倍,MCF-7细胞RSK4蛋白表达水平也最高,与其他3种细胞株相比,差异有统计学意义,P<0.001。RT-PCR法和蛋白质印迹法检测结果一致,具有高度相关性,r=0.766,P<0.001。结论:人乳腺癌细胞株MCF-7为高表达RSK4基因的细胞,MDA-MB-231细胞为低表达RSK4基因的细胞,两者均可作为人乳腺癌细胞中研究RSK4基因的实验材料。     

大黄酸对肿瘤细胞EGFR和HER-2靶点的作用及其作用机制

一目的研究传统中药大黄中葸醌类化合物之一大黄酸对乳腺癌细胞表皮生长因子受体家族EGFR和HER-2靶点的作用及其作用机制。方法通过WesternBlot检测乳腺癌MCF-7、SKBR-3、MDA-MB-231、和MCF-7／ADR细胞中EGFR和HER-2的蛋白表达水平。用MTT方法检测大黄酸对乳腺癌细胞的增殖抑制作用。用RT-PCR方法检测大黄酸对EGFR和HER-2转录水平的影响。通过Western Blot方法研究大黄酸对乳腺癌细胞HER-2、P-HER-2、EGFR、P-EGFR蛋白表达的影响并研究相关的作用机制。结果MCF-7、SKBR-3、MDA-MB-231、和MCF-7／ADR 4株乳腺癌细胞系均显示EGFR高表达。关于HER-2,仅见SKBR-3细胞高表达;而MDA-MB-231和MCF-7／ADR细胞显示中等程度表达,MCF-7细胞低表达。大黄酸对乳腺癌MCF-7、SKBR-3、MDA-MB-231、和MCF-7／ADR细胞增殖抑制的IC50分别为28μg／ml、24μg／ml、24μg／ml和〉100μg／ml。Western Blot研究结果表明大黄酸对HER-2、P-HER-2和P-EGFR蛋白表达有抑制作用,并且随着剂量的增加抑制作用增强,但对EGFR蛋白表达没有明显抑制作用。RT-PCR研究发现大黄酸抑制HER-2的mRNA的水平,从转录水平上发挥对HER-2蛋白表达的抑制作用。进一步研究表明大黄酸通过抑制表皮生长因子家族EGFR和HER-2的酪氨酸激酶磷酸化,进而抑制RAS-RAF-MEK-ERK信号通路,抑制肿瘤细胞增殖,而对NF-κB和AKT诱导的细胞凋亡通路没有明显作用。结论传统中药大黄中的葸醌类化合物大黄酸能够抑制表皮生长因子家族EGFR和HER-2酪氨酸激酶的磷酸化,从而抑制了MAPK信号通路的磷酸化,达到抑制肿瘤细胞增殖的作用。同时大黄酸可以从转录水平抑制HER-2的蛋白表达。     

5-氮杂-2’-脱氧胞苷联合曲古菌素A对乳腺癌SKBR-3细胞中性激素受体基因表达的影响

目的:探讨5-氮杂-2’-脱氧胞苷(5-aza-2’-deoxycytidine,5-aza-dC)联合曲古菌素A(trichostatin A,TSA)能否诱导性激素受体阴性的乳腺癌细胞系SKBR-3同时重新表达功能性雌激素受体α(estrogen receptor alpha,ERα)和雄激素受体(androgen receptor,AR),并进一步研究诱导前后SKBR-3细胞对乳腺癌内分泌治疗敏感性的变化.方法:分别采用RT-PCR法和免疫细胞化学法检测5-aza-dC联合TSA诱导前后SKBR-3细胞和阳性对照MCF-7细胞中ERα、AR、孕激素受体(progestone receptor,PR)、雌激素调节蛋白pS2与前列腺特异性抗原(prostate specific antigen,PSA) mRNA和蛋白的表达水平;采用CCK-8法检测各内分泌治疗药物对5-aza-dC联合TSA诱导后重新表达ERα和AR的乳腺癌SKBR-3细胞增殖能力的影响.结果:5-aza-dC联合TSA能够诱导激素受体阴性的乳腺癌SKBR-3细胞同时重新表达ERα和AR,它们的下游产物PR、pS2以及PSA也相应表达;诱导后SKBR-3细胞中ERα和AR的表达量明显低于在MCF-7细胞中的表达量(P＜0.05).5-aza-dC联合TSA能明显抑制SKBR-3细胞的增殖能力(P＜0.05),加入雌激素类药物17β-雌二醇(17β-estradiol,E2)后细胞的增殖能力略有上升,但差异无统计学意义(P＞0.05).在加入E2的基础上,分别再加入雌激素拮抗剂他莫昔芬(tamoxifen,TAM)或孕激素醋酸甲地孕酮(megestrol acetate,MA)后,二者都能使肿瘤细胞的增殖能力明显下降(P＜0.05);联合加入TAM和MA后,肿瘤细胞的增殖能力进一步明显下降(P＜0.05).结论:5-aza-dC联合TSA能够诱导乳腺癌细胞系SKBR-3同时恢复表达功能性的ERα和AR,抑制肿瘤细胞的生长,同时使SKBR-3细胞恢复了对激素的依赖性和内分泌治疗的敏感性.     

ING4基因表达对乳腺癌细胞生长抑制作用的研究

目的：研究人ING4基因对乳腺癌细胞MCF-7的生长抑制作用。方法：通过实时荧光定量-PCR（real-time fluorogentic quantitative-PCR，RFQ-PCR）检测3种乳腺癌细胞株中ING4 mRNA的表达水平；ING4基因的表达质粒转染MCF-7细胞；MTT法和FCM法检测ING4基因对MCFM细胞增殖的影响；Annexin-V／PI双染法观察细胞凋亡情况；RT-PCR法分析转染／NG4后p53、p21及bax基因的转录表达情况。结果：在3种乳腺癌细胞MCF-7、MDA-MB-435和MDA-MB-231中ING4的表达均明显低于正常乳腺组织。转染ING4表达载体的MCF-7细胞较对照组细胞生长速度减慢（P〈0．05）；细胞周期中G1期比例增加，S期比例减少；细胞凋亡率升高。p53的表达未见明显变化，而p21和bax表达显著上调。结论：ING4与乳腺癌的发生发展具有一定相关性；高表达ING4基因能够抑制人乳腺癌细胞MCF-7的生长。     

NP方案联合不同间隔时间放疗对乳腺癌MCF-7细胞作用研究

目的:研究化疗方案顺铂(DDP) 盖诺(NVB)联合不同间隔时间放射治疗对人乳腺癌MCF-7细胞辐射敏感性影响。方法:采用细胞集落形成方法观察DDP NVB方案联合不同间隔时间放疗对细胞的杀伤作用,流式细胞仪对各组周期分布和凋亡情况进行分析比较,并观察凋亡形态特征。结果:MCF-7细胞化放疗间隔不同时间后细胞SF值差异随照射剂量增加而逐渐明显,8Gy时12小时组最低,0小时组居中,48、72小时组最高。化放疗间隔时间不同引起MCF-7细胞SF值不同与NP作用后不同间隔时间G2/M期阻滞比例不同有一定相关性,NP化疗可诱导细胞凋亡,而且随时间增加呈不可逆转增加,但化疗后不同时间凋亡差异同SF值差异间并未呈现相关性。结论:NP不同时机顺序联合放疗对MCF-7细胞辐射敏感性有一定影响,这种差异同NP作用后不同时间细胞周期分布、凋亡等作用有关。     

沉默长链非编码RNA HOTAIR对直肠腺癌细胞放射敏感性的影响

目的 探讨沉默长链非编码RNA HOTAIR对直肠腺癌细胞株SW480和HCT116细胞增殖、放射敏感性和凋亡的影响。方法 通过实时荧光定量PCR检测直肠腺癌细胞株中lncRNA HOX转录反义RNA (lncRNA HOTAIR)的表达水平。应用RNA干扰技术沉默HOTAIR的表达,分析其在细胞增殖、细胞放射敏感性和凋亡中的作用。对细胞株进行梯度剂量照射后,检测其放射敏感性和细胞凋亡情况。结果 直肠腺癌细胞株SW480和HCT116中的lncRNA HOTAIR的相对表达水平明显高于直肠黏膜细胞系。克隆形成试验结果显示,与对照siRNA转染组比较,在细胞株SW480和HCT116中siRNA-HOTAIR的放射增敏比分别为1.58和1.33。沉默直肠腺癌细胞中的lncRNA HOTAIR可增加直肠腺癌SW480细胞株的凋亡率及放射敏感性。结论 直肠腺癌中lncRNA HOTAIR的表达水平与细胞放射敏感性之间存在相关性,其有可能是预测直肠腺癌细胞放射敏感性的指标之一。放射联合应用siRNA-HOTAIR对直肠腺癌细胞株SW480和HCT116有抑制细胞增殖、诱导凋亡和放射增敏的作用。     

lncRNA RP3-340N1.2 在乳腺癌组织中的表达及其对MCF-7 细胞增殖和迁移的影响

目的:观察长链非编码RNA（long non-coding RNA,lncRNA）RP3-340N1.2 在乳腺癌组织中的表达及其对乳腺癌MCF-7 细胞增殖和迁移的影响,并探讨其可能的作用机制。方法：收集湖北医药学院附属人民医院肿瘤中心2017 年1 月至2017 年9月13 例行乳腺癌根治术切除的癌组织及相应的癌旁组织标本,采用qRT-PCR检测13 例乳腺癌组织及癌旁组织、乳腺癌细胞株和正常乳腺上皮细胞中RP3-340N1.2 的表达差异。使用Lipofectamine 3000 转染试剂分别将RP3-340N1.2 质粒（实验组）和阴性对照质粒（对照组）转染至乳腺癌MCF-7 细胞,CCK-8 法和Transwell 迁移实验检测RP3-340N1.2 过表达对MCF-7 细胞增殖、迁移能力的影响。qRT-PCR检测RP3-340N1.2 过表达对miR-134-5p 和OPCML mRNA表达的影响,Western blotting 检测OPCML相关蛋白的表达水平。结果：RP3-340N1.2 在乳腺癌组织中表达明显低于癌旁组织（P<0.01）,其在乳腺癌细胞株中表达水平明显低于正常乳腺上皮细胞（P<0.01）。上调RP3-340N1.2 的表达抑制MCF-7 细胞的增殖和迁移能力（均P<0.05）。过表达RP3-340N1.2 后MCF-7 细胞中miR-134-5p 表达水平明显降低（P<0.01）、OPCML mRNA 和蛋白的表达水平升高（P<0.01）,而周期调控蛋白CDK4、Cyclin D2 的表达水平下调,细胞迁移调控蛋白Vimentin 和N-cadherin 蛋白表达下调（均P<0.01）。结论：RP3-340N1.2 在乳腺癌组织和细胞株中低表达,上调RP3-340N1.2 的表达可引起miR-134-5p 的表达降低、OPCML mRNA表达升高,从而抑制乳腺癌细胞的增殖和迁移能力。     

Activity of retinoic acid receptor-γ selectively binding retinoids alone and in combination with interferon-γ in breast cancer cell lines

Retinoids modulate several cell functions and especially inhibit the growth of a wide variety of cells including breast cancer. Retinoic acid receptor-γ (RAR-γ) has been shown to mediate the antiproliferative activity of retinoids. To further test this hypothesis we examined the effects of different RAR-γ selectively binding retinoids (CD2325, CD2247, CD666 and CD437) on breast cancer cell lines. With exception of CD2247, all retinoids inhibited proliferation of MCF-7, SKBR-3, T47D and ZR-75-1 breast cancer cell lines, similar to the natural compound all-trans retinoic acid (ATRA). In addition, all 4 compounds were able to act synergistically with interferon-γ (IFN-γ) in all breast cancer cell lines including the retinoid-resistant BT-20 and 734-B lines. In functional transactivation assays we demonstrated that only in the MCF-7 cell line, TPA-mediated AP-1 activity was suppressed only by ATRA and CD2325, whereas in SKBR-3, another RA-sensitive breast cancer cell line, it was not. The synergistic antiproliferative activity involving retinoids and IFN-γ could not be explained by an enhanced anti-AP-1 activity. No correlation was found between expression of RARs and cellular retinoic acid binding proteins (CRABPs) and antiproliferative effects of the retinoids. RAR-γ selectively binding retinoids are potent inhibitors of breast cancer cell proliferation, alone and in combination with IFN-γ. For this reason and because of a possible low toxicity, as compared with retinoic acid, we speculate that these RAR-γ selective binding retinoids might be of clinical importance. Int. J. Cancer 71:497-503, 1997. © 1997 Wiley-Liss Inc.     

MYC-binding lncRNA EPIC1 promotes AKT-mTORC1 signaling and rapamycin resistance in breast and ovarian cancer

AKT-mTORC1 (mammalian target of rapamycin complex 1) signaling pathway plays a critical role in tumorigenesis and can be targeted by rapamycin. However, the underlying mechanism of how long noncoding RNA (lncRNAs) regulate the AKT-mTORC1 pathway remains unclear. EPIC1 (epigenetically-induced lncRNA 1) is a Myc-binding lncRNA, which has been previously demonstrated to be overexpressed in multiple cancer types. In a pathway analysis including 4962 cancer patients, we observed that lncRNA EPIC1 expression was positively correlated with the AKT-mTORC1 signaling pathway in more than 10 cancer types, including breast and ovarian cancers. RNA-seq analysis of breast and ovarian cancer cells demonstrated that EPIC1-knockdown led to the downregulation of genes in the AKT-mTORC1 signaling pathway. In MCF-7, OVCAR4, and A2780cis cell lines, EPIC1 knockdown and overexpression, respectively, inhibited and activated phosphorylated AKT and the downstream phosphorylation levels of 4EBP1 and S6K. Further knockdown of Myc abolished the EPIC1′s regulation of AKT-mTORC1 signaling; suggested that the regulation of phosphorylation level of AKT, 4EBP1, and S6K by EPIC1 depended on the expression of Myc. Moreover, EPIC1 overexpressed MCF-7, A2780cis, and OVCAR4 cells treated with rapamycin showed a significant decreasing in rapamycin mediated inhibition of p-S6K and p-S6 comparing with the control group. In addition, Colony Formation assay and MTT assay indicated that EPIC1 overexpression led to rapamycin resistance in breast and ovarian cancer cell lines. Our results demonstrated the lncRNA EPIC1 expression activated the AKT-mTORC1 signaling pathway through Myc and led to rapamycin resistance in breast and ovarian cancer.     

Cytotoxicity of a natural anthraquinone (Aloin) against human breast cancer cell lines with and without ErbB-2: topoisomerase IIalpha coamplification

In the present study the cytotoxic activity of aloin, a natural anthracycline from Aloe plant, is reported against two human breast cancer cell lines; without (MCF-7) and with (SKBR-3) erbB-2-topoIIalpha coamplification. MCF-7cell line was shown to be more sensitive to aloin than SKBR-3 demonstrated by MTT and clonogenic assays, from which IC50 and 50% ICF values are reported to be 60 microg/ml, respectively, in the former cell line and as high as 150 and 80 microg/ml, respectively, in the latter, which are still far below the maximum tolerated dose of the compound. The effect of aloin is suggested to be brought about by more than one mechanism depending on the dose level and tumor phenotype. This was demonstrated by flow cytometric analysis, fluorescence microscopy and western blot analysis, which revealed that aloin at higher concentrations caused a reduction in the proportion of cells undergoing mitosis by induction of apoptosis, inhibition of topo II alpha protein expression and downregulation of cyclin B1 protein expression in MCF-7 cell line, whereas erbB-2 protein expression was not affected. Topo IIalpha protein expression was mildly downregulated in SKBR-3 cell line at higher concentrations only.     

LncRNA-ATB promotes trastuzumab resistance and invasion-metastasis cascade in breast cancer

Trastuzumab resistance is leading cause of mortality in HER2-positive breast cancers, and the role of TGF-β-induced epithelial-mesenchymal transition (EMT) in trastuzumab resistance is well established, but the involvement of lncRNAs in trastuzumab resistance is still unknown. Here, we generated trastuzumab-resistant breast cancer cells with increased invasiveness compared with parental cells, and observed robust epithelial–mesenchymal transition (EMT) and consistently elevated TGF-β signaling in these cells. We identified long noncoding RNA activated by TGF-β (lnc-ATB) was the most remarkably upregulated lncRNA in TR SKBR-3 cells and the tissues of TR breast cancer patients. We found that lnc-ATB could promote trastuzumab resistance and invasion-metastasis cascade in breast cancer by competitively biding miR-200c, up-regulating ZEB1 and ZNF-217, and then inducing EMT. In addition, we also found that the high level of lnc-ATB was correlated with trastuzumab resistance of breast cancer patients. Thus, these findings suggest that lncRNA-ATB, a mediator of TGF-β signaling, could predispose breast cancer patients to EMT and trastuzumab resistance.     

诱导建立乳腺癌MCF-7放射耐受细胞亚株的实验研究

  目的  探索诱导并建立人乳腺癌MCF-7放射耐受细胞亚株的体外实验方法。  方法  体外培养MCF-7细胞株, 应用梯度递增的X线对MCF-7进行诱导照射, 照射剂量达到59Gy时, 得到放射耐受细胞亚株(MCF-7R), 扫描电镜和透射电镜观察亲本株MCF-7与放射耐受细胞亚株MCF-7R细胞超微结构, 流式细胞仪检测其细胞周期分布, 集落形成实验检测其放射敏感性, 并计算存活分数, 多靶单击模型拟合细胞存活曲线。  结果  与MCF-7相比, MCF-7R外形及细胞器均出现明显改变; G₂/M期比例明显降低; (13.32%vs.9.43%)放射敏感性参数SF₂即照射2 Gy时的细胞存活分数升高34%(P < 0.001), 准域剂量Dq值由2.261 Gy升高至3.695 Gy(P < 0.05), 平均致死剂量D_o值由1.215 Gy升高至1.834 Gy(P < 0.05)。  结论  照射剂量梯度递增法是可行的建立人乳腺癌放射耐受细胞亚株的方法, 得到的放射耐受亚株细胞形态及细胞生物学特性与亲本株细胞相比较有明显差异。      

lncRNA 178030.2通过TRPS1对三阴性乳腺癌细胞紫杉醇耐药的影响与机制研究

目的:探讨lncRNA 178030.2通过TRPS1对三阴性乳腺癌细胞紫杉醇耐药的影响与机制。方法:采用逐步增加剂量间歇作用的方法诱导三阴性乳腺癌紫杉醇耐药细胞系并命名为MDA-MB-231/R。采用定量PCR和Western blot检测耐药细胞和亲本细胞中lncRNA 178030.2和TRPS1的表达;应用Lipofectamine 2000将lncRNA 178030.2高表达质粒pcmv-178030.2和对照质粒pcmv转染至MDA-MB-231细胞,分别为高表达组和对照组,定量PCR检测lncRNA 178030.2水平的变化,分别用定量PCR和Western blot检测TRPS1 mRNA及蛋白水平的变化;RIP实验检测lncRNA 178030.2是否与TRPS1相结合;MTT法检测MDA-MB-231细胞对紫杉醇的敏感性及细胞增殖。应用Lipofectamine 2000将lncRNA 178030.2小干扰RNA si178030.2和对照siNC转染至MDA-MB-231/R细胞,分别为干扰组和对照组,定量PCR检测lncRNA 178030.2水平的变化,分别用定量PCR和Western blot检测TRPS1 mRNA及蛋白水平的变化;MTT法检测MDA-MB-231/R细胞对紫杉醇的敏感性及细胞增殖。应用Lipofectamine 2000将TRPS1过表达质粒pcmv-TRPS1和对照质粒pcmv转染至MDA-MB-231细胞,分别为高表达组和对照组,分别用定量PCR和Western blot检测两组细胞中TRPS1 mRNA及蛋白水平的表达情况,然后再分别应用Lipofectamine 2000将lncRNA 178030.2高表达质粒pcmv-178030.2转染入两组细胞,MTT法检测MDA-MB-231细胞对紫杉醇的敏感性及细胞增殖。结果:成功构建在3 μg/ml紫杉醇中稳定生长的三阴性乳腺癌耐药细胞系MDA-MB-231/R。定量PCR结果显示:lncRNA 178030.2在紫杉醇耐药细胞系MDA-MB-231/R中的表达明显高于在其亲本细胞MDA-MB-231中的表达;定量PCR和Western blot显示:TRPS1 mRNA和蛋白在紫杉醇耐药细胞系MDA-MB-231/R中的表达明显低于在其亲本细胞系MDA-MB-231中的表达;与对照组相比,高表达lncRNA 178030.2组的MDA-MB-231细胞中TRPS1表达下降,细胞对紫杉醇的敏感性降低,细胞增殖增强,且lncRNA 178030.2确实可以与TRPS1相结合;与对照组相比,低表达lncRNA 178030.2组的MDA-MB-231/R细胞中TRPS1表达升高,细胞对紫杉醇的敏感性升高,细胞增殖减弱;在MDA-MB-231细胞中过表达TRPS1后再过表达lncRNA 178030.2,其促进紫杉醇耐药、促进细胞增殖的作用也明显减弱。结论:lncRNA 178030.2促进三阴性乳腺癌细胞MDA-MB-231的紫杉醇耐药,促进细胞增殖,其发生机制可能与下调TRPS1的表达有关。     

Pyrotinib Sensitizes 5-Fluorouracil-Resistant HER2⁺ Breast Cancer Cells to 5-Fluorouracil

5-Fluorouracil (5-FU) is a widely used chemotherapeutic agent for breast cancer. However, acquired chemoresistance leads to a loss of its efficacy; methods to reverse are urgently needed. Some studies have shown that pyrotinib, an ErbB receptor tyrosine kinase inhibitor, is effective against HER2+ breast cancer. However, whether pyrotinib sensitizes 5-FU-resistant breast cancer cells to 5-FU is unknown. We hypothesized that the combination of pyrotinib and 5-FU would show synergistic antitumor activity, and pyrotinib could reverse 5-FU resistance in HER2+ breast cancer cells in vitro and in vivo. Our data showed that pyrotinib inhibited the growth of 5-FU-resistant SKBR-3/FU and MDA-MB-453/FU cell lines and the parental cell lines. 5-FU remarkably suppressed the growth of SKBR-3 and MAD-MB-453 cells. However, SKBR-3/FU and MADMB-453/FU cells showed resistance to 5-FU. A combination of pyrotinib and 5-FU resulted in the synergistic inhibition of the growth of the 5-FU-resistant SKBR-3/FU and MDA-MB-453/FU cell lines and the parental cell lines. Pyrotinib decreased significantly the IC50 values of 5-FU and the thymidylate synthase (TS) mRNA expression levels in the 5-FU-resistant SKBR-3/FU and MDA-MB-453/FU cell lines and the parental cell lines and increased significantly the intracellular concentration of 5-FU in SKBR-3/FU and MDA-MB-453/FU cells. In addition, pyrotinib reduced the ABCG2 mRNA and protein expression levels in SKBR-3/FU and MDA-MB- 453/FU cells and downregulated the protein expression levels of pAKT, pHER2, and pHER4 in all four cell lines. After TS or ABCG2 in 5-FU-resistant breast cancer cells was knocked down, the sensitivity of SKBR-3/ FU and MDA-MB-453/FU cells to 5-FU was restored. Moreover, in vivo experiments demonstrated that pyrotinib in combination with 5-FU more effectively inhibited SKBR-3/FU tumor growth than either pyrotinib or 5-FU alone. In conclusion, our findings suggest that pyrotinib could restore sensitivity of 5-FU-resistant HER2+ breast cancer cells to 5-FU through downregulating the expression levels of TS and ABCG2.     

Comparative analysis of xanafide cytotoxicity in breast cancer cell lines

Xanafide, a DNA-intercalating agent and topoisomerase II inhibitor, has previously demonstrated comparable cytotoxicity to the parent drug amonafide (NSC 308847). The current study was conducted to investigate further the anti-proliferative effects of xanafide in human breast cancer cell lines, in vitro and in vivo. The in vitro activity of xanafide against MCF-7, MDA-MB-231, SKBR-3 and T47D cell lines was compared to that of paclitaxel, docetaxel, gemcitabine, vinorelbine and doxorubicin. In MCF-7, xanafide demonstrated comparable total growth inhibition (TGI) concentrations to the taxanes and lower TGI values than gemcitabine, vinorelbine and doxorubicin. MCF-7 (oestrogen receptor (ER)+/p53 wild-type) was the most sensitive cell line to xanafide. MDA-MB-231 and SKBR-3 exhibited similar sensitivity to xanafide. T47 D (ER+/p53 mutated), showed no response to this agent. The in vivo activity of xanafide was further compared to that of docetaxel in MCF-7 and MDA-MB-231 cell lines using the hollow fibre assay. Xanafide was slightly more potent than docetaxel, at its highest dose in MCF-7 cell line, whereas docetaxel was more effective than xanafide in MDA-MB-231 cell line. Our results show that there is no relationship between sensitivity of these cell lines to xanafide and cellular levels of both isoforms of topoisomerase II and suggest that ER and p53 status and their crosstalk may predict the responsiveness or resistance of breast cancer patients to xanafide.     

Enhanced chemosensitization in multidrug-resistant human breast cancer cells by inhibition of IL-6 and IL-8 production

Drug resistance remains a major hurdle to successful cancer treatment. Many mechanisms such as overexpression of multidrug-resistance related proteins, increased drug metabolism, decreased apoptosis, and impairment of signal transduction pathway can contribute multidrug resistance (MDR). Recent studies strongly suggest a close link between cytokines and drug resistance. To identify new targets involved in drug resistance, we established a multidrug-resistant human breast cancer cell line MCF-7/R and examined the cytokine profile using cytokine antibody array technology. Among 120 cytokines/chemokines screened, IL-6, IL-8, and 13 other proteins were found to be markedly increased in drug-resistant MCF-7/R cell line as compared to sensitive MCF-7/S cell line, while 7 proteins were specifically reduced in drug-resistant MCF-7/R cells. Neutralizing antibodies against IL-6 and IL-8 partially reversed the drug resistance of MCF-7/R to paclitaxel and doxorubicin, while a neutralizing antibody against MCP-1 had no significant effect. Inhibition of endogenous IL-6 or IL-8 by siRNA technology significantly enhanced drug sensitivity of MCF-7/R cells. Furthermore, overexpression of IL-6 or IL-8 expression by transfection increased the ADM resistance in MCF-7/S cells. Our data suggest that increased expression levels of IL-6 and IL-8 may contribute to MDR in human breast cancer cells.