Med19基因敲减增加乳腺癌细胞化疗敏感性及其可能的机制

目的:研究中介体(mediator,Med) 19对乳腺癌化疗敏感性的影响并分析其分子机制.方法:选用多柔比星(adriamycin,ADM)耐药的人乳腺癌细胞MCF-7/ADM和亲本细胞MCF-7(NC组),采用慢病毒载体介导RNA干扰方法构建Med9稳定低表达的MCF-7/ADM与MCF-7细胞株(KD组),并用Real-time PCR和Western blotting方法验证干扰效果.CCK-8法检测慢病毒介导的Med19敲减前后两种细胞对ADM、顺铂(cisplatin,DDP)和紫杉醇(taxinol,TAX)药物敏感性的变化.Real-time PCR和Western blotting检测Med19敲减对多药耐药基因1(multidrug resistance 1,MDR1)和细胞凋亡基因Bcl2、Bax及Caspase-3、active Caspase-3的表达的影响.流式细胞术检测敲减Med19及ADM处理对细胞凋亡的影响.结果:与MCF-7相比,MCF-7/ADM细胞对ADM、DDP和TAX均具有耐药性.成功构建Med19稳定低表达的MCF-7/ADM与MCF-7细胞株,并且其对ADM、DDP、TAX的敏感性增加,药物作用IC50显著降低(均P＜0.05).MCF-7/ADM细胞Med19 mRNA和蛋白表达显著高于MCF-7细胞,Med19的敲减可降低MCF-7/ADM细胞中MDR1 mRNA与蛋白表达水平(均P＜0.05)并可增加MCF-7/ADM及MCF-7细胞中凋亡相关active Caspase-3、Bax的蛋白表达,降低Bc12的蛋白表达(均P＜0.05).此外,与NC及NC+ ADM组相比,KD组及KD+ ADM组凋亡水平显著增加(均P＜0.05).结论:Med19高表达参与乳腺癌化疗耐药,其机制可能与Med19调节MDR1的表达并影响细胞凋亡有关.     

慢病毒载体过表达SATB1蛋白对乳腺癌细胞侵袭性的影响

目的构建SATB1基因过表达的慢病毒载体,检测其在MCF-7乳腺癌细胞中的表达及对癌细胞侵袭性的影响。方法应用DNA重组技术,将SATB1基因插入到含有绿色荧光蛋白基因的慢病毒表达载体质粒GV287中,获得重组载体,经测序鉴定后转染293T细胞产生慢病毒载体GV287-SATB1,用GV287-SATB1转染人乳腺癌MCF-7,Western blot分析转染前后SATB1表达情况,穿膜实验检测MCF-7细胞的侵袭性。结果成功构建人MCF-7细胞SATB1基因过表达慢病毒载体,MCF-7细胞转染慢病毒载体后SATB1蛋白表达水平显著上调,细胞的侵袭性显著增强。结论 SATB1基因过表达的慢病毒载体,可在MCF-7乳腺癌细胞中过表达SATB1蛋白,并显著增强MCF-7细胞的侵袭性。     

沉默HuR的表达增加人乳腺癌耐药MCF-7/Adr细胞对多柔比星的敏感性

目的：研究RNA干扰人抗原R（human antigen R,HuR）基因的表达对人乳腺癌耐药细胞株MCF-7/Adr对多柔比星（doxorubicin）敏感性的影响。 方法： 构建靶向 HuR基因 的shRNA表达质粒（pGenesil-siHuR）,稳定转染至MCF-7/Adr细胞,real-time PCR检测细胞中 MDR1 mRNA的表达,Western blotting检测MCF-7/Adr细胞中由 MDR1 基因编码的P糖蛋白（P-glycoprotein,P-gp）的表达,MTT法检测pGenesil-siHuR 转染后MCF-7/Adr细胞在多柔比星作用后的存活率和IC50,流式细胞术检测MCF-7/Adr细胞的凋亡率。 结果： 与未转染的MCF-7/Adr细胞比较,pGenesil-siHuR质粒转染MCF-7/Adr细胞中 MDR1 mRNA的表达水平明显减低\[（0184±0.029） vs （1.203±0.026）,P<0.01\],P-gp表达水平明显降低。pGenesil-siHuR质粒转染MCF-7/Adr细胞后,MCF-7/Adr细胞对多柔比星的IC50从未转染的(148.2±2.3）nmol/L降至(42.9±0.4）nmol/L;经多柔比星处理后,pGenesil-siHuR质粒转染组MCF-7/Adr细胞的凋亡率明显上升\[（34.6±1.1）% vs （1.1±0.2）%,P<001\]。 结论： RNA干扰HuR的表达能抑制 MDR1基因 的表达,增加耐药乳腺癌MCF-7/Adr细胞对多柔比星的敏感性。     

慢病毒介导的siRNA干扰乳腺癌MCF-7细胞VEGF-C表达的实验

目的研究慢病毒载体介导的siRNA对乳腺癌MCF-7细胞系VEGF-C表达的敲减作用。方法构建慢病毒VEGF-C/siRNA载体,转染乳腺癌MCF-7细胞,采用Real-time PCR检测MCF-7细胞在转染前后VEGF-C的mRNA表达,计算其转染效率和VEGF-C敲减率。结果慢病毒VEGF-C/siRNA转染效率超过80%,其VEGF-C的mRNA表达敲减率达50%。结论慢病毒VEGF-C/siRNA转染率高,能有效敲减VEGF-C的mRNA表达。     

siRNA逆转乳腺癌细胞系MCF-7/ADR耐药

背景与目的：肿瘤的多药耐药性常导致乳腺癌化疗失败,多约耐药基因1(multidrug resistance 1、mdr1)编码的P-糖蛋白(P-glycoprotein．P-gP)过度表达是重要的耐药机制。本研充拟探讨siRNA抑制耐药乳腺癌细胞系MCF-7／ADR mdr1基因表达的可行性。方法：选择耐阿毒素人乳腺癌细胞系MCF-7／ADR技其敏感细胞系MCF-7作为研究对象,将预先设计的siRNA包装入质粒载体,然后转化质粒刮大肠杆菌中,经过克隆、扩增、纯化后转染到MCF-7／ADR细胞中,潮霉素筛选,流式细胞仪检测P-gP的表达率,实时定量PCR俭测,mdr1基因的表达率,并对转染后的细胞作阿霉素耐药实验。结果：流式细胞仪榆测结果显示,MCF-7／ADR细胞经特异性siRNA作用后,P-gP的表达率由99．8％下降到12．3％：实时相对定量PCR检测结果显示．MCF-7／ADR细胞经特异性siRNA作用后其Ct值由25．22增加到30．64．阿霉素耐药实验显示,转染siRNA的MCF-7／ADR细胞IC50为0．51μmol／L,而术转染组的IC50为17．88μmol／L。结论：siRNA能引发人乳腺癌多耐药细胞系MCF-7／ADR内mdr1基因沉默,从而为siRNA作为一种可能的治疗手段提供了理论依据。     

microRNA-21对乳腺癌MCF-7/ADR细胞株多柔比星耐药性影响的研究

目的:探讨microRNA-21(miR-21)基因表达改变对乳腺癌细胞多柔比星化疗耐药的影响.方法:人工合成miR-21模拟序列或干扰序列,以脂质体为载体,转染乳腺癌细胞MCF-7及其多柔比星耐药细胞株MCF-7/ADR;应用荧光定量RT-PCR检测miR-21的表达;应用MTT法检测细胞转染前后对多柔比星的耐药性;应用流式细胞仪检测细胞凋亡;应用蛋白质印迹法检测PTEN、BAX及Bcl-2蛋白的表达.结果:MTT检测结果示,MCF-7及其耐药株MCF-7/ADR细胞多柔比星半数抑制浓度(IC50)分别为(0.28±0.03)和(17.5±0.12) μmol/L.miR-21表达上调,MCF-7细胞对多柔比星的IC50明显增高为(3.65±0.12) μmol/L;miR-21表达下调,MCF-7/ADR细胞对多柔比星的IC50明显降低为(7.53±0.11) μmol/L.流式细胞分析显示,miR-21下调后MCF-7/ADR细胞凋亡率明显增加.同时,细胞内PTEN、BAX蛋白表达水平增加,Bcl-2表达降低.结论:miR-21在乳腺癌化疗耐药中具有重要作用,抑制miR-21的表达可以逆转乳腺癌多柔比星耐药细胞株对多柔比星的耐药性.     

Snail促进乳腺癌MCF-7细胞移植瘤对多柔比星的耐药及其机制

目的:探讨Snail在乳腺癌MCF-7细胞移植瘤对多柔比星耐药中的作用及其可能的机制。方法:构建Snail基因真核表达载体pcDNA3.1-Snail,转染至MCF-7细胞,筛选稳定表达Snail的MCF-7/Snail细胞,以转染空质粒pcDNA3.1的MCF-7细胞(MCF-7/pcDNA)为对照。构建小鼠MCF-7/Snail及MCF-7/pcDNA细胞移植瘤模型,注射多柔比星,观测移植瘤生长,计算抑瘤率。免疫组织化学方法检测移植瘤组织中Snail、多药耐药基因-1(multidrug resistance-1,MDR-1)和基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)的表达。结果:成功构建pcDNA3.1-Snail表达载体,转染MCF-7细胞后获得MCF-7/Snail和MCF-7/pcDNA细胞,并制备小鼠移植瘤。多柔比星治疗后,MCF-7/Snail细胞移植瘤的瘤重明显高于MCF-7/pcDNA细胞移植瘤[(1.413±0.674)g vs(1.257±0.576)g,P<0.05],多柔比星对MCF-7/Snail移植瘤抑瘤率明显低于MCF-7/pcDNA移植瘤(18.42%vs30.18%,P<0.05),MCF-7/Snail细胞移植瘤的组织中Snail、MDR-1、MMP-9的表达均显著高于MCF-7/pcDNA移植瘤(408.08±20.39vs67.67±16.56,363.50±26.56vs55.08±12.23,396.25±16.03vs56.92±7.35;均P<0.01),且Snail与MDR-1和MMP-9的表达均呈正相关(r1=0.89,P<0.01;r2=0.81,P<0.01)。结论:Snail促进乳腺癌MCF-7细胞移植瘤对多柔比星的耐药,其机制与增强MDR-1和MM9-9表达有关。     

微小RNA-451在乳腺癌细胞中的表达及其与耐药的关系

目的:研究人类微小RNA-451(Homo sapiens micro RNA-451,hsa-miR-451)在乳腺癌细胞中的表达及其与多柔比星(adriamycin,ADM)耐药的关系。方法:用实时荧光定量PCR一步法检测乳腺癌亲本细胞MCF-7和耐ADM细胞MCF-7/ADM中hsa-miR-451的表达;利用脂质体分别将成熟miR-451的模拟物(mimics)及阴性对照转染MCF-7/ADM细胞,然后采用实时荧光定量PCR法检测细胞中多药耐药基因1(multi-drug resistance gene 1,MDR1)mRNA的表达,蛋白质印迹法检测细胞中P-糖蛋白(P-glycoprotein,P-gp)的表达,MTT法检测不同浓度ADM作用下的细胞增殖情况。结果:miR-451在MCF-7/ADM耐药细胞中低表达,与亲本细胞MCF-7相比明显降低(P<0.05)。MCF-7/ADM细胞转染miR-451mimics后,MDR1 mRNA和P-gp蛋白的相对表达量比阴性对照转染组均明显下降(P<0.05);而miR-451 mimics转染组细胞对ADM的敏感性增加,其半数抑制浓度(half inhibition concentration,IC50)值与阴性对照转染组细胞相比差异有统计学意义(P<0.05)。结论:在乳腺癌耐药细胞MCF-7/ADM中miR-451异常低表达,可能通过作用于MDR1/P-gp参与乳腺癌细胞耐药的发生和发展。     

川芎嗪逆转肿瘤多药耐药性及其机制的研究

目的:探讨川芎嗪(tetrameth-ylpyrazine,TMP)逆转人乳腺癌MCF-7/ADM细胞对多柔比星(adriamycin,ADM)的耐药及其P-糖蛋白(P-glycopro-tein,P-gp)表达的影响。方法:MTT法测定细胞的药敏性,荧光分光光度法检测细胞内多柔比星浓度的变化,流式细胞术检测耐药细胞凋亡率的变化,流式细胞术观测细胞P-gp的表达。结果:非细胞毒性剂量(320mg/L)川芎嗪能显著降低MCF-7/ADM的IC50(P=0·0031),逆转倍数为2·13倍;且能明显增加耐药细胞ADM的浓度(P=0·0042)和凋亡率(P=0·0026);320mg/L川芎嗪使耐药细胞的P-gp表达率由(90·6±0·41)%降低至(69·1±1·65)%。结论:川芎嗪具有部分逆转人乳腺癌MCF-7/ADM细胞对多柔比星的耐药性,其逆转机制可能与抑制该细胞P-gp的表达有关。     

干扰iASPP基因对转染MCF-7细胞凋亡变化的观察

目的:观察iASPP基因干扰RNA转染乳腺癌细胞MCF-7后的干扰效果及细胞凋亡变化。方法:设计特异性siRNA序列,将序列克隆至PGCsilencerTM H1/Neo/GFP质粒中,用脂质体将重组子转染至MCF-7细胞中,用RT-PCR方法检测i ASPP的表达,蛋白质印迹法检测蛋白表达的变化,流式细胞仪检测细胞凋亡的情况。结果:iASPP干扰质粒转染MCF-7细胞后,iASPP的mRNA表达和蛋白表达减少40%～50%;p53蛋白相对表达量由转染阴性质粒的0.37增加到转染干扰质粒的0.64;细胞凋亡率由原来的17.8%和16.2%分别增加到53.5%和51.3%。结论:抑制内源性i ASPP能有效地恢复乳腺癌细胞MCF-7中p53的抑癌功能,为乳腺癌的治疗提供新的思路。     

抑制CC类趋化因子配体5基因表达对乳腺癌细胞增殖能力的影响

目的 探讨抑制CC类趋化因子配体5(CCL5)基因表达对人乳腺癌细胞增殖能力的影响.方法 用特异性CCL5 RNA干扰(RNAi)序列慢病毒载体感染人乳腺癌细胞MCF-7和MDA-MB-231,分别为KD1组和KD2组;另在MCF-7和MDA-MB-231细胞中分设阴性病毒载体感染的阴性对照组(NC1组和NC2组)和未感染组(CON1组和CON2组).采用实时定量逆转录聚合酶链反应(RT-PCR)检测转染病毒后乳腺癌细胞中CCL5的表达,四甲基偶氮唑蓝(MTT)比色法和流式细胞术(FACS)分析细胞的增殖情况,平板克隆形成实验观察细胞的克隆形成能力.结果 CCL5 RNAi慢病毒可显著降低MCF-7和MDA-MB-231细胞中CCL5基因的表达.MTF法检测结果显示,在不同的培养时间,MCF-7和MDA-MB-231细胞的KD组、NC组与CON组细胞培养上清A值差异均无统计学意义(均P＞0.05).FACS分析结果显示,KD1组、NC1组和CON1组的增殖指数(PI)值分别为0.48±0.03、0.43±0.01和0.45±0.02;KD2组、NC2组和CON2组的PI值分别为0.48±0.02、0.44±0.05和0.47±0.02(两两比较,均P＞0.05).荧光显微镜下观察显示,KD组的克隆体积及每克隆的细胞数明显小于NC组和CON组.KD1组和KD2组克降数目(0.34±0.08和0.33±0.10)明显少于NC1组(0.81±0.12)、NC2组(0.97±0.09)、CON1组(0.92±0.12)和CON2组(1.04±0.07),差异有统计学意义(P＜0.05).结论 CCL5基因的表达下调对乳腺癌MCF-7和MDA-MB-231细胞群体倍增时间无明显影响,但可显著降低细胞的克隆形成能力,从而使肿瘤细胞的恶性增殖受到抑制.

Abstract：

Objective To investigate the effect of suppression of CCI5 ligand gene on the proliferation of human breast cancer cells. Methods A lentiviral vector carrying a short interfering RNA (siRNA) targeting CCL5 was transfected into human breast cancer cell line MCF-7 and MDA-MB-231 cells.The expression of CCL5 mRNA in the cells was detected by real-time PCR. The proliferation of MCF-7 and MDA-MB-231 cells was assessed by MTT assay and FACS assay, and the colony formation ability of both cell lines were measured, respectively. Results Real time PCR showed a good knockdown effect of CCL5 in both cell-lines. Colony-forming assay showed that the ability of colony formation of MCF-7/CCL5-siRNA and MDA-MB-231/CCL5-siRNA was decreased markedly. The colony number of MCF-7/CCL5-siRNA group was (0. 34 ± 0. 08), significantly lower than 0. 81 ± 0. 12 in the MCF-7/CCL5-N group and 0.92 ± 0.12 in the MCF-7 group (P ＜ 0. 05). The colony number of MDA-MB-231/CCL5-siRNA group was 0. 33 ± 0. 10,significantly lower than 0.97 ±0.09 in the MDA-MB-231/CCL5-N group and 1.04 ±0.07 in the MDA-MB-231 group (P ＜0.05). However, MTT assay revealed that the proliferation of MCF-7/CCL5-siRNA cells was not significantly different from that of MCF-7/CCL5-N or MCF-7 cells, respectively (P ＞0.05), and the same result was found in MDA-MB-231 cells. FACS assay showed that the proliferation index (PI) of groups MCF-7/CCL5-siRNA, MCF-7/CCL5-N and MCF-7 were 0.48 ± 0. 03, 0. 43 ± 0. 01 and 0.45 ±0. 02. The PI of groups MDA-MB-231/CCL5-siRNA, MDA-MB-231/CCL5-N and MDA-MB-231 cells were 0. 48 ± 0.02, 0.44 ± 0.05 and 0. 47 ± 0. 02. There was no statistical difference among them ( P ＞ 0.05 ).Conclusion The down-regulation of CCL5 gene in human breast cancer cells may significantly suppress their colony formation ability, rather than affecting their population doubling time to some extent.     

人BRMS1干扰质粒的构建及其有效性的鉴定

目的:构建针对人BRMS1基因的RNA干扰表达载体并检测其有效性,为后续研究提供基础。方法:设计合成针对人BRMS1的特异性si RNA前体寡核苷酸,依次通过退火,连接,构建含前体si RNA的重组质粒pcDNA6.2-GW/EmGFP-BRMS1-si-RNA。经测序鉴定后,通过脂质体介导将其与pDsRed2-N1-BRMS1质粒(包含BRMS1红色荧光融合蛋白的载体)共转染人胚肾293细胞。分别用倒置荧光显微镜和流式细胞术观察转染细胞中红色荧光蛋白的强度,检测干扰效果。结果:确定了两对针对人BRMS1的si RNA片段。经测序鉴定,两种含前体si RNA的质粒均构建成功。共转染后,倒置荧光显微镜下观察可见,两种si R-NA均能明显降低细胞中BRMS1的表达;用流式细胞仪检测证实,si RNA1及si RNA2分别使BRMS1表达下降67.33%和76.67%,与阴性对照组比较差异有统计学意义,P<0.05。结论:成功构建了针对人BRMS1基因的si RNA表达质粒,为进一步利用RNA干扰技术研究BRMS1功能与作用机制奠定了基础。     

细胞分裂周期蛋白42高表达在雌激素诱导的乳腺癌细胞耐药性增强过程中的作用

目的 观察细胞分裂周期蛋白42(Cdc42)在雌激素作用下的变化,探讨细胞内物质运输的改变在雌激素引发的乳腺癌细胞耐药中的意义.方法 分别以100 ng/ml 17β雌二醇(E2)处理MCF-7细胞,以Cdc42的小干扰RNA(siRNA) Stealth Select RNAiTM siRNA转染MCF-7细胞.采用四甲基偶氮唑蓝法检测细胞的药物敏感性,流式细胞术检测细胞内阿霉素(ADM)的蓄积量,实时荧光定量聚合酶链反应检测细胞的Cdc42 mRNA表达,Western blot法检测细胞活化的Cdc42蛋白及总Cdc42蛋白表达量.结果 E2处理后,ADM对MCF-7细胞的半数抑制浓度(IC50)由(0.098±0.011) μg/ml增高到(0.134±0.130)μg/ml(P＜0.05),细胞内ADM的相对含量则由7.253±0.310下降为3.233±0.313(P＜0.05),而Cdc42 mRNA、活化Cdc42蛋白及总Cdc42蛋白的表达量均显著增加(P＜0.05).Cdc42 siRNA转染后,ADM对MCF-7细胞的IC50下降到(0.057±0.017)μg/ml(P＜0.05),细胞内ADM的相对含量增高为11.217±0.521(P＜0.05),而Cdc42 mRNA、活化Cdc42蛋白及总Cdc42蛋白则均有显著下降(P＜0.05).结论 雌激素可以诱导乳腺癌细胞耐药性增强,其机制可能是通过上调Cdc42基因的转录、表达和活化,加速胞内物质运输速度,使得化疗药物无法在细胞内聚集.

Abstract：

Objective To investigate the changes of Cdc42 expression under estrogen stimulation, and to explore the signaling pathway of intracellular material transportation caused by estrogen. Methods MTT was used to test the drug sensitivity of cells. Real-time PCR was used to evaluate the expression of Cdc42 mRNA. The amount of ADM accumulated in MCF-7 cells was detected by flow cytometry. The protein levels of active-Cdc42 and Total-Cdc42 were measured by Western blot. Results IC50 of ADM in MCF-7 cells was increased from (0.098±0.011)μg/ml to (0.134±0.130)μg/ml (P<0.05) after estrogen stimulation. The amount of ADM accumulated in MCF-7 cells was reduced from 7.253±0.310 to 3.233±0.313 (P<0.05). All of Cdc42 mRNA, active-Cdc42 protein and total-Cdc42 protein were increased (P<0.05). After the treatment with siRNA, the IC50 of ADM in siRNA group was decreased to (0.057±0.017)μg/ml (P<0.05) compared with that in the control group. The amount of accumulated ADM was significantly increased in the siRNA group, and all the expression levels of Cdc42 mRNA, active-Cdc42 protein and total-Cdc42 protein were decreased in the siRNA group (P<0.05). Conclusions Estrogen enhances the drug resistance in breast cancer cells. The mechanism of this effect may be via the enhancing Cdc42 expression and decreasing the accumulation of chemotherapeutic drugs in the cancer cells.     

靶向VEGF的不对称siRNA抑制乳腺癌MCF-7细胞的增殖

目的:探讨靶向血管内皮生长因子(vascular endothelial growth factor,VEGF)基因的siRNA对乳腺癌MCF-7细胞的抑制效果。方法:设计靶向VEGF的4种小干扰RNA(small interfering RNA,siRNA),包括对称siRNA(siRNA21/21,siRNA23/23)与不对称siRNA(asymmetric siRNA,aiRNA;aiRNA21/23,aiRNA19/21)。siRNA转染入MCF-7细胞后,MTT及流式细胞术检测MCF-7细胞增殖及凋亡情况,RT-PCR、ELISA法检测MCF-7细胞中VEGF基因和蛋白的表达。结果:与对照组相比,aiRNA21/23、siRNA23/23、aiRNA19/21、siRNA21/21这4种siRNA都能有效抑制VEGF mRNA的表达[(71.4±5.01)%、(40.0±3.11)%、(37.2±2.79)%、(11.1±0.99)%vs(2.4±0.11)%,P<0.01],且抑制MCF-7细胞的增殖[(44.7±5.38)%、(38.5±5.67)%、(33.6±2.18)%、(33.1±3.18)%vs(2.2±0.28)%,P<0.01],其中以不对称aiRNA21/23的抑制效果最明显。与对照组比较,aiRNA21/23显著促进MCF-7细胞的凋亡[(49.9±4.02)%vs(4.7±0.91)%,P<0.01]。结论:靶向VEGF基因的siRNA可抑制MCF-7细胞的增殖、促进细胞凋亡,尤以不对称siRNA的效果最明显。     

Adenovirus-delivered CIAPIN1 small interfering RNA inhibits HCC growth in vitro and in vivo

Hepatocellular carcinoma (HCC) is an aggressive cancer with a poor prognosis. The specific cellular gene alterations responsible for hepatocarcinogenesis are not well known. Cytokine-induced antiapoptotic molecule (CIAPIN1), a recently reported antiapoptotic molecule which plays an essential role in mouse definitive hematopoiesis, is considered a downstream effecter of the receptor tyrosine kinase-Ras signaling pathway. However, the exact function of this gene in tumors is not clear. In this study, we reported that CIAPIN1 is highly expressed in HCC as compared with non-tumor hepatic tissue (P < 0.05). We employed adenovirus-mediated RNA interference technique to knock down CIAPIN1 expression in HCC cells and observed its effects on HCC cell growth in vitro and in vivo. Among the four HCC and one normal human liver cell lines we analyzed, CIAPIN1 was highly expressed in HCC cells. Knock down of CIAPIN1 could inhibit HCC cell proliferation by inhibiting the cell cycle S-phase entry. Soft agar colony formation assay indicated that the colony-forming ability of SMMC-7721 cells decreased by approximately 70% after adenovirus AdH1-small interfering RNA (siRNA)/CIAPIN1 infection. In vivo experiments showed that adenovirus AdH1-siRNA/CIAPIN1 inhibited the tumorigenicity of SMMC-7721 cells and significantly suppressed tumor growth when injected directly into tumors. These results suggest that knock down of CIAPIN1 by adenovirus-delivered siRNA may be a potential therapeutic strategy for treatment of HCC in which CIAPIN1 is overexpressed.     

miR-490-3p逆转MCF-7／ADM细胞对阿霉素耐药性的影响

【摘 要】目的 探讨miR-490-3p在阿霉素（ADM）耐药乳腺癌细胞系MCF-7／ADM中的表达情况及其对MCF-7／ADM细胞增殖、凋亡和ADM耐药的影响。方法 以野生型人乳腺癌细胞系MCF-7及其耐药型细胞系MCF-7／ADM为研究对象,用实时荧光定量PCR（qPCR）比较两种细胞中miR-490-3p的表达水平,将miR-490-3p的过表达载体pcDNA1（+）miR-490-3p瞬时转染至MCF-7／ADM细胞（过表达组）后采用qPCR检测转染效果,同时设pcDNA3.1（-）空载体对照组和空白对照组;采用MTT法测定转染pcDNA3.1（+）miR-490-3p对各组MCF-7/ADM细胞增殖能力的影响,测定ADM对MCF-7／ADM细胞的增殖抑制率并计算半数抑制浓度（IC50）及逆转倍数以评价对ADM敏感性的变化;分别于瞬时转染48 h后用Annexin V／FITC流式细胞术检测各组MCF-7/ADM细胞的凋亡率,Western blotting检测耐药蛋白P-糖蛋白（P-gp）,乳腺癌耐药蛋白（BCRP）和多药耐药相关蛋白1（MRP1）的表达,荧光分光光度计检测胞内ADM药物浓度,流式细胞仪检测P-gp活性。结果 MCF-7／ADM细胞中miR-490-3p的表达水平为0.24±0.07,显著低于野生型MCF-7细胞的1.02±0.03（P＜0.05）;过表达组瞬时转染24～96 h的miR-490-3p水平持续升高,均高于空载体对照组和空白对照组（P＜0.05）;过表达组的增殖抑制率随转染时间的延长而增加,转染24、48、72和96 h的增殖抑制率依次为（17.52±1.87）％、（31.67±2.79）％、（45.09±1.88）％和（61.82±2.52）％,高于空载体对照组（P＜0.05）;ADM对过表达组的IC50值为（11.27±2.34）μg／ml,低于空白对照组的和空载体对照组（P＜0.05）,且过表达组相对于空白对照组和空载体对照组的逆转倍数分别为3.35倍和3.39倍;与其余两组相比,过表达组的MCF-7/ADM细胞早、晚期凋亡率和细胞内药物浓度均升高,P-gp、BCRP和MRP1表达及P-gp活性均降低,差异均有统计学意义（P＜0.05）。结论 上调miR-490-3p可逆转MCF-7／ADM细胞对ADM的耐药性,可能通过降低耐药蛋白表达及抑制P-gp活性有关,且升高其水平可抑制细胞增殖并诱导凋亡。     

Shh和Gli-1在人乳腺癌MCF-7耐药细胞株中的表达及意义

目的 研究Shh和Gli-1在人乳腺癌耐药株中的表达情况,探讨Hedgehog信号通路与乳腺癌耐药的关系。方法 高浓度间歇诱导法建立人乳腺癌耐药细胞株MCF-7／PTX,MTT法检测紫杉醇（PTX）对MCF-7与MCF-7／PTX细胞的半数抑制浓度（IC50）。实时定量PCR（QPCR）检测MCF-7、MCF-7／PTX细胞中Shh、Gli-1 mRNA的表达。Western blotting检测MCF-7、MCF-7／PTX细胞中Shh、Gli-1蛋白的表达。结果 PTX 对 MCF-7细胞的IC50为（0.10±0.02）mg／L,对 MCF-7／PTX细胞的 IC50为（5.30±0.01）mg／L;耐药指数为53.0。Shh mRNA在MCF-7、MCF-7／PTX细胞中的表达量分别为0.78±0.12和1.45±0.56（P＜0.01）;Gli-1 mRNA在MCF-7、MCF-7／PTX细胞中的表达量分别为1.86±0.02和3.56±0.26（P＜0.01）。Shh 蛋白在MCF-7、MCF-7／PTX细胞中的表达量分别为0.58±0.06和1.03±0.22（P＜0.01）;Gli-1 蛋白在MCF-7、MCF-7／PTX细胞中的表达量分别为1.17±0.12和2.78±0.09（P＜0.01）。结论 人乳腺癌耐药细胞株MCF-7／PTX高表达Shh、Gli-1,化疗药物可能通过上调Hedgehog信号通路相关蛋白及基因介导乳腺癌耐药,针对该信号通路的靶向治疗将是克服乳腺癌耐药的一个新的选择。     

Enhanced therapeutic effect of Adriamycin on multidrug resistant breast cancer by the ABCG2-siRNA loaded polymeric nanoparticles assisted with ultrasound

The overexpression of the breast cancer resistance protein (ABCG2) confers resistance to Adriamycin (ADR) in breast cancer. The silencing of ABCG2 using small interfering RNA (siRNA) could be a promising approach to overcome multidrug resistance (MDR) in cancer cells. To deliver ABCG2-siRNA effectively into breast cancer cells, we used mPEG-PLGA-PLL (PEAL) nanoparticles (NPs) with ultrasound-targeted microbubble destruction (UTMD). PEAL NPs were prepared with an emulsion-solvent evaporation method. The NPs size was about 131.5 ± 6.5 nm. The siRNA stability in serum was enhanced. The intracellular ADR concentration increased after the introduction of siRNA-loaded NPs. After intravenous injection of PEAL NPs in tumor-bearing mice, the ABCG2-siRNA-loaded NPs with UTMD efficiently silenced the ABCG2 gene and enhanced the ADR susceptibility of MCF-7/ADR (ADR resistant human breast cancer cells). The siRNA-loaded NPs with UTMD + ADR showed better tumor inhibition effect and good safety in vivo. These results indicate that ADR-chemotherapy in combination with ABCG2-siRNA is an attractive strategy to treat breast cancer.