【作者】 纪晓惠；

【导师】 王士杰； 耿翠芝；

【作者基本信息】 河北医科大学 ， 外科学， 2014， 博士

【摘要】 目的：乳腺癌是全球女性最常见的恶性肿瘤，严重危害着女性的生命和健康。近年来我国乳腺癌的发病率持续上升,且呈低龄化趋势。乳腺癌是典型的激素依赖性肿瘤，长期以来，以他莫西芬（tamoxifen, TAM）为代表的内分泌治疗凭借其低毒、经济，效应作用持久成为雌激素受体（estrogen receptor, ER）阳性乳腺癌患者综合治疗中最重要的治疗手段之一。然而，目前临床上约40%ER阳性乳腺癌患者对抗雌激素治疗产生耐药，具体机制尚不明确。因此，对于乳腺癌相关基因及其在TAM耐药发生机制中的研究成为乳腺癌研究的热点问题。有研究表明，在ER阳性的乳腺癌中，配对盒基因（paired box gene-2，PAX2）和乳腺癌扩增因子（amplified in breast cancer-1, AIB1）能够在TAM治疗过程中调控人类表皮生长因子受体（human epidermal growth factorreceptor-2, HER-2）基因的转录水平，进而调控HER-2的蛋白表达，该机制有可能是ER阳性乳腺癌TAM治疗耐药的重要原因之一。为揭示ER阳性乳腺癌患者TAM耐药的作用机制及相关影响因素，本课题首先应用免疫组织化学链霉菌抗生物素蛋白-过氧化酶（streptavidin peroxdaseconjugated method, S-P）法检测术后ER阳性并接受正规TAM治疗的乳腺癌患者标本中PAX2和AIB1的蛋白表达，结合随访结果，回顾性分析两者的表达强度对ER阳性乳腺癌患者TAM疗效的影响;其次采用逆转录聚合酶链反应（reverse transcription polymerase chain reaction, RT-PCR）技术和免疫组织化学S-P法分别从转录水平和蛋白水平检测PAX2和AIB1在ER阳性乳腺癌患者中乳腺癌组织及乳腺非肿瘤组织的表达情况，分析乳腺癌组织中PAX2和AIB1的表达与其临床生物学行为及ER、孕激素受体PR（progesterone receptor, PR）、HER-2表达的关系，以进一步确定PAX2、AIB1是否为判断ER阳性乳腺癌患者抗雌激素治疗效果的敏感指标。为了进一步研究PAX2和AIB1在ER阳性乳腺癌患者抗雌激素治疗中的作用机制，本课题应用RT-PCR法检测ER阳性和ER阴性乳腺癌细胞系在雌激素刺激下PAX2、AIB1及HER-2的表达，以及TAM敏感和TAM耐药乳腺癌细胞系在TAM作用下PAX2、AIB1及HER-2的表达情况；采用siRNA技术和质粒转染技术分别对TAM敏感乳腺癌细胞系和TAM耐药乳腺癌细胞系进行对应的PAX2、AIB1基因沉默及基因过表达，并分析HER-2及相应蛋白的表达情况以明确ER阳性乳腺癌组织中PAX2，AIB1对HER-2表达的调控作用，为合理解释ER阳性乳腺癌患者TAM耐药提供依据。方法：1采用免疫组化S-P法检测167例ER阳性TAM规范治疗且资料完整的乳腺癌患者中PAX2和AIB1蛋白表达，分析两者的表达强度、及与TAM治疗后复发或转移的关系，以明确其对ER阳性乳腺癌患者TAM疗效的影响。2采用RT-PCR技术和免疫组化S-P法分别从转录水平和蛋白水平检测70例ER阳性乳腺癌组织和乳腺非肿瘤组织中PAX2和AIB1的表达情况，分析它们与乳腺癌患者年龄、绝经情况、肿物大小、病理学类型、临床分期、组织学分级、腋淋巴结转移状况、ER、PR等临床生物学行为的关系，尤其分析两基因之间及其与HER-2蛋白表达的关系，进一步确定PAX2、AIB1是否为判断ER阳性乳腺癌患者抗雌激素治疗效果的敏感指标及其是否通过HER-2的表达进行调节。3采用RT-PCR技术从mRNA水平检测雌激素刺激ER阳性乳腺癌细胞系MCF-7和ER阴性乳腺癌细胞系MDA-MB-231前后细胞内PAX2，AIB1，HER-2的表达及细胞增殖情况；1‰的无水乙醇和1μM TAM作用于ER阳性TAM敏感乳腺癌细胞系MCF-7和TAM耐药乳腺癌细胞系MCF-7/TAMR24小时后用Weston blot检测，以获得TAM敏感及耐药乳腺癌细胞系中PAX2和AIB1及HER-2基因的基础蛋白表达状况。4应用siRNA技术分别将PAX2siRNA转染至MCF-7，AIB1siRNA转染至MCF-7/TAMR细胞中，观察沉默PAX2和AIB1两基因后HER-2及相关蛋白的表达和细胞增殖的情况；反之应用质粒转染技术分别将PAX2质粒转染至MCF-7/TAMR，AIB1质粒转染至MCF-7细胞中，观察上调PAX2、AIB1两基因后HER-2及相关蛋白表达和细胞增殖的情况。结果：第一部分：PAX2和AIB1蛋白表达与ER阳性乳腺癌患者TAM耐药关系的研究1随访结果：167例ER阳性、术后接受TAM规范治疗的乳腺癌患者5年内无瘤生存共110例；术后5年内出现局部复发或远位转移共57例，平均无病生存期（disease-free survival, DFS）45个月，平均总生存期（overallsurvival, OS）57个月。2PAX2和AIB1蛋白表达强度在不同HER-2蛋白表达水平组间（低表达组和高表达组）有统计学差异（χ2=8.401, p=0.004；χ2=12.398，p=0.000），提示：HER-2过表达组中，PAX2蛋白表达率较低，而AIB1蛋白表达率较高。3乳腺癌患者术后复发转移状况与PAX2、AIB1蛋白表达的关系：167例ER阳性行TAM治疗的乳腺癌患者中，5年内出现复发转移患者的PAX2蛋白表达较低，而AIB1蛋白表达较高，且差异有统计学意义（χ2=4.198, p=0.040; χ2=5.336, p=0.021），提示：PAX2高表达及AIB1低表达患者复发转移率较低，是较好的预测TAM疗效的指标。第二部分：PAX2、AIB1在乳腺癌组织中的表达及其与乳腺癌临床特性关系的研究1乳腺癌组织和乳腺非肿瘤组织中PAX2和AIB1基因mRNA水平的表达各70例乳腺癌组织中的AIB1mRNA测值1.7390±0.7687高于乳腺非肿瘤组织中的测值1.2742±0.6836，二者差异有统计学意义（Z=-2.031，p=0.030），而两种组织中PAX2mRNA的差异无统计学意义（Z=-0.184，p=0.830）。2PAX2, AIB1的mRNA和蛋白表达与临床资料的相关分析分别按患者年龄（≤50岁/＞50岁）、月经状况（绝经/未绝经）、肿瘤大小（≤2cm/＞2cm组）、临床分期（Ⅰ/Ⅱ/Ⅲ期）、病理学类型、组织学分级（Ⅰ^Ⅱ/Ⅲ级）及腋窝淋巴结转移状况（0个/1³个/＞3个）对70例乳腺癌患者的PAX2,AIB1基因表达进行分层分析，发现PAX2mRNA和蛋白表达的高低与乳腺癌患者上述临床因素无关（p＞0.05），而AIB1mRNA表达与组织学分级和腋窝淋巴结转移状况有关，AIB1蛋白表达仅与腋窝淋巴结转移状况有关，组间差异有统计学意义，与其它临床因素无关。3PAX2, AIB1mRNA和蛋白表达与HER-2，ER，PR表达的关系HER-2（-）患者19例，HER-2（+）⁺⁺患者32例和HER-2（+++）患者19例，各组间PAX2mRNA和蛋白表达随着乳腺癌患者HER-2蛋白的高表达而减低，差异有统计学意义（p=0.037; χ2=7.614, p=0.022），而不同HER-2水平AIB1mRNA差异无统计学意义（p=0.054）, AIB1的蛋白表达却随着HER-2蛋白的增高而增高，差异有统计学意义（χ2=11.564，p=0.003）。ER（+）⁺⁺乳腺癌患者32例，ER（+++）乳腺癌患者38例, PR（-）⁺⁺乳腺癌患者46例, PR（+++）乳腺癌患者24例，统计学分析后发现不同ER/PR表达水平的乳腺癌患者中PAX2和AIB1的基因和蛋白表达均无统计学差异（p>0.05）。4PAX2与AIB1蛋白表达的关系53例AIB1（-）⁺⁺的乳腺癌患者中PAX2高表达者19例，其高表达率为35.85%;17例AIB1（+++）的乳腺癌患者中PAX2高表达者1例，其高表达率为5.88%，不同AIB1蛋白表达水平间PAX2的蛋白表达差异有统计学意义，且随着AIB1表达的增加PAX2表达减低，二者呈负相关（χ2=5.664，p=0.017；r=-0.306）。5随访结果：70例患者中失访12例，复发或转移6例，其中死亡3例，OS平均为46个月，DFS为44个月。随访资料完整的58例患者中，26例规范应用TAM治疗，转移2例，其中1例死亡，OS平均为46.5个月，DFS平均为46.7个月。而26例TAM治疗患者中AIB1高表达9例，其中转移2例，转移率为22.22%；AIB1低表达17例均无转移，提示AIB1高表达患者的复发转移风险高（χ2=4.093，p=0.043）。第三部分：PAX2和AIB1表达与乳腺癌TAM治疗耐药机制的实验研究1雌激素刺激ER阳性乳腺癌细胞系MCF-7后，PAX2的mRNA表达水平与刺激前相比显著降低，AIB1及HER-2反之；而雌激素刺激ER阴性乳腺癌细胞系MDA-MB-231后PAX2、AIB1、HER-2的mRNA表达水平与刺激前相比未见明显变化。提示：PAX2和AIB1参与了HER-2相关的雌激素依赖性传导通路，乳腺癌细胞增殖过程伴随着PAX2的mRNA表达率降低和AIB1及HER-2的mRNA表达水平的提高。2在TAM敏感型乳腺癌细胞系MCF-7和TAM耐药型乳腺癌细胞系MCF-7/TAMR中，均可检测到PAX2、AIB1及HER-2的蛋白表达，二种细胞中AIB1蛋白表达差异无统计学意义，MCF-7细胞中PAX2蛋白表达量较MCF-7/TAMR细胞低，而HER-2蛋白表达反之，差异有统计学意义（p＜0.05）。提示：TAM耐药的原因可能与PAX2和HER-2的表达相关。TAM刺激MCF-7细胞后AIB1表达显著升高而PAX2、HER2表达降低，差异有统计学意义（p＜0.05）。而TAM刺激MCF-7/TAMR细胞后PAX2、AIB1蛋白表达变化无意义，仅HER-2蛋白表达升高，差异具有统计学意义（p＜0.05）。3PAX2siRNA转染MCF-7细胞,AIB1siRNA转染MCF-7/TAMR细胞MCF-7细胞系转染PAX2siRNA后显示，PAX2siRNA+TAM处理组MCF-7细胞中HER-2mRNA表达和蛋白表达水平比PAX2siRNA+Vehicle处理组明显增加，而AIB1的蛋白表达降低（p<0.05），而细胞增殖活性则无明显抑制。提示PAX2基因的沉默会阻碍TAM对HER-2表达和细胞增殖的抑制，使HER-2转录和蛋白表达增加，而且PAX2表达的降低，逆转了TAM对AIB1上调作用。MCF-7/TAMR细胞系转染AIB1siRNA后显示，AIB1siRNA+TAM处理组MCF-7/TAMR细胞中HER-2的mRNA表达和蛋白表达水平比AIB1siRNA+Vehicle处理组降低，并且PAX2蛋白表达水平和细胞增殖活性也明显降低（p＜0.05），结果提示，MCF-7/TAMR细胞中沉默AIB1恢复了TAM下调HER-2转录和HER-2蛋白表达的作用，AIB1的高表达可能抑制乳腺癌细胞TAM的抗雌激素活性。4PAX2质粒扩增至MCF-7/TAMR细胞，AIB1质粒扩增至MCF-7细胞MCF-7/TAMR细胞系转染PAX2质粒后显示PAX2质粒+TAM处理组与空载质粒+TAM处理组相比MCF-7/TAMR细胞中HER-2mRNA表达水平和蛋白表达水平明显减低，AIB1蛋白的相对表达量减少，细胞增殖活性降低（p<0.05）。提示：PAX2表达的增加逆转了TAM对MCF-7/TAMR细胞HER2蛋白表达的促进作用，使其恢复对TAM的敏感性，增加PAX2可以逆转TAM耐药状况。MCF-7细胞系转染AIB1质粒后，AIB1质粒+TAM处理组较AIB1质粒+Vehicle组MCF-7细胞中HER-2mRNA表达水平和蛋白表达水平明显增高，细胞增殖活性明显增高而PAX2蛋白表达水平降低（p<0.05），提示：AIB1表达升高并未改变TAM抑制PAX2表达的作用，但上调AIB1逆转了MCF-7细胞中TAM对HER2的抑制作用，使细胞增殖活性增加，AIB1表达增高引起TAM耐药。结论：1在ER阳性的乳腺癌患者中，PAX2蛋白高表达AIB1蛋白低表达患者5年内复发转移率较低，是较好的预测预后的指标；PAX2蛋白高表达在HER-2蛋白过表达组中较低，而AIB1蛋白高表达在HER-2蛋白过表达组中较高，提示PAX2,AIB1与HER-2过表达及TAM治疗耐药相关。2PAX2基因在乳腺癌组织与乳腺非肿瘤组织之间表达无差异，PAX2mRNA及蛋白表达与年龄、月经状况、肿瘤大小、临床分期、组织学分级、腋窝淋巴结转移、病理类型、ER/PR表达水平均无显著相关性。3乳腺癌组织中AIB1基因表达高于乳腺非肿瘤组织，AIB1mRNA表达与组织学分级、腋窝淋巴结转移情况正相关，而AIB1蛋白表达仅与腋窝淋巴结转移情况正相关，与年龄、月经状况、肿瘤大小、临床分期、病理类型、ER/PR表达水平均无显著相关性。4PAX2，AIB1在ER阳性的乳腺癌细胞中均表达，在HER-2高表达的TAM耐药株中PAX2表达较TAM敏感的细胞株低，而二者AIB1表达水平相当。PAX2过表达增加乳腺癌细胞对TAM的敏感性，AIB1过表达则减低了其敏感性；预示了PAX2和AIB1可能作为乳腺癌内分泌治疗耐药的预测指标。5PAX2是TAM治疗过程中HER-2的转录抑制因子，针对PAX2的特异激活剂有望成为乳腺癌内分泌治疗耐药的新靶点。AIB1与PAX2相互调节，并可能通过竞争性结合HER-2上游的调节因子，调控HER-2的转录，从而影响雌激素受体阳性乳腺癌TAM治疗的敏感性。6PAX2表达的增强及AIB1表达的降低均有助于改善TAM耐药状态，PAX2和AIB1有望成为TAM耐药患者新的基因治疗靶点。更多还原

【Abstract】 Objective: Breast cancer is the most common malignant tumor of femalein the world, which seriously threatening those health and life. The incidence ofbreast cancer keeps on rising annually in China and more young females arefaced with it. Breast cancer is typical hormonal dependent tumor, andendocrine therapy, its representative drug tamoxifen（TAM）, has become themost important and successful one in the combined therapies for estrogenreceptor-positive（ER+） breast cancers due to its low toxicity, economy andlong curative effect. However, it was proved that40%ER-positive tumorsbecame resistant induced by long-term tamoxifen therapy, leading to progressionof breast cancer even death. However, the molecular mechanisms of tamoxifenresistance were not completely understood. So it was hot that studies on theresistant mechanism of TAM and its related gene of breast cancer.Some researchs had shown that paired box gene-2（PAX2）and amplifiedin breast cancer-1（AIB1） regulated the transcription of human epidermalgrowth factor receptor-2（HER-2） and the protein expression of HER-2duringthe treatment of tamoxifen for ER+breast cancers,which could account forTAM resistance. To explore the resistance mechanism of TAM and the relativefactors, we first detected the protien expressions of PAX2and AIB1withimmunohistochemistry（IHC） in postoperative ER（+） breast cancer patientstreated with TAM. Combining the follow-up results, we retrospectivelyanalyzed the relationship between PAX2/AIB1and the curative effect ofTAM.Secondly we detected the expressions of PAX2and AIB1in the tumortissues and the nontumorous tissues of breast on the transcriptional and proteinlevel by using reverse transcription-polymerase chain reaction （RT-PCR） andIHC.The relatetionships among the expressions of PAX2/AIB1in ER（+）breast cancers, the clinic pathological characteristics and the expression of ER,PR and HER-2were analyzed, to futher determine if PAX2/AIB1weresensitive indicators for prognosing the effect of antiestrogen therapy. Forfinding out the role of PAX2/AIB1on the antiestrogen treatment in ER+breastcancers,we used RT-PCR and Western blot method to detect the expressions ofPAX2, AIB1and HER-2in ER （+）/ER（-） breast cell lines （MCF-7andMDA-MB-231） stimulated with estrogen and TAM sensitive/TAM resistantbreast cell lines （MCF-7and MCF-7/TAMR） treated with or without TAM.PAX2siRNA and AIB1siRNA were transiently transfected to MCF-7andMCF-7/TAMR respectively, while plasmid of PAX2and AIB1weretransfected to MCF-7/TAMR and MCF-7to explore the regulationmechanisms among PAX2/AIB1, HER-2and TAM resistance.Methods:1The protein expressions of PAX2and AIB1in167postoperative ER（+）breast cancers treated with TAM were detected by IHC, and the relationshipbetween PAX2/AIB1and the recurrence or metastasis after TAM treatmentwas analyzed, to determine if PAX2/AIB1influenced the TAM therapeuticeffect.2The mRNA and protein expression of PAX2and AIB1in70breast tumorand nontumorous tissues were detected by using RT-PCR and IHC. Therelationships between PAX2/AIB1and the clinic pathological characteristicswere analyzed, including age, menses, tumor size, clinical stages,pathological type, histological grades, the metastatic state of the axillarylymph nodes, ER, progestogen receptor（PR） and HER-2in70breast cancers,to futher determine if PAX2/AIB1regulated the expression of HER-2andinfluenced the effect of antiestrogen therapy.3The mRNA expressions of PAX2, AIB1and HER-2were measuredwith RT-PCR in ER （+） MCF-7and ER （-） MDA-MB-231breast cancer celllines stimulated with or without estrogen respectively, so did TAM sensitvecell lines MCF-7and TAM resistant cell lines MCF-7/TAMR stimulated by0.1%anhydrous alcohol and1μM tamoxifen for24h to get the basicexpressions of PAX2, AIB1and HER-2in those cell lines. 4PAX2siRNA and AIB1siRNA were transiently transfected to MCF-7and MCF-7/TAMR respectively, while plasmid of PAX2and AIB1weretransfected to MCF-7/TAMR and MCF-7to observe the expression ofPAX2/AIB1/HER-2and cell proliferation explore the regulation mechanismsamong PAX2/AIB1, HER-2and TAM resistance.Results：The first part: Protein expression of PAX2/AIB1and the relationshipsbetween them and the TAM resistance of ER+breast cancers1Follow-up results: Among167breast cancer patients,110casesappeared tumor-free survival, while57cases appeared local recurrence ormetastasis within5years after therapy, with the mean disease-free survival（DFS） of45months and the mean overall survival （OS） of57months.2Among the different expression level of HER-2（low-expression andover-expression）, the difference of PAX2and AIB1protein expression weresignificant （χ2=8.401, p=0.004; χ2=12.398, p=0.000）．In the group of protienover-expression of HER-2, the protein over-expression rate of PAX2waslower, while that of AIB1was higher.3Relationships between the protein expression of PAX2, AIB1andrecurrence or metastasis of breast cancers:Among167cases,57cases appeared recurrence or metastasis, while110cases were disease-free survival within5years. After statistical analysis,recurrence or metastasis rates had significant difference between differentgroups of PAX2（χ2=4.198, p=0.040） and AIB1（χ2=5.336, p=0.021）. Breastcancers with over-expression of PAX2and low-expression of AIB1had lowerrecurrence or metastasis rate, so PAX2and AIB1might be sensitive prognosticindex for prognosing TAM treatment effect.The second part: Expression of PAX2/AIB1in breast cancers and therelationships between them and the clinic characteristics1Expression of PAX2/AIB1on the mRNA level in breast cancer andnontumorous tissuesThe value of AIB1mRNA in breast cancer and nontumorous tissues were （1.7390±0.7687） and （1.2742±0.6836）, which the difference wassignifant（Z=-2.031,p=0.030）. While there had no significant difference ofPAX2mRNA between those tissues （Z=-0.184, p=0.830）.2The relationship between the mRNA and protein expressions ofPAX2/AIB1and clinic pathological characteristics of70breast cancersAccording to the age（<50years and≥50years）, the menstrual status（premenopause and postmenopause）, tumor size （≤2cm,>2cm）, the clinicalstage （stage I,II and III）, the pathological types （infiltrating ductal carcinoma,infiltrating lobular carcinoma and others）, the histological grade （grade I^IIand III） and the metastatic state of the axillary lymph nodes （negative,1³，and＞3）, the mRNA and protein expression of PAX2had no significantdifference among groups of those factors（p>0.05）．The mRNA expression ofAIB1had positive correlation with the histological grades and the metastaticstate of the axillary lymph nodes, while the protein expression of AIB1onlyhad positive correlation with the latter.3The relationships between the expression of PAX2/AIB1and HER-2,ER, PR on mRNA and protein levelThere were19,32and19cases of HER-2（-）, HER-2（+⁺+） and HER-2（+++） respectively. The mRNA and protein expression of PAX2had negativecorrelation with the expression of HER-2and the difference of each group wassignificant（p=0.037; χ2=7.614, p=0.022）. The mRNA expression of AIB1hadno significant difference between different expression of HER-2, while theprotein expression of AIB1increased with the rising expression of HER-2（χ2=11.564，p=0.003）.There were32,38,46and24cases of ER （+⁺+）, ER （+++）, PR （+⁺+）and PR （+++） respectively. It was showed that the mRNA and proteinexpression of PAX2and AIB1had no relationships with the expressions of ERand PR after statistical analysis.4The relationships between protein expression of PAX2and AIB1The over-expression rate of PAX2was35.85%（19/53） in the low-expression of AIB1（-）⁺⁺, and that was5.88%（1/17） in the over-expression of AIB1（+++）, which had the significant difference. The protein expression ofPAX2had negative correlation with that of AIB1（χ2=5.664；p=0.017；r=-0.306）.5Follow-up results: In all the70patients there were12cases out ofcontact and6cases with recurrence or metastasis including2cases dead. TheDFS was44months and OS was46months. So there were26cases treatedwith TAM after operation including2cases with metastasis, whose DFS was46.7months and OS was46.5months.Among the26cases,9cases with over-expression of AIB1had2casesoccuring metastasis and17cases with low-expression of AIB1had nometastasis, which suggested that patients with over-expression of AIB1hadhigh risk of recurrence and metastasis（χ2=4.093；p=0.043）.The third part:The empirical study on the roles of PAX2/AIB1on theresistance mechanism of TAM1The mRNA expressions of AIB1/HER-2increased and that of PAX2decreased in the ER （+） breast cell line MCF-7after estrogen stimulation,while there had no significant changes in ER （-） breast cell line MDA-MB-231after estrogen stimulation. It suggested that PAX2, AIB1and HER-2took partin the estrogen dependent signal conduction path and the cell proliferationswere companied by the decreased expression of PAX2and the increased thatof AIB1and HER-2.2PAX2, AIB1and HER-2could be detected in both MCF-7（TAMsensitive cell lines） and MCF-7/TAMR （TAM resistant cell lines） and theexpression of AIB1between these two cell lines had no significant difference（p>0.05）. But the expression of PAX2in MCF-7was higher than that inMCF-7/TAMR,while the expression of HER-2was opposite. It suggested thatthe resistant mechanism of TAM might be related with the expression of PAX2and HER-2.The expression of AIB1visibly increased and that of PAX2/HER-2decreased in MCF-7treated with TAM, and the difference was significant （p＜0.05）. While only HER-2expression increased in MCF-7/TAMR treated with TAM, and the changes of PAX2/AIB1had no significant difference.3MCF-7transiently transfected by PAX2siRNA and MCF-7/TAMRtransiently transfected by AIB1siRNA （down-regulation of PAX2/AIB1）After MCF-7was transiently transfected by PAX2siRNA, the mRNA andprotein expression of HER-2in PAX2siRNA+TAM group increased morethan those of HER-2in PAX2siRNA+Vehicle one, while the proteinexpression of AIB1decreased （p<0.05）, and the cell proliferation was notobviously depressed. It suggested that down-regulation of PAX2in MCF-7cell line inhibited the role of TAM on depressing the HER-2expression andcell proliferation, while it reversed up-regulation of AIB1induced by TAM.After MCF-7/TAMR was transiently transfected by AIB1siRNA, themRNA and protein expression of HER-2, the protein expression of PAX2andthe cell proliferation in AIB1siRNA+TAM group decreased more than thosein AIB1siRNA+Vehicle one（p<0.05）. It suggested that down-regulation ofAIB1in MCF-7/TAMR cell line refreshed the role of TAM on depressing theHER-2expression and the over-expression of AIB1would inhibit theantiestrogen of TAM in breast cancer cell.4MCF-7/TAMR transfected by plasmids of PAX2and MCF-7transfected by those of AIB1（up-regulation of PAX2/AIB1）After MCF-7/TAMR was transfected by plasmids of PAX2, the mRNAand protein expression of HER-2, the expression of AIB1and the cellproliferation in PAX2plasmid+TAM group decreased more than those inPAX2plasmid+Vehicle one （p<0.05）. It suggested that up-regulation of PAX2reversed the role of TAM on promoting the expression of HER-2inMCF-7/TAMR cell and refreshed the sensitivity of TAM. Augument of PAX2would reverse the TAM resistance.After MCF-7/TAMR was transfected by plasmids of AIB1, the mRNAand protein expression of HER-2and the cell proliferation in AIB1plasmid+TAM group increased more than those in AIB1plasmid+Vehicle one, whilethe expression of PAX2decreased（p<0.05）. It suggested that up-regulation ofAIB1didn’t change the role of TAM on depressing the expression of PAX2 but reversed the role of TAM on depressing the expression of HER-2inMCF-7cell. Augument the expression of AIB1would induce TAM resistance.Conclusions:1Among the ER（+） breast cancers treated with TAM, the patients withover-expression of PAX2and low-expression of AIB1had lower recurrenceor metastasis rate within5years. PAX-2and AIB1were good index forprognosis. PAX2and AIB1were related with HER-2expression and TAMresistance.2The mRNA expression of PAX2had no significant difference betweentumor and nontumorous breast tissues. Expression of PAX2had no significantrelationships with age, menses, tumor size, clinical stage, histological grade,metastatic state of the axillary lymph nodes, pathology type and ER/PR onmRNA and protein level.3The mRNA expression of AIB1in breast cancers were higher than thatin nontumorous tissues. The mRNA expression of AIB1had positivecorrelation with the histological grade and the metastatic state of the axillarylymph nodes and the protein expression of AIB1only had positive relationshipwith the latter, but had no significant relationships with other factors.4PAX2and AIB1were all detected in both MCF-7and MCF-7/TAMRcells. The expression of PAX2in MCF-7was higher than that in MCF-7/TAMR, while the expression of AIB1had no difference in these two cell lines.Over-expression of PAX2would increase the TAM sensitivity of cell, whilethe over-expression of AIB1would decrease it.5PAX2was the inhibitor of HER-2transcription on the therapy of TAM.The specific activation of PAX2would be a new target for curing TAMresistance. AIB1had cooperation with PAX2, which regulated the HER-2transcription by competively binding cis-regulatory element of HER-2andthus influeced the sensitivity of TAM.6Raising the expression of PAX2and depressing the expression of AIB1might improve the resistant condition of TAM, so PAX2and AIB1might benovel therapeutic targets.更多还原