【作者】 徐红梅；

【导师】 赵子琴； 朱虹光；

【作者基本信息】 复旦大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 前言乳腺癌是女性最常见的恶性肿瘤之一,在世界上我国虽属女性乳腺癌的低发国,但近年来乳腺癌的发病率明显增高,严重威胁着妇女的健康,因此研究乳腺癌的发病机制,寻找新的肿瘤分子标志物对乳腺癌的诊断和治疗都是非常有必要的。肿瘤的发生、发展包含多基因表达谱的协同改变。基因的表达除决定于遗传学因素,即DNA序列所传递的信息之外,还决定于表观遗传学因素。在后基因组时代,表观遗传学在阐明肿瘤发病机制中的作用日益重要。表观遗传学改变是一种不涉及核苷酸序列变化的可遗传的基因表达方式的改变,包含DNA甲基化、基因组印记、染色质组蛋白修饰以及非编码RNA调控等方式。基因组印记是目前生物医学领域研究的热点,基因组印记(genomic imprinting)决定性状的遗传不遵循孟德尔定律,表现为亲源依赖性的单等位基因表达,在胚胎的生长、发育中起重要作用。DNA甲基化是影响表观遗传机制的主要因素之一,与基因表达抑制、基因功能缺失有关。对印记基因的分离鉴定及其作用机制的研究结果表明DNA甲基化是基因组印记发生和维持的主要机制。越来越多的研究表明印记基因异常表达或印记状态改变是某些恶性肿瘤重要的分子生物学特征,有可能成为肿瘤早期诊断的指标及治疗的新靶点。SLC22A18(solute carrier family 22,member 18)亦称为TSSC5/ORCTL2/IMPT1/BWR1A,是位于人染色体11p15.5的父系印记基因,cDNA全长1.5 kb,蛋白分子量43KD,编码跨膜转运体相似蛋白,影响药物敏感性、细胞代谢。SLC22A18蛋白水平的调节可能是通过泛素化-蛋白酶体途径。近年来研究表明SLC22A18印记异常和基因变异与多种肿瘤相关如Wilm′S瘤、肝母细胞瘤、肺癌、肝细胞肝癌和乳腺癌,值得关注的是国外有报道SLC22A18在乳腺癌中突变率甚低,印记获得参与了乳腺癌的发生,说明表观遗传学改变可能参与了SLC22A18表达的调控。目前国内外尚未见关于SLC22A18在乳腺癌中的表达及其调控机制的系统研究,因此本课题目的是研究印记基因SLC22A18在乳腺癌中的表达及其遗传学和表观遗传学调控机制,重点阐明5′端CpG岛异常甲基化对SLC22A18基因表达调控的影响,为寻找新的乳腺癌诊断标记物及评估肿瘤的风险,发展新的治疗靶点提供研究基础。第一部分SLC22A18在乳腺癌组织中表达的研究目的研究印记基因SLC22A18在乳腺浸润性导管癌组织和乳腺良性病变中mRNA和蛋白水平的表达,分析SLC22A18的表达与乳腺癌临床病理特征之间的相关性,从而探讨SLC22A18基因在乳腺癌发生发展中的作用。方法应用实时荧光定量逆转录聚合酶链反应(Realtime RT-PCR)方法和免疫组织化学方法检测56例乳腺浸润性导管癌和对应癌旁乳腺组织,以及20例乳腺良性病变组织中SLC22A18 mRNA和蛋白的表达情况,并分析SLC22A18表达与乳腺癌临床病理特征之间的关系。结果SLC22A18在56例乳腺浸润性导管癌组织中mRNA表达量低于癌旁组织(z=-4.15,P＜0.01);56例乳腺浸润性导管癌组织中SLC22A18 mRNA表达量低于20例乳腺良性病变(z=-2.76,P＜0.01)。48例乳腺浸润性导管癌组织中SLC22A18 mRNA表达量低于8例早期浸润癌(z=-2.19,P＜0.05)。SLC22A18蛋白在20例乳腺良性病变、56例乳腺浸润性导管癌组织中表达阳性率分别为95%、60.7%,两组阳性表达率差异有统计学意义(x~2=8.21,P＜0.01)。SLC22A18在48例乳腺浸润性导管癌和8例早期浸润癌中蛋白阳性表达率差异无统计学意义(P＞0.05)。SLC22A18 mRNA和蛋白表达与患者年龄、肿瘤大小、组织学分级、TNM分期及淋巴结转移均无相关性(P＞0.05)。小结SLC22A18表达下调可能与乳腺癌的发生有关,有可能成为一个潜在的乳腺癌诊断的分子标记物。第二部分乳腺癌中SLC22A18基因突变、杂合性缺失和印记的研究目的研究印记基因SLC22A18在乳腺浸润性导管癌组织中的突变、杂合性缺失情况及其印记状态,探讨突变、杂合性缺失和印记改变对SLC22A18基因表达的影响。方法用聚合酶链式反应-单链构象多态性分析法(PCR-SSCP)、聚合酶链反应-限制性酶切片段长度多态性分析法(PCR-RELP)及DNA测序方法分析56例乳腺浸润性导管癌及对应癌旁乳腺组织中SLC22A18的突变、杂合性缺失及印记状态,分析SLC22A18基因突变、杂合性缺失和印记改变与基因表达之间的关系。结果PCR-SSCP电泳结果发现56例乳腺浸润性导管癌中有1例SLC22A18基因外显子9出现异常泳动条带,DNA测序结果显示第9外显子1087位胞嘧啶缺失,导致阅读框发生移码突变,1110位出现终止密码TGA;外显子7未出现异常泳动带,提示外显子7无突变发生。56例配对乳腺癌组织标本中,41%(23/56例)乳腺癌旁组织为杂合性个体,其对应癌组织有34%(19/56)为杂合性个体,因此有17.4%(4/23例)乳腺癌发生了杂合性缺失(LOH)。26.3%(5/19例)的乳腺癌组织发生了SLC22A18印记获得(GOI),与之相对应的远癌组织中也有10.5%(2/19例)也获得了印记;8例乳腺早期浸润癌组织中有2例获得了印记。4例发生杂合性缺失的乳腺癌组织和19例杂合子乳腺癌组织中SLC22A18 mRNA表达,两组间比较差异有统计学意义(P＜0.05);19例杂合子样本中,5例印记获得和14例无印记获得样本中SLC22A18 mRNA表达,两组间相比较差异有统计学意义(P＜0.05);杂合性缺失和印记获得与SLC22A18蛋白表达之间无相关性(P＞0.05)。小结乳腺癌组织中存在印记基因SLC22A18突变、杂合性缺失和印记获得,SLC22A18基因LOH和GOI与基因转录水平的表达有一定的相关性,可能参与了乳腺癌的发生,SLC22A18印记的重新获得可能是乳腺癌发生的早期表现,印记改变有可能成为乳腺癌早期诊断的指征。第三部分乳腺癌组织中SLC22A18基因启动子甲基化状态的研究目的研究乳腺浸润性导管癌组织中印记基因SLC22A18启动子区甲基化状态,探讨其对的SLC22A18 mRNA和蛋白表达的影响,及其与临床病理特征之间的关系。方法应用甲基化特异性聚合酶链反应(MSP)检测56例乳腺浸润性导管癌及对应癌旁组织中SLC22A18基因启动子区的甲基化状态。结果56例乳腺浸润性导管癌及对应癌旁组织中,SLC22A18启动子区的甲基化发生率分别为73.2%(41/56)和39.3%(22/56),差异有统计学意义(P＜0.01);56例乳腺浸润性导管癌中SLC22A18基因启动子区甲基化率与20例乳腺良性病变中甲基化率35%(7/20例)相比,差异有统计学意义(P＜0.01);56例乳腺浸润性导管癌中甲基化组SLC22A18 mRNA表达量低于非甲基化组(0.76±0.69比2.08±1.58,P＜0.01)。56例乳腺浸润性导管癌中甲基化组和非甲基化组SLC22A18蛋白阳性表达率有差别(51.2%比86.7%,P＜0.05)。5例获得印记的乳腺癌组织中,SLC22A18启动子区CpG岛均呈部分甲基化状态。SLC22A18基因启动子区甲基化频率与患者年龄、肿瘤大小、淋巴结转移、组织病理学分级及临床TNM分期无相关性(P＞0.05)。小结SLC22A18基因启动子甲基化与乳腺癌发生有一定的关联,SLC22A18基因表达下调与其启动子区CpG岛异常甲基化相关,启动子甲基化与印记状态有一定关联。第四部分5-aza-2′-deoxycytidine和Trichostatin A对乳腺癌细胞SLC22A18基因的表达、启动子甲基化及细胞增殖的影响目的为了进一步研究DNA甲基化是乳腺癌中SLC22A18表达调控的主要机制,用DNA甲基转移酶抑制剂5-aza-2′-deoxycytidine(5-aza-dc)和/或组蛋白去乙酰化酶抑制剂Trichostatin A(TSA)处理乳腺癌细胞株,检测SLC22A18基因表达及其启动子区CpG岛甲基化状态的变化,同时观察药物处理对乳腺癌细胞增殖和细胞周期的影响。方法用5-aza-dc和/或TSA处理乳腺癌细胞株,5-aza-dc 2.5μmol/L处理96小时,TSA 100ng/ml处理12小时,或5-aza-dc 84h和TSA12h联合处理。药物处理后,用WST-1法检测细胞增殖情况,流式细胞术检测细胞周期时相分布。Realtime RT-PCR和Western blot检测处理前后SLC22A18 mRNA和蛋白的表达情况,MSP检查药物处理前后SLC22A18基因启动子甲基化的状态,从而探讨SLC22A18基因启动子甲基化对其mRNA和蛋白表达的影响及5-aza-dc和TSA对细胞增殖和细胞周期的影响。结果MDA-MB-231乳腺癌细胞中存在SLC22A18基因启动子异常甲基化;5-aza-dc、5-aza-dc/TSA能逆转MDA-MB-231细胞中SLC22A18基因启动子的甲基化状态,使SLC22A18基因mRNA和蛋白表达增加,5-aza-dc/TSA联合作用明显;5-aza-dc和/或TSA后,可抑制乳腺癌细胞增殖,细胞周期阻滞于S期。小结SLC22A18基因表达与其启动子区CpG岛的异常甲基化呈负相关,DNA异常甲基化是SLC22A18基因表达下调的主要调控机制;5-aza-dc和TSA联合诱导可使去甲基化作用加强,恢复基因表达,还可抑制乳腺癌细胞增殖,影响细胞周期。第五部分染色质免疫沉淀技术研究DNMT3b和HDAC1与SLC22A18基因启动子区DNA相结合的情况目的应用染色质免疫沉淀(ChIP)技术分析DNA甲基转移酶DNMT3b和组蛋白去乙酰化酶HDAC1与SLC22A18基因启动子区DNA相结合的情况。方法5-aza-dc和/或TSA作用于乳腺癌细胞株MDA-MB-231后,用甲醛溶液交联固定蛋白-DNA复合体,利用DNMT3b和HDAC1抗体通过染色质免疫沉淀技术处理该复合体,纯化回收ChIP产物,并经PCR扩增出SLC22A18启动子区DNA片段,进而分析SLC22A18启动子区域与特异性蛋白DNMT3b和HDAC1相结合的情况。结果ChIP结果显示DNMT3和HDAC1与MDA-MB-231细胞中SLC22A18启动子DNA相结合,5-aza-dc/TSA和5-aza-dc可以明显抑制DNMT3b与SLC22A18启动子的结合,而TSA不抑制DNMT3b的活性。5-aza-dc/TSA和TSA可以抑制HDAC1与SLC22A18启动子的结合,而5-aza-dc不抑制HDAC1的活性。5-aza-dc和/或TSA不影响DNMT3b和HDAC1蛋白的表达。小结DNMT3b和HDAC1与SLC22A18启动子DNA相结合,参与形成抑制复合体,修饰组蛋白,改变染色体结构,阻止转录因子与其特异性识别位点结合,从而抑制SLC22A18转录和表达。第六部分SLC22A18基因对乳腺癌细胞增殖影响的初步研究目的通过细胞转基因技术,瞬时过表达SLC22A18,观察SLC22A18过表达对乳腺癌细胞MDA-MB-231生长和细胞周期时相的影响。方法构建pIRES2-EGFP-SLC22A18真核表达质粒;采用脂质体介导法,将SLC22A18基因瞬时转染体外培养的MDA-MB-231细胞,用Realtime RT-PCR、Western blot法鉴定基因表达情况,WST-1法检测细胞生长情况,流式细胞术观察细胞周期时相。结果成功构建pIRES2-EGFP-SLC22A18真核表达质粒,瞬时转染MDA-MB-231细胞后,SLC22A18蛋白表达增强,细胞增殖能力有所下降,与pIRES2-EGFP空载体细胞相比,pIRES2-EGFP-SLC22A18细胞S期比率下降。小结SLC22A18基因瞬时过表达可抑制乳腺癌细胞MDA-MB-231生长,细胞周期发生改变。结论本课题系统研究了印记基因SLC22A18在乳腺癌中的表达及其遗传学和表观遗传学调控机制。SLC22A18在乳腺癌中表达下调;SLC22A18在乳腺癌中突变率甚低;SLC22A18基因杂合性缺失和印记获得与其mRNA水平表达下降相关;启动子异常甲基化是SLC22A18在乳腺癌中低表达的重要调控机制;DNMT3b和HDAC1与SLC22A18启动子区DNA相结合,参与形成抑制复合体,影响SLC22A18的转录和表达;印记基因SLC22A18有可能成为一个潜在的乳腺癌诊断的分子标记物。创新点1.目前国内外尚未见关于SLC221A8在乳腺癌中的表达及其调控机制的系统研究,本课题较为全面、系统的研究了SLC22A18在乳腺癌中的表达及其遗传学和表观遗传学调控机制,证实印记基因SLC22A18在乳腺癌中表达下调,启动子异常甲基化是SLC22A18在乳腺癌中表达下调的主要调控机制。2.分析了SLC22A18在乳腺癌中低表达与印记状态改变相关,证实SLC22A18印记的重新获得可能是乳腺癌发生的早期表现,印记改变有可能成为乳腺癌早期诊断的指征。3.印记基因SLC22A18启动子异常甲基化和印记改变有可能成为一个潜在的乳腺癌诊断标记物。4.建立和应用染色质免疫沉淀技术,从转录水平分析了DNMT3b和HDAC1与SLC22A18启动子相结合后,导致染色质结构变化,抑制SLC22A18转录和表达。更多还原

【Abstract】 Breast cancer is the most common disease among women worldwide.Although China has the low morbidity of breast cancer,its yearly increasing rate is 1%-2% higher than the average rate in the world.Over the past ten years,the ever-increasing incidence has made breast cancer one of the most frequent malignancies among Chinese women.The precise molecular mechanism of breast cancer is not well understood.Many studies have provided evidence of genetic alterations in breast cancers such as point mutations,loss of heterozygosity(LOH),and homozygous deletions in many tumor suppressor genes,which are associated with carcinogenesis.More recently,it has also been reported that epigenetic plays an important role in the development and progression of human breast cancer.The field of epigenetic includes DNA methylation,genomic imprinting and chromatin remodelling.Genomic imprinting is a form of epigenetic inheritance that distinguishes maternal and patemal alleles. Genomic imprinting plays a critical role in fetal growth,cell proliferation and adult behavior.There are numerous reports of tumors showing imprinted genes also involved in carcinogenesis.It is now well established that DNA methylation is involved in regulating gene transcription repression.Evidences indicate that DNA methylation is the main mechanism of genomic imprinting.Recently,more and more study show the aberrant expression and the variation imprinting status may be a critical biomarker in carcinogenesis.SLC22A18(solute carrier family 22,member 18 ),a paternally imprinted gene is located in chromosome 11p15.5,also known as TSSC5,BWR1A,IMPT1 and ORCTL2,encodes an effiux transporter-like protein with ten transmembrane domains, whose regulations may contribute to drug sensitivity,cellular metabolism and growth. SLC22A18 mRNA expression is observed in the fetal kidney and liver,and also in multiple adult tissues such as the liver,kidney,prostate and colon.The protein is found on the apical membrane surfaces of the proximal tubules in human kidney.It has been speculated that it may be involved in resistance in export of genotoxic substances whose retention may increase the risk of tumor formation.More recently, TSSC5 has been shown to be a substrate for a conserved E3 ubiquitin ligase RING105, and the manipulation of RING 105 may assist in restoring TSSC5 function and normal growth control in cancer.Structural mutations,deletion and abnormal imprinting patterns of SLC22A18 have been identified in a number of sporadic tumors,including Wilm’s tumor,hepatoblastomas,hepatocelluar carcinomas and breast cancer.These resluts indicated that SLC22A18 may play a role in tumorigenesis.The molecular mechanisms that lead to inactivation of the SLC22A18 gene remain poorly understood.Because approximately 80%of breast tumors were diagnosed as infiltrating ductal carcinomas(IDCs),we searched for genetic alterations,mutation and allelic loss,and the methylation status of SLC22A18 gene in patients with IDCs in China.Part 1 Expresstion of imprinted gene SLC22A18 in infiltrating ductal breast carcinomaPurpose To investigate the expression of imprinted gene SLC22A18 in breast cancer and breast benign diseases.Then the expression and its clinical relevance in breast caner were explored.Methods Real-time quantitative reverse transcriptase-polymerase chain reaction(Realtime RT-PCR) and immunohistochemistry(IHC) was applied on 56 cases of infiltrating ductal breast carcinoma(IDC),56 cases of corresponding adjacent non-cancerous tissues and 20 benign breast tissues in order to detect mRNA and protein expression of SLC22A18 gene.Statistical analysis was carried out to analyze the correlation between SLC22A18 gene expression and various clinical parameters in these breast cancer patients.Results SLC22A18 mRNA expression in 56 IDC tissues was lower than that in all corresponding adjacent non-cancerous tissues(z=-4.15,P＜0.01).SLC22A18 mRNA expression was lower in breast cancer cases,when compared with that in benign cases(z=-2.76,P＜0.01).SLC22A18 mRNA expression in 48 IDCs was lower than that in 8 dural carcinoma in situ(part of IDC)(z=-2.19,P＜0.05).There was a decreased or completely diminished SLC22A18 protein expression in breast cancer.A significant difference of SLC22A18 protein expression was also observed in IDC and benign groups(P＜0.01).Neither mRNA nor protein expression of SLC22A18 gene correlated with clinicopathologic parameters such as age of patients, size of tumor,clinical stage,pathologic subtype,histologic grade or lymph node metastasis(P＞0.05).Conclusions Our results indicate that SLC22A18 expression is markedly down-regulated in IDC.Decreased expression of SLC22A18 gene may play an important role in the carcinogenesis of IDC.PartⅡMutation,loss of heterozygosity and genomic imprinting of SLC22A18 in human breast cancerPurpose To clarify the role of SLC22A18 in breast cancer,we examined the gene for mutation,loss of heterozygosity,and genomic imprinting of SLC2218 in IDC,and explore the relationship between mutation,deletion,genomic imprinting and SLC22A18 expression.Methods Single strand conformation polymorphism analysis of polymerase chain reaction products(PCR-SSCP),polymerase chain reaction-restricted fragments length polymorphism(PCR-RELP) and DNA sequencing were used to detect the mutation,deletion,and genomic imprinting of SLC2218 in 56 paired IDC tissues. Then the molecular mechanism of breast cancer was explored.Results Of the 56 paired IDC,only 1 showed abnormal SSCP band shift of exon9.DNA sequencing showed C deletion at 1087 locus,resulting in a stop codon TGA.No band shifts of exon 7 were found in 56 IDCs and corresponding adjacent non-cancerous tissues.We screened 56 paired samples and identified 23 heterozygotes of SLC22A18 in corresponding adjacent non-cancerous tissues,and 19 heterozygotes in IDCs.So loss of heterozygosity(LOH) occurred in 4(17.3%) of 23 informative IDCs.SLC22A18 showed a gain of imprinting(GOI) in 5/19(26.3%) IDCs.GOI was also demonstrated in 2/19(10.5%) corresponding adjacent non-cancerous tissues.2 of 8 dutal carcinoma in situ(part of IDC) also gained imprinting.In the specimens with SLC22A18 DNA heterozygote,SLC22A18 mRNA expression in 4 LOH of IDCs was lower than that in 19 heteozygotes in IDCs(P＜0.05).SLC22A18 mRNA expression in GOI samples was lower than that in 14 no GOI samples(P＜0.05).LOH and GOI did not correlat to SLC22A18 protein expression.Conclusions The phenomena of mutation,LOH and GOI of SLC22A18 were occurred in IDCs.LOH and GOI were related to the transcriptional expression.LOH and GOI of SLC22A18 may be involved in breast carcinogenesis and prognosis.PartⅢSLC22A18 promoter aberrant methylation in breast cancerPurpose To study the methylation status of imprinted gene SLC22A18 in IDCs, and the correlation between methylation status and clinical characteristics in IDCs. Further,to investigate the expression and promoter methylation in IDCs.Methods The methylation status at the promoter regions of SLC22A18 gene was examined by methylation-specific polymerase chain reaction(MSP) in the specimens of IDC from 56 patients.Results By employing MSP we determined the frequency of aberrant methylation for SLC22A18 in 56 IDCs samples and their corresponding adjacent non-cancerous tissues.The percentages of methylation for SLC22A18 in 56 IDCs were 73.2%and in the corresponding adjacent non-cancerous tissues,39.3%, respectively.The frequency of methylation of SLC22A18 in 56 IDCs was higher than that in the corresponding adjacent non-cancerous tissues(73.2%versus 39.3%,P＜0.01).The mRNA and protein expression of SLC22A18 gene in methylation group of IDCs was significantly reduced when compared with that of the unmethylation group of IDCs(P＜0.01,P＜0.05).The frequency of methylation of SLC22A18 was not correlated with clinicopathologic parameters such as age of patients,size of tumor, clinical stage,pathologic subtype,histologic grade or lymph node metastasis(P＞0.05).Conclusions The reduced expression of SLC22A18 was associated with aberrant DNA methylation in the promoter region.Promoter methylation was related to status of imprinting.Detection of promoter methylation is potentially usedful as epigenetic marker in tumor progress.PartⅣEffects on the growth inhibition of human breast carcinoma cell and reversion of SLC22A18 methylation by 5-aza-2’-deoxycytidine and Trichostatin APurpose In order to further study whether DNA methylation is the direct cause of aberrant SLC22A18 expression,breast cancer cell line MDA-MB-231 was treated with DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine(5-aza-dC) and histone deacetylase inhibitor Trichostatin A(TSA).Meanwhile,we observed the changes of cell growth and cell cycle after treatment.Methods Cell proliferation,cell cycle,the level of methylation and expression of SLC22A18 in breast cancer cell line MDA-MB-231 after treatment with 5-aza-dc and/or TSA was observed under WST-1 method,FCM,MSP,Realtime RT-PCR and Western blot.Results Treatment with 5-aza-dc alone or 5-aza-dc/TSA on breast cancer cell lineMDA-MB-231 could reverse aberrant hypermethylation of the SLC22A18 promoter and increase SLC22A18 mRNA and protein expression.Combination of 5-aza-dc and TSA could further reverse hypermethylation of the promoter and up-regulated the expression.The results suggesting that up-regulation of SLC22A18 expression was the direct effect of demethylation.We also found that the proliferation of breast cancer cells was suppressed and the cell cycle was arrested in S stage after treatment with 5-aza-dc and TSA.Conclusions SLC22A18 promoter aberrant methylation was associated with a decease its expression.Aberrant DNA methylation may be the main mechanism of down-regulation of SLC22A18 in IDC.The combination of 5-aza-dc and TSA could further reverse promoter methylation and increase gene expression.PartⅤThe relationship ofDNMT3b,HDAC1 and SLC22A18 promoter DNA by chromatin immunopreeipitationPurpose To establish effective chromatin immnuoprecipitation(CHIP) and identify the interaction between the chromatin DNA of MDA-MB-231 cells and DNMT3b,HDAC 1 protein by Chip analysis.Methods The MDA-MB-231 cells were treated by 5-aza-dc and/or TSA.Then the MDA-MB-231 cells were treated by formaldehyde so that a complex composed of protein and chromatin DNA could stably exit in vivo.The DNA-protein complex was precipitated with and an appropriate amount of anti-DNMT3b and HDAC1 antibody. The DNA-protein complex was eluted and purified to extract the specific DNA.The specific DNA was amplified using PCR.The PCR products were was loaded directly onto 1.5%agarose gels,stained with ethidium bromide and visualized under UV illumination.Results ChIP assay performed with specific DNMT3b and HDAC1 antibody showed that DNMT3b and HDAC1 were associated with the SLC22A18 promoter in MDA-MB-231 cells.Treatment with 5-aza-dc/TSA and 5-aza-dc,but not TSA, reduced the level of association of DNMT3b with SLC22A18 promoter.Thus, recruitment of DNMT3b to methylated SLC22A18 promoter deceases upon demethylation of the promoter.Chip assay performed with specific HDAC 1 antibody showed that HDAC1 was associated with the SLC22A18 promoter in MDA-MB-231 cells.The association was significantly reduced by TSA or TSA/5-aza-dc treatment and was unaffected by 5-aza-dc alone.The DNMT3b and HDAC1 protein expression of SLC22A18 was unaffected by 5-aza-dc and/or TSA treatment.Conclusions DNMT3b and HDAC1 forming a complex,which bound to methylated CpG sites and made chromatin structure from open to close,blocked transcriptional factors to bind to theirs specific binding sites and then suppress SLC22A18 expression.PartⅥThe proliferation of breast cancer cells after transfection of SLC22A18Purpose To elucidate the effect of SLC22A18 overexpression on the breast cancer cells.Methods Constructed vector plRES2-EGFP-SLC22A18,then the vector was transiently transfected into MDA-MB-231 cells.Cell proliferation,cell cycle and expression of SLC22A18 were observed under WST-1 method,FCM,Real-time RT-PCR and Western blot.Results The plasmid plRES2-EGFP- SLC22A18 was constructed successfully. After transiently transfected the vector into MDA-MB-231,the mRNA and protein expression of SLC22A18 were decreased.The ability of cell proliferation was down-regulated.The cell cycle changed,the percentage of S stage was lower than the control.Conclusions The proliferation of breast cancer cell line MDA-MB-231 was inhibited after transiently transfected the vector plRES2-EGFP-SLC22A18.Conclusions1.Decreased expression of SLC22A18 gene may play an important role in the carcinogenesis of IDC.2.The phenomena of mutation,LOH and GOI of SLC22A18 were occurred in IDCs. LOH and GOI were related to the transcriptional expression.LOH and GOI of SLC22A18 may be involved in breast carcinogenesis and prognosis.3.The reduced expression of SLC22A18 was associated with aberrant DNA methylation in the promoter region.Promoter methylation was related to status of imprinting.Detection of promoter methylation is potentially usedful as epigenetic marker in tumor progress.4.Aberrant DNA methylation may be the main mechanism of down-regulation of SLC22A18 in IDC.5.DNMT3b and HDAC1 were associated with the SLC22A18 promoter in MDA-MB-231 cells.DNMT3b and HDAC1 forming a complex,which bound to methylated CpG sites and made chromatin structure from open to close,blocked transcription factors to bind to theirs specific binding sites and then suppress SLC22A18 expression.6.The proliferation of breast cancer cell line MDA-MB-231 was inhibited after transiently transfected the vector plRES2-EGFP- SLC22A 18.Originalities of this workFor the first time,the genetic and epigenetic mechanisms involved in imprinting gene SLC22A18 down-regulation in breast cancer were studied.The reduced expression of SLC22A18 in IDC was associated with aberrant DNA methylation in the promoter region.DNA methylation and GOI of SLC22A18 may be involved in breast carcinogenesis and prognosis.ChIP assay was established.DNMT3b and HDAC1 forming a complex,which bound to methylated CpG sites and made chromatin structure from open to close,blocked transcription factors to bind to theirs specific binding sites and then suppress SLC22A18 expression.更多还原