目的检测19例肾细胞癌患者癌组织及癌旁组织Ras相关区域家族蛋白1A(RASSF1A)基因启动子区甲基化情况并检测其mRNA表达水平,探索两者之间的联系。方法收集肾细胞癌患者癌组织及相应癌旁组织19份,分别提取其基因组DNA,以甲基化特异性PCR(Methylation special PCR,MSP)法检测RASSF1A基因启动子区甲基化情况,并以QPCR法检测了RASSF1A基因的mRNA表达水平。结果19例中有10例肾细胞癌患者癌组织存在高甲基化,mRNA表达水平表达降低,二者之间存在显著负相关性(r=-0.8734,P<0.01)。结论肾细胞癌中RASSF1A基因启动子区存在高甲基化,并抑制该基因的表达。
目的研究多药耐药基因ABCG2启动子区甲基化状态与其在乳腺癌组织中表达的关系,探讨ABCG2基因表达的表观遗传学机制。方法采用甲基化特异性PCR(Methylation specific PCR,MSP)检测15例乳腺癌组织及配对癌旁组织中ABCG2启动子区-359～-353两特异位点的甲基化情况,并经MSP产物测序检测位点的甲基化状态;用Q-PCR检测ABCG2基因mRNA的表达水平;并应用统计学Spearman等级相关性方法分析二者的相关关系。结果15例乳腺癌组织中有13例(86.67%)ABCG2基因启动子区的特异位点存在高甲基化(P<0.05),MSP产物的测序验证了特异位点的甲基化,并且mRNA表达水平相对增高,二者之间存在显著正相关性(r2=0.6842,P<0.05)。结论ABCG2基因的启动子区-359～-353位点存在高甲基化,可促进该基因在乳腺癌组织中的表达。
目的研究乳腺癌细胞(MCF-7)经米托蒽醌诱导耐药后其甲基化结合蛋白(Methyl-DNA-binding protein,MBD)的表达变化,并分析在MCF-7细胞经诱导产生多药耐药性中与甲基化结合蛋白的关系。方法利用耐米托蒽醌的MCF-7耐药株,并用未染毒的野生型MCF-7为对照,通过蛋白印迹法检测不同浓度米托蒽醌处理的细胞组乳腺癌耐药蛋白(breast cancer resistance protein,BCRP)和甲基化结合蛋白MBD1、MBD2及MBD4的蛋白表达水平。结果跟野生型MCF-7比较,随着染毒浓度的增大,乳腺癌耐药蛋白BCRP的表达逐渐增加,而MBD1、MBD2、MBD4的表达均呈降低趋势,且MBD4的降低尤为明显,与对照组比较,其MBD4蛋白表达水平分别为(0.910±0.049)、(0.835±0.065)、(0.708±0.082)、(0.660±0.145)(P<0.05)。结论在米托蒽醌诱导产生耐药的MCF-7细胞株中,MBDs的表达,尤其是MBD4,有可能参与了肿瘤耐药的形成。
Objective To filtrate breast cancer resistance protein （BCRP）-mediated resistant agents and to investigate clinical relationship between BCRP expression and drug resistance. Methods MTT assay was performed to filtrate BCRP-mediated resistant agents with BCRP expression cell model and to detect chemosensitivity of breast cancer tissue specimens to these agents. A high performance liquid chromatography （HPLC） assay was established, and was used to measure the relative dose of intracellular retention resistant agents. RT-PCR and immunohistochemistry （IHC） were employed to investigate the BCRP expression in breast cancer tissue specimens. Results MTT assay showed that the expression of BCRP increased with the increasing resistance of 5-fluorouracil （5-Fu） （P〈0.05, n=3） in the cell model, while HPLC assay indicated that the intracellular retention dose of 5-Fu was significantly correlated with the expression of BCRP （t=-0.897, P〈0.05, n=3）. A total of 140 breast cancer tissue specimens were collected. BCRP-positive expression was detected in forty-seven specimens by both RT-PCR and IHC. As shown by MTT assay subsequently, the resistance index （RI） of 47 BCRP-positive breast cancer tissue specimens to 5-Fu was 7-12 times as high as that of adjacent normal tissue samples. BCRP expression was related to 5-Fu resistance （R2=0.8124, P〈0.01）. Conclusion Resistance to 5-Fu can be mediated by BCRR Clinical chemotherapy for breast cancer patients can be optimized based on BCRP-positive expression.
The aim of this study is to investigate epigenetic mechanism of ABCG2 induced drug-resistance. It is not only expatiate for drug-resistance regulation mechanism in all-round, but also to provide scientific experimental basis for selecting target to reverse its drug-resistance. Apply methylation-specific PCR （MSP） to have tested methylation of ABCG2 promoter region -359 to -353 specific positions in breast cancer tissues and paired adjacent tissue of 22 cases and test their methylation positions with MSP products for sequencing; and adopt fluorescent quantitation RT-PCR to test expression level DNMT1, DNMT3A, DNMT3B and ABCG2; to make analysis on relationship between them with statistical spearman correlation. Specific positions of ABCG2 gene promoter region of 18 cases among the 22 cases with breast cancer （18/22, 82%） existed high methylation （P〈0.05）, MSP products sequencing proved methylation of the specific position, and mRNA expression level was relative higher in remarkable positive correlation （P〈0.05） ABCG2, DNMT1, DNMT3A, DNMT3B mRNA expression levels in breast cancer tissues were obviously higher than adjacent tissues （P〈0.01）, and DNMT3B expression level was obviously higher than DNMT1 and DNMT3A （P〈0.01） in negative correlation with ABCG2 gene expression （P=0.001）. -359 to -353 positions of promoter regions of ABCG2gene existed high methylation capable to push expression of this gene in beast cancer tissue. DNMT3B is involved in expression regulation in ABCG2 gene, and provides new scientific basis for drug-resistance target as reverse ABCG2 induction
Yuan Jianhui Zhou Jianmeng JiNana Xu Xinyun Liu Jianjun Ke Yuebin Cheng Jinquan． Zhuang Zhixiong
