【作者】 王麟；

【导师】 金万宝；

【作者基本信息】 中国医科大学 ， 药理学， 2007， 博士

【摘要】 散发性乳腺癌中BCRP表达与ERα基因启动子甲基化相关性研究目的探讨乳腺癌耐药相关蛋白（breast cancer resistance protein,BCRP）在散发性乳腺癌组织中的表达特征及其与雌激素受体α（Estrogen Receptorα,ERα）蛋白表达和ERα基因启动子甲基化的相关性,通过细胞实验观察DNA甲基转移酶抑制剂5’-氮杂-脱氧胞苷酸（5’-Aza-2’-deoxycytidine,5’-Aza-dC）及17-β雌二醇（17β-Estradiol,E₂）处理前后ERα阴性乳腺癌细胞株MDA-MB-435s ERα基因启动子甲基化及ERα、BCRP mRNA表达变化情况,探讨乳腺癌中BCRP表达与雌激素信号的相关性及乳腺癌内分泌治疗耐药与多药耐药间可能存在的内在联系,初步阐明去甲基化药物在乳腺癌内分泌治疗与化学治疗中的可能意义。材料与方法采用免疫组织化学（immunohistochemistry,IHC）染色法,检测146例散发性乳腺癌和22例乳腺纤维腺瘤组织中BCRP、ERα、PR、HER-2、P53蛋白的表达,分析BCRP表达与临床病理学参数之间的相关性;通过甲基化特异性PCR（Methylation specific PCR,MSP）法检测60例乳腺浸润性导管癌肿瘤及14例乳腺纤维腺瘤组织中ERα基因启动子区四个CpG岛密集区域甲基化情况,探讨乳腺浸润性导管癌组织中ERα基因启动子区甲基化特征及其与ERα和BCRP表达的相关性;通过MSP、逆转录PCR（Reverse transcript PCR,RT-PCR）等方法检测5’-Aza-dC和E₂处理前后ER阴性MDA-MB-435s乳腺癌细胞株ERα基因启动子甲基化及ERα、BCRP mRNA变化情况,探讨去甲基化药物对于BCRP表达的可能影响及其机制。结果1.散发性乳腺癌中BCRP表达与临床病理参数相关性:（1） BCRP在乳腺癌组织中表达情况:乳腺癌中BCRP表达阳性率60.3%,纤维腺瘤中BCRP表达阳性率31.8%,两者相比有显著性差异（X²=6.301,P=0.012）;（2）BCRP表达与乳腺癌临床指标相关性:在乳腺癌组织中,BCRP表达水平与患者年龄、病理类型、临床分级及淋巴结转移均无关;（3）乳腺癌组织中BCRP与ERα、PR表达的相关性:结合ERα、PR表达与绝经情况分组,未绝经且ERα阳性患者乳腺肿瘤组织中BCRP表达阳性率（38.5%）显著低于未绝经ERα阴性（71.9%）和已绝经ERα阳性（60.3%）及已绝经ERα阴性患者（63.0%）（X²=9.301,P=0.007）;绝经与未绝经PR阳性或阴性患者之间BCRP表达均未见显著性差别;（4）乳腺癌组织中BCRP与HER-2、P53表达的相关性:HER-2阳性表达乳腺癌中BCRP表达阳性率显著高于HER-2阴性乳腺癌（81.0%vs.46.6%,X²=17.321,P=0.001）,P53阳性组与阴性组间BCRP表达未见显著差异。2.浸润性导管癌中ERα基因启动子甲基化特征及其与ERα、BCRP表达相关性:（1）乳腺癌与纤维腺瘤组织中的ERα表达与基因启动子甲基化:乳腺浸润性导管癌组织中ERα表达阳性率与纤维腺瘤比较未见显著性差异（48.3%vs.71.4%,X²=2.429,P=0.119）;乳腺癌组织甲基化发生率显著性高于纤维腺瘤组织（83.3%vs.28.6%,X²=17.260,P=0.001）;（2）乳腺癌中ERα基因启动子甲基化与ERα表达相关性:ERα阴性浸润性导管癌组织中ER2、ER3、ER4三个检测区域的甲基化发生率（63.3%,58.1%,83.9%）均显著高于ERα阳性组织（24.1%,17.2%,13.8%）,以ERα表达水平与甲基化区域检出个数做Spearman相关性分析,二者呈显著负相关（r=-0.713,P=0.001）;ERα阴性浸润性导管癌组织中ER4甲基化发生率显著高于其它三个检测区域（X²=8.321,P=0.004）;（3）乳腺癌中ERα基因启动子甲基化与BCRP蛋白表达的相关性:以BCRP表达水平与甲基化区域检出个数做Spearman相关性分析,二者呈较弱的正相关趋势（r=0.254,P=0.050）;（4）乳腺癌中ERα基因启动子甲基化与临床病理参数的相关性:ERα基因启动子甲基化水平与乳腺癌患者年龄、临床分级、淋巴结转移及HER-2表达情况无关;孕激素受体阳性患者ERα基因启动子区甲基化水平显著低于孕激素受体阴性患者（X²=9.598,P=0.002）。3.5’-Aza-dC联合E₂对MDA-MB-435s细胞ERα基因启动子甲基化及ERα、BCRPmRNA表达的影响（1）5’-Aza-dC对ERα基因启动子区甲基化的影响:采用10、50、100ng/ml终浓度的5’-Aza-dC处理MDA-MB-435s细胞,10ng/ml浓度处理后ER1、ER2、ER4三个检测区域发生了去甲基化,50、100ng/ml终浓度处理后MDA-MB-435s细胞ERα基因启动子四个甲基化检测区域均发生了去甲基化;（2）5’-Aza-dC对ERαmRNA表达的影响:溶剂及单纯3nmol/ml E₂处理组MDA-MB-435s细胞均未见明显PCR扩增阳性条带;单独采用100ng/ml终浓度的5’-Aza-dC处理MDA-MB-435s细胞96h后ERαmRNA相对表达量增加了16.3倍（0.814±0.031 vs.0.050±0.006）;分别采用10、50、100ng/ml终浓度的5’-Aza-dC联合E₂处理MDA-MB-435s细胞96h后,10、50、100ng/ml浓度组ERαmRNA相对表达量分别增加了11.3（0.563±0.007 vs.0.050±0.006）、13.2（0.661±0.008 vs.0.050±0.006）、16.0（0.799±0.051 vs.0.050±0.006）倍;100ng/ml 5’-Aza-dC联合E₂组与单纯100ng/ml 5’-Aza-dC处理组间比较差别无统计学意义（t=1.358,P=0.232）,10、50、100ng/ml 5’-Aza-dC分别联合3nmol/ml E₂处理组间ERαmRNA表达水平随5’-Aza-dC浓度增大而增加,差别具有显著性（F=85.182,P=0.001）;（3）17-β-雌二醇对5’-Aza-dC处理前后BCRPmRNA表达的影响:MDA-MB-435s细胞单纯100ng/ml 5’-Aza-dC和3nmol/ml E₂处理组BCRP mRNA表达水平与溶剂组无显著性差异（F=3.256,P=0.425）;10、50、100ng/ml 5’-Aza-dC联合3nmol/ml E₂处理MDA-MB-435s细胞96h后,三组BCRPmRNA相对表达量分别降低至溶剂对照组的81.5%（0.551±0.0177 vs.0.676±0.028）、51.7%（0.350±0.024 vs.0.676±0.028）,42.8%（0.289±0.077 vs.0.676±0.028）,各处理组间BCRP mRNA表达水平随5’-Aza-dC浓度增大而降低,差别具有显著性（F=67.232,P=0.001）。结论1.BCRP在乳腺癌组织中表达显著高于正常乳腺组织。BCRP在乳腺癌组织中表达水平患者与年龄、病理类型、临床分期、淋巴结转移等无关,在绝经前与ERα表达负相关、与HER-2表达正相关,与PR、p53表达未见相关性。2.乳腺癌组织中ERα基因启动子甲基化水平显著高于乳腺良性病变。ERα阴性患者乳腺癌组织ERα基因启动子甲基化发生率显著高于ERα阳性患者,ERα蛋白表达水平与其基因启动子区甲基化位点个数呈高度负相关,乳腺癌组织中ERα失表达与其基因启动子甲基化有关,靠近编码区启动子序列甲基化更易引起ERα失表达。3.5’Aza-dC可有效诱导MDA-MB-435s细胞ERα基因启动子去甲基化,并恢复其ERαmRNA表达;E₂对野生型MDA-MB-435s细胞BCRP表达无影响,但可显著下调ERα基因去甲基化MDA-MB-435s细胞的BCRP mRNA表达,表明ERα甲基化状态可能影响BCRP表达调控,提示ERα阴性乳腺癌易产生化疗耐药的机制可能与缺少雌激素信号对多药耐药蛋白表达调控有关。更多还原

【Abstract】 ObjectiveTo discuss the relationship of the expression of breast cancer resistance protein （BCRP） and Estrogen receptor alpha（ERα） with the methylation feature of promoter of ERαgene in breast cancer.Investigate the changes of methylation state of promoter of ERαand the level of ERαand BCRP mRNA in ERαnegative MDA-MB-435s breast cancer cell line before or after treated with methyltransferases inhibitor 5’-Aza-deoxycytidine（5’-Aza-dC）.To investigate the correlation between the expression of BCRP and estrogen signaling and observe the possible effect of demethylation drugs on antihormone therapy to breast cancer,to elucidate the possible internal relation between endocrine therapy resistance and multidrug resistance as well as different pathological profiles in breast cancer.MethodsThe protein expression of BCRP,ER,PR,HER-2 and P53 in 146 sporadic breast cancers and 22 fibroadenoma tissues were examined using immunohistochemistry.The correlations of BCRP with the clinical data as well as the pathological parameters were analyzed by correlate test.the methylation states of four CpG compact sites in promoter region of ERαgene in 60 infitrating ductal carcinomas（IDCs） and 14 fibro adenomas（FAs） were detected by methylation specific PCR（MSP）,correlation analysis between methylation of promoter of ERαgene and its’ expression as well as clinical parameter was performed.The methylation state of promoter of ERαand the level of ERαand BCRP mRNA in ERαnegative MDA-MB-435s breast cancer cell line before or after be treated with 5’-Aza-dC by MSP and RT-PCR.ResultsThe positive immunostaining rate of BCRP in breast cancer was significantly higher than that in fibroadenoma tissues（60.3%vs.31.8%,X²=6.301,P=0.012）.The expression of BCRP in breast cancer tissues was not associated with histological types, age,clinical stage or lymph node metastases state,but was significantly lower in ERαpositive pre menopause patients（38.5%,X²=9.301,P=0.007）.The expression of BCRP in HER-2 positive pateins were significantly higher than that in HER-2 negative pateins （81.0%vs.46.6%,X²=17.321,P=0.001）,but no correlation with P53 or PR.The expression of ERαprotein in IDCs didn’t show statistical difference compared with that in AFs;the methylation rate in IDCs were significantly higher than that in AFs（83.3%vs.28.6%,X²=17.260,P=0.001）;the methylation rates of checking site ER2,ER3,ER4 in ERαnegative IDCs（63.3%,58.1%,83.9%） were totally higher than that in ERαpositive IDCs（24.1%,17.2%,13.8%）;The methylation frequency of the promoter region of ERαgene was dramatically negative correlated with expression of ERα（r=-0.713,P=0.001） but modest positive correlated with expression of BCRP （r=0.254,P=0.050） in IDCs;the methylation rate of ER4 was significantly higher than that of other three check sites in ERαnegative IDCs（X²=8.321,P=0.004）;the methylation frequency of the promoter region of ERαgene in progesterone receptor （PR）positive IDCs was significantly lower than that in PR negative IDCs（X²=9.598, P=0.002）,but not correlated with age,clinical stage,lymph node metastases or HER-2 state.Three of four methylation detection site were demethylated in MDA-MB-435s cell line which after been treated with 10 ng/ml 5’-Aza-dC and all the four sites were demethylated in that treated with 50 or 100ng/ml 5’-Aza-dC for 96h.The expression of ERαmRNA was increased for 16.3 fold in MDA-MB-435s after been treated only with 100 ng/ml 5’-Aza-dC for 96h（0.814±0.031 vs.0.050±0.006）;After been exposed to 10, 50 or 100ng/ml 5’-Aza-dC respectively in combination with 3nmol/l E₂ for 96h,the expressions of ERαmRNA in MDA-MB-435s were correspondingly increased for 11.3 fold（0.563±0.007 vs.0.050±0.006）,13.2 fold（0.661±0.008 vs.0.050±0.006） and 16.0 fold（0.799±0.051 vs.0.050±0.006）;the expressions of ERαmRNA increased followed the concentration of 5’-Aza-dC.Positive stain band of BCRP mRNA were present in every group,the expression of ERαmRNA didn’t changed in MDA-MB-435s after been treated singly with 100 ng/ml 5’-Aza-dC or 3nmol/ml E₂ for 96h（F=3.256, P=0.425）;After been exposed to 10,50 or 100ng/ml 5’-Aza-dC respectively in combination with 3nmol/l E₂ for 96h,the expressions of BCRP mRNA in MDA-MB-435s were correspondingly decreased to 81.5%（0.551±0.0177 vs.0.676±0.028）,51.7%（0.350±0.024 vs.0.676±0.028）,42.8%（0.289±0.077 vs.0.676±0.028） when compared with the control group,the expressions of BCRP mRNA decreased contrast with the concentration of 5’-Aza-dC.Conclusion1.the protein level of BCRP in sporadic breast cancer was higher than that in fibroadenoma tissues and negatively correlated with ERαstatues in pre-menopause pateints but positively correlated with HER-2 statues all the menopause state;The expression of BCRP in breast cancer tissues was not associated with histological types, age,clinical stage or lymph node metastases state.2.The aberrant methylation of the promoter region of ERαgene was detected in IDC and this aberrant methylation contribute to the loss of ERαexpression;the effect of the methylation on protein expression depends on the frequency of methylation or specific methylated CpG Island;PR expression was correlated with methylation of the promoter region of ERαgene.3.5’Aza-dC can not only efficaciously induced demethylation of the promoter of ERαgene but also restore the ERαexpression in a concentration dependent manner in ERαnegative breast cancer cell line.When used in combine with 5’Aza-dC,E₂ dramatically induced BCRP Expression down regulation in ERαnegative breast cancer cell line.These datas sugest that methylation of promoter of ERαgene might contrubite to MDR development in ERαnegative breast cancer due to Estrogen signaling deficiency caused BCRP regulation malfunction更多还原