The correlation or serum estradiol concentrstion and uterine estrogen receptor (ER) gene expression (ERn and ERc quantitated by Dextrsn Coat Charcoal assay and ER mRNA by Northern blotting) was studied during the rat ...The correlation or serum estradiol concentrstion and uterine estrogen receptor (ER) gene expression (ERn and ERc quantitated by Dextrsn Coat Charcoal assay and ER mRNA by Northern blotting) was studied during the rat estrous cycle and early Pregnant stage (dl-d10). The ER gone expression was up - regulated by estrogen and the levels of ER mRNA synchronized with the changes of ER protein, suggesting that estrogen influenced the trsnscriPtional step of the ER gene. Post-coitum ER expression increased with the serum estrsdiol progressively, reached a peak on d4-ds (Just before implantation), but drastically dropped to the nadir on d6-d7 (during implantation) and then recovered. It was of interest to discover that ER mRNA level in the nonimplantstion sites (NIS) of uterus was much higher than that in the implantstion sites (IS).展开更多
他莫昔芬(tamoxifen,TAM)作为雌激素受体阳性(estrogen receptor,ER+)乳腺癌的一线化疗药物使大多数患者受益,但原发性和继发性耐药问题严重影响临床治疗效果。深入研究ER+乳腺癌TAM耐药机制,改善治疗效果是当前亟待解决的问题。抑癌因...他莫昔芬(tamoxifen,TAM)作为雌激素受体阳性(estrogen receptor,ER+)乳腺癌的一线化疗药物使大多数患者受益,但原发性和继发性耐药问题严重影响临床治疗效果。深入研究ER+乳腺癌TAM耐药机制,改善治疗效果是当前亟待解决的问题。抑癌因子NDRG2(N-myc downstream regulated gene 2,NDRG2)在肿瘤发生发展中发挥重要作用,但是否参与ER+乳腺癌TAM耐药尚不清楚。本研究旨在探明NDRG2在ER+乳腺癌TAM耐药中发挥的作用和机制。通过RT-PCR与免疫印迹分析对比TAM敏感型和耐药型ER+乳腺癌细胞发现,NDRG 2的mRNA转录水平和蛋白质翻译水平在TAM耐药细胞中表达显著下调,且与耐药能力负相关(P<0.001);CCK-8细胞毒性实验和软琼脂克隆形成实验证实,在耐药细胞中过表达NDRG2可显著降低TAM药物半抑制浓度IC 50和软琼脂克隆形成率(P<0.001),逆转耐药表型。分子机制上,X-box结合蛋白1(X-box binding protein 1,XBP1)mRNA剪切实验与内质网相关降解(endoplasmic-reticulum associated degradation,ERAD)报告蛋白的结果显示,过表达NDRG2可增强耐药细胞中剪切型XBP1s mRNA转录与ERAD报告蛋白CD3ε-YFP表达(P<0.001),引发耐药细胞内质网强应激反应;免疫印迹检测结果显示,过表达NDRG2可显著提高耐药细胞中内质网应激感受器肌醇需要激酶1α(inositol requiring enzyme 1,IRE1α)的磷酸化水平及其下游因子,例如内质网EIP辅助因子(endoplasmic reticulum-localized DnaJ 4,ERdj4)、PKR蛋白激酶的细胞抑制剂(cellular Inhibitor of the PKR protein kinase,P58 IPK)、α甘露糖苷酶样应激蛋白(er degradation enhancingαmannosidase likeprotein,EDEM)和蛋白质二硫键异构酶家族A成员5(protein disulfide isomerase family a member 5,PDIA5)的表达水平(P<0.001)。小鼠异种移植瘤研究进一步证实,在耐药细胞中过表达NDRG2可增强TAM治疗效果,显著抑制耐药移植瘤生长(P<0.001)。以上研究结果表明,通过提高耐药细胞中NDRG2表达,增强TAM治疗引发的内质网强烈应激,可逆转ER+乳腺癌细胞耐药性,改善TAM治疗效果。研究结果为解决ER+乳腺癌TAM耐药问题提供了新的思路和有价值的潜在药物靶点。展开更多
文摘The correlation or serum estradiol concentrstion and uterine estrogen receptor (ER) gene expression (ERn and ERc quantitated by Dextrsn Coat Charcoal assay and ER mRNA by Northern blotting) was studied during the rat estrous cycle and early Pregnant stage (dl-d10). The ER gone expression was up - regulated by estrogen and the levels of ER mRNA synchronized with the changes of ER protein, suggesting that estrogen influenced the trsnscriPtional step of the ER gene. Post-coitum ER expression increased with the serum estrsdiol progressively, reached a peak on d4-ds (Just before implantation), but drastically dropped to the nadir on d6-d7 (during implantation) and then recovered. It was of interest to discover that ER mRNA level in the nonimplantstion sites (NIS) of uterus was much higher than that in the implantstion sites (IS).
文摘他莫昔芬(tamoxifen,TAM)作为雌激素受体阳性(estrogen receptor,ER+)乳腺癌的一线化疗药物使大多数患者受益,但原发性和继发性耐药问题严重影响临床治疗效果。深入研究ER+乳腺癌TAM耐药机制,改善治疗效果是当前亟待解决的问题。抑癌因子NDRG2(N-myc downstream regulated gene 2,NDRG2)在肿瘤发生发展中发挥重要作用,但是否参与ER+乳腺癌TAM耐药尚不清楚。本研究旨在探明NDRG2在ER+乳腺癌TAM耐药中发挥的作用和机制。通过RT-PCR与免疫印迹分析对比TAM敏感型和耐药型ER+乳腺癌细胞发现,NDRG 2的mRNA转录水平和蛋白质翻译水平在TAM耐药细胞中表达显著下调,且与耐药能力负相关(P<0.001);CCK-8细胞毒性实验和软琼脂克隆形成实验证实,在耐药细胞中过表达NDRG2可显著降低TAM药物半抑制浓度IC 50和软琼脂克隆形成率(P<0.001),逆转耐药表型。分子机制上,X-box结合蛋白1(X-box binding protein 1,XBP1)mRNA剪切实验与内质网相关降解(endoplasmic-reticulum associated degradation,ERAD)报告蛋白的结果显示,过表达NDRG2可增强耐药细胞中剪切型XBP1s mRNA转录与ERAD报告蛋白CD3ε-YFP表达(P<0.001),引发耐药细胞内质网强应激反应;免疫印迹检测结果显示,过表达NDRG2可显著提高耐药细胞中内质网应激感受器肌醇需要激酶1α(inositol requiring enzyme 1,IRE1α)的磷酸化水平及其下游因子,例如内质网EIP辅助因子(endoplasmic reticulum-localized DnaJ 4,ERdj4)、PKR蛋白激酶的细胞抑制剂(cellular Inhibitor of the PKR protein kinase,P58 IPK)、α甘露糖苷酶样应激蛋白(er degradation enhancingαmannosidase likeprotein,EDEM)和蛋白质二硫键异构酶家族A成员5(protein disulfide isomerase family a member 5,PDIA5)的表达水平(P<0.001)。小鼠异种移植瘤研究进一步证实,在耐药细胞中过表达NDRG2可增强TAM治疗效果,显著抑制耐药移植瘤生长(P<0.001)。以上研究结果表明,通过提高耐药细胞中NDRG2表达,增强TAM治疗引发的内质网强烈应激,可逆转ER+乳腺癌细胞耐药性,改善TAM治疗效果。研究结果为解决ER+乳腺癌TAM耐药问题提供了新的思路和有价值的潜在药物靶点。
文摘目的:探讨黑素瘤抗原(melanoma antigen gene,MAGE)-A11对雌激素受体(estrogen receptor,ER)介导的乳腺癌MCF-7细胞增殖的影响。方法:利用RT-PCR及Western blotting筛选出ER表达阳性的人乳腺癌MCF-7细胞作为模式细胞,采用基因转染、RT-PCR和Western blotting检测MAGE-A11对17β-雌二醇(17β-E)诱导的ER下游靶基因Efp表达的影响,采用免疫共沉淀法检测MCF-7细胞中MAGE-A11和ER蛋白的相互作用,采用MTT法和克隆形成实验分别检测MAGE-A11及17β-E处理对MCF-7细胞生存率和细胞克隆形成数的影响。结果:ER阳性MCF-7细胞经17β-E处理24 h后,下游靶基因Efp的mRNA(2.97±0.16 vs 1.71±0.09,P<0.05)和蛋白表达水平显著升高(2.65±0.12 vs 0.92±0.06,P<0.05);转染MAGE-A11的MCF-7细胞经17β-E 24 h处理后,其Efp的mRNA(4.01±0.19 vs 2.97±0.16,P<0.05)及蛋白表达(3.52±0.15 vs 2.65±0.12,P<0.05)更显著增加。免疫共沉淀结果显示,外源性MAGE-A11与ER之间存在相互作用。MCF-7细胞经17β-E处理后细胞增殖率显著增加[(152±6.7)%vs(108±4.8%),P<0.05],转染MAGE-A11的MCF-7细胞经17β-E处理后细胞增殖率更显著增加[(181±8.6)%vs(152±6.7)%,P<0.05];17β-E处理后MCF-7细胞克隆形成数显著增多[(77±5)vs(18±2)个,P<0.05],转染MAGE-A11的MCF-7细胞经17β-E处理后细胞的克隆形成数更显著增加[(125±6)vs(77±5)个,P<0.05)。结论:在ER阳性的乳腺癌MCF-7细胞中,MAGE-A11可通过与ER的相互作用增强ER介导的Efp的表达,从而促进细胞增殖,MAGE-A11可能成为ER阳性乳腺癌内分泌治疗耐药的靶基因。
