【作者】 王晓；

【导师】 孙靖中；

【作者基本信息】 山东大学 ， 外科学， 2008， 博士

【摘要】 乳腺癌是一种常见的恶性肿瘤,抑癌基因和癌基因的异常表达是乳腺癌发生的重要机制。WWOX(WW domain containing oxidoreductase)基因是跨越了整个常见染色体脆性位点FRA16D的一个新的抑癌基因,2000年由Bednarek等应用鸟枪基因测序技术结合对感兴趣对应的转录子进行分离并分析的方法鉴定出的一个新基因,位于染色体16q23.3-24.1。研究发现,多种癌细胞中出现WWOX外显子5-8或6-8的异常转录,提示WWOX是另一种具抑制肿瘤生长的基因,在基因组和转录水平影响这个基因的异常可能与致癌性有关。研究发现在乳腺癌、卵巢癌和非小细胞性肺癌中存在着WWOX的表达降低,WWOX基因在多种肿瘤组织及细胞系中出现频发杂合性丢失、mRNA异常转录子及WWOX蛋白表达异常等证明WWOX属于抑癌基因。WWOX蛋白含有414个氨基酸,其氨基末端有两个WW结构域,而WW结构域的功能主要与蛋白之间的相互作用有关。在WWOX蛋白的中心部位有一短链氧化还原酶功能域(short-chain dehydrogenase/reductase domain,SRD),SRD具有特异性酶的功能。因此,WWOX可能在雌二醇和雌激素受体相互作用的调节中发挥了作用。抑癌基因在基因组和转录水平的异常表达是肿瘤发生的常见原因,WWOX基因失活机制可能有:①WWOX等位基因杂合子缺失及纯合子缺失;②WWOX基因mRNA转录缺失或严重降低,导致表达缺失或降低;③DNA调控区CpG岛甲基化。WWOX基因与脆性三联组氨酸(FHIT)基因的mRNA表达有相关性。文献报道胰腺癌中WWOX基因的表达缺失与DNA调控区CpG岛的甲基化有关。近年来肿瘤基因甲基化的研究之所以受到肿瘤研究者的重视,原因在于:(1)基因调控区域的CpG岛的甲基化与各种基因(包括抑癌基因)表达的调控异常密切相关;(2)特异性CpG岛的甲基化容易在癌症患者个体以及高危人群的体液和组织中检测到,因此特异性基因甲基化模式的研究有助于肿瘤诊断和预后预测;(3)甲基化可以被特异性抑制剂所逆转,提示这一类型的抑制剂可以应用于临床治疗。研究发现肼苯哒嗪能作为去甲基化药物恢复T淋巴细胞功能相关抗原、诱导出P16基因启动子甲基化的乳腺癌细胞系的P16基因mRNA和蛋白质表达,在肿瘤治疗中显示出良好的应用前景。本研究目的:①通过检测乳腺癌和癌旁乳腺组织中WWOX蛋白的表达,并与ER、PR、HER-2/neu、绝经状态及临床分期等临床病理学指标进行分析;通过逆转录酶联聚合反应(RT-PCR)检测乳腺癌MDA-MB-231、MCF-7细胞系以及乳腺癌标本WWOX基因mRNA的表达;②应用甲基化特异性PCR(methylation specific PCR,MSP)检测乳腺癌中WWOX基因表达异常与DNA调控区(启动子及第一外显子)CpG岛甲基化状态的关系;③应用肼苯哒嗪对MDA-MB-231、MCF-7细胞进行处理,以5-杂氮-2′-脱氧胞苷(5-aza-2’-deoxycytidine,5-Aza-CdR)作为阳性对照,观察肼苯哒嗪对于WWOX基因CpG岛去甲基化、mRNA表达及癌细胞增殖活性的作用。现将主要内容简要介绍如下。第一部分乳腺癌WWOX蛋白表达及其临床意义目的检测乳腺癌组织及细胞株中WWOX蛋白表达,并分析其与ER、绝经状态及临床分期等指标间的关系。方法应用免疫组织化学SP法检测56例乳腺正常组织、12例DCIS和87例乳腺癌组织WWOX蛋白的表达,并与雌激素受体(ER)、孕激素受体(PR)、HER-2/neu、绝经状态及肿瘤分期等临床病理学资料进行分析。应用免疫细胞化学法检测MDA-MB-231、MCF-7细胞中WWOX蛋白的表达。结果1.乳腺癌组织中WWOX蛋白表达显著降低(62.1%),与乳腺正常组织(28.6%)相比,差异有统计学意义(P=0.001);2.乳腺癌组织WWOX表达缺失与ER状态关系密切,39.2%的ER阴性病例和28.8%的ER阳性病例中WWOX蛋白表达缺失,差异有统计学意义(P=0.026);3.乳腺癌WWOX蛋白表达与绝经状态相关,20.3%的绝经前病人和57.2%的绝经后病人中WWOX蛋白表达缺失,差异有统计学意义(P=0.003);4.晚期乳腺癌病人WWOX表达降低或缺失的比例增高,Ⅰ、Ⅱ、Ⅲ期病人中WWOX蛋白表达缺失的比例分别为23.1%、28.6%和46.2%,差异有统计学意义(P=0.001);5.MDA-MB-231细胞WWOX蛋白染色明显降低,而MCF-7细胞呈现强染色;6.WWOX蛋白表达与PR、HER-2/neu及腋窝淋巴结转移等指标的关系无统计学意义(P＞0.05)。结论乳腺癌中广泛存在着WWOX蛋白的表达缺失,其异常表达与ER状态、绝经状态和肿瘤临床分期关系密切,提示WWOX基因可能通过激素受体信号途径在乳腺癌的发生和发展中发挥作用。乳腺癌MDA-MB-231细胞WWOX蛋白低表达,而MCF-7细胞高表达。第二部分乳腺癌组织和细胞株WWOX基因调控区(启动子和第一外显子)CpG岛甲基化状态的研究目的探讨乳腺癌组织和MDA-MB-231、MCF-7细胞株中WWOX基因调控区(启动子和第一外显子)CpG岛甲基化状态,并分析WWOX基因mRNA表达与CpG岛甲基化状态的关系。方法选取乳腺癌细胞株MDA-MB-231、MCF-7细胞、20例乳腺癌组织和10例癌旁组织为研究对象,通过WWOX基因DNA提取,应用CpGenomeDNA修正盒进行DNA亚硝酸盐修饰,采用甲基化特异性PCR(methylation specificPCR,MSP)方法检测乳腺癌组织和细胞系WWOX基因CpG岛甲基化状态;通过逆转录聚合酶链反应(RT-PCR)技术检测WWOX基因mRNA表达,探讨WWOX基因CpG岛甲基化与mRNA表达的关系。结果1.乳腺癌癌旁组织中WWOX基因DNA调控区CpG岛无甲基化条带表达;2.20例乳腺癌组织中,11例中出现WWOX基因启动子CpG岛甲基化扩增,甲基化率为55%,其中4例为完全甲基化(仅甲基化引物扩增出目的基因),7例为部分甲基化(甲基化引物和非甲基化引物均扩增出目的基因)。9例中出现WWOX基因第一外显子CpG岛甲基化扩增,甲基化率为45%,其中4例为完全甲基化,5例为部分甲基化;3.乳腺癌组织和癌旁组织中WWOX基因mRNA相对含量的平均值分别为1.18±0.11、2.1±0.12,差异有统计学意义(P＜0.05);4.WWOX基因调控区CpG岛甲基化的乳腺癌组织中WWOX mRNA表达显著降低,与癌旁组织相比,差异有统计学意义(P＜0.001);5.WWOX基因调控区CpG岛甲基化与非甲基化的乳腺癌组织中mRNA表达水平分别为0.62±0.08、1.81±0.21,差异有统计学意义(P＜0.01);6.MDA-MB-231细胞WWOX基因启动子、第一外显子CpG岛MSP扩增出阳性条带,而非甲基化PCR扩增阴性;MBA-MD-231细胞中,WWOX mRNA表达显著降低(0.03±0.02);7.MCF-7细胞未出现WWOX基因CpG岛甲基化阳性条带,而非甲基化扩增阳性。WWOX mRNA高表达(1.52±0.14)。结论乳腺癌组织和细胞系存在着不同程度的WWOX基因DNA调控区(启动子和外显子)CpG岛甲基化,是导致WWOX基因表达缺陷的原因之一。WWOX基因调控区CpG岛甲基化的乳腺癌组织WWOX mRNA表达明显降低,提示DNA甲基化是乳腺癌WWOX基因mRNA表达缺陷的重要机制。第三部分肼苯哒嗪对乳腺癌细胞WWOX基因CpG岛去甲基化作用的实验研究目的探讨肼苯哒嗪能否发挥去甲基化作用,诱导乳腺癌MDA-MB-231、MCF-7细胞mRNA及蛋白表达,并抑制细胞增殖。方法肼苯哒嗪处理MDA-MB-231、MCF-7细胞,以5-Aza-CdR作为阳性对照,MSP检测WWOX基因CpG甲基化状态;RT-PCR分析药物处理组、对照组WWOX基因mRNA及蛋白表达变化;采用细胞计数法绘制细胞生长曲线,噻唑蓝(MTT)法测定细胞抑制率,分析肼苯哒嗪处理前后乳腺癌细胞的生长变化。结果1.MDA-MB-231细胞WWOX基因启动子、第一外显子甲基化PCR扩增阳性,非甲基化PCR扩增阴性;经10μmol/L肼苯哒嗪处理后,MDA-MB-231细胞WWOX基因MSP不能扩增出阳性条带,而非甲基化扩增阳性;WWOX基因mRNA表达明显高于对照组,差异有统计学意义(P＜0.001);WWOX蛋白表达增强;细胞的增殖速度减慢,对数生长期均延迟24h,与对照组相比差异有统计学意义(P＜0.001)2.1.0,2.0,4.0,6.0μmol/L浓度的5-Aza-CdR处理后,WWOX基因MSP亦不能扩增出阳性条带,非甲基化扩增阳性;不同浓度5-Aza-CdR处理后,MDA-MB-细胞WWOX基因mRNA表达明显升高,但WWOX mRNA表达相对值与5-Aza-CdR的浓度无相关关系;细胞的增殖速度减慢,与对照组相比差异有统计学意义(P＜0.001)。3.肼苯哒嗪及5-Aza-dc处理前后,MCF-7细胞WWOX基因启动子和第一外显子CpG岛非甲基化扩增阳性,无甲基化扩增;WWOX mRNA表达差异无统计学意义(P＞0.05);细胞生长无显著性差异(P＞0.05)。结论肼苯哒嗪可以发挥去甲基化作用,诱导MDA-MB-231细胞WWOX基因mRNA及蛋白表达,并抑制细胞增殖活性。1.0、2.0、4.0、6.0μmol/L等浓度的5-Aza-CdR处理MDA-MB-231细胞后,在去甲基化、恢复WWOX基因mRNA和蛋白表达以及降低癌细胞增殖等方面作用无显著性差异。乳腺癌中广泛存在着WWOX基因的表达缺失,其异常表达与ER状态、绝经状态和肿瘤临床分期关系密切,提示WWOX基因可能是通过激素受体信号途径在乳腺癌的发生和发展中发挥作用。乳腺癌组织中存在不同程度的WWOX基因启动子和第一外显子CpG岛的完全甲基化和部分甲基化,且WWOX基因mRNA表达降低,提示CpG岛甲基化是乳腺癌WWOX基因mRNA表达缺陷的重要机制。MDA-MB-231细胞中,WWOX基因启动子和第一外显子CpG存在着完全甲基化,WWOX基因mRNA及蛋白表达明显降低。MCF-7细胞无WWOX基因CpG岛甲基化,WWOX基因mRNA及蛋白高表达。10μmol/L肼苯哒嗪和1.0、2.0、4.0、6.0μmol/L的5-Aza-CdR对MDA-MB-231细胞可以起到去甲基化作用,处理后WWOX基因启动子和第一外显子CpG岛甲基化扩增条带消失,WWOX基因mRNA重新表达,WWOX蛋白染色显著增强,且细胞增殖活性明显降低。我们的研究证明DNA启动子和第一外显子CpG岛甲基化是WWOX基因表达缺失的重要机制,肼苯哒嗪可以恢复MDA-MB-231细胞WWOX基因mRNA表达并抑制细胞增殖,有望成为新的乳腺癌治疗方法。更多还原

【Abstract】 The WWOX（WW domain containing oxidoreductase）gene is a tumor suppressor gene that is detected in chromosome 16q,spanning the common fragile site FRA16D. Aberrations of WWOX at either genome and expression level or both were common in various neoplasms including breast,ovarian,bladder,esophageal and prostate carcinomas.WWOX proteins contain two WW domains in the NH₂-terminal region and a short chain dehydrogenase/reduetase（SDR）domain in the central region of the protein.The WW domains are involved in protein-protein interactions.While the WWOX SDR domain encode an amino acid sequence homologous to steroid oxidoreductases,WWOX expression level in normal tissue was higher in hormonally regulated tissues,such as testis,prostate,and ovary,suggesting that WWOX may play an important role in hormones regulated cancers and that WWOX protein may be an enzyme involved in steroid metabolism.Usually CpG islands are located in the promoter,exon 1 and 3’ side of DNA of tumors.Expression of many tumor suppressor genes is down-regulated in cancer by epigenetic mechanisms,and CpG-rich islands of gene promoter regions are frequently methylated in cancers.A role for epigenetic mechanisms of WWOX transcriptional regulation was suggested for hemopoietic and pancreatic neoplasms, and WWOX promoter methylation status was associated with lack of expression. Studies indicate that aberrant transcriptions and expression of tumor suppress gene contribute to the carcinogenesis of breast cancer.The potential mechanisms include allelic loss and/or loss of homozygosity,dysfunction of transcription of WWOX mRNA,and hypermethylation of CpG islands DNA regulatory sites.WWOX gene maps at the location of FRA16D,one of the most active common chromosomal fragile sites.The WWOX gene is similar to the FHIT gene in the both genes encompass very active common fragile sites,both genes show frequent allelic loss and/or loss of homozygous deletion region in several human cancers,and both frequently show aberrant transcripts.A recent study showed WWOX and FHIT expression was strongly correlated,suggesting that genes at common fragile sites are likely to be coordinately inactivation in cancer,and promoter hypermthylation including the position of site-specific methylation of WWOX gene may play an impotant role in pancreatic tumor development.The renaissance of methylation studies has generated considerable interests for cancer researchers because:（1）methylation of CpG islands in gene regulatory regions,in combination with chromatin remodeling,is involved in downregulation of expression of genes,including tumor suppressor genes;（2）methylation of specific CpG islands in easily detected in tissues and body fluids of individuals with cancer, or at high risk for cancer development,so that specific gene methylation patterns can be useful in diagnosis or prognosis;and（3）epigenetic and chromatin remodeling marks can be reversed by specific agents or inhibitiors,suggesting such inhibitors as therapeutic agents.Methylation of regulatory regions of many genes has been reported in cancer cells,but which of these methylation marks will be most useful in diagnostic or prognostic clinical trials,or as surrogate markers in preclinical/clinical prevention and therapy trials of specific cancers,is only beginning to be defined.In the present study,WWOX protein expression pattern was analyzed in patients with normal breast and in those with breast carcinoma.We correlated WWOX protein expression with staging of breast carcinoma and with a few clinicopathological parameters as well.And this study aimed to determine if DNA methylation is a mechanism of WWOX（WW domain containing oxidoreductase）inactivation in breast cancer and MAB-MD-231,MCF-7 cell line.The expression of WWOX mRNA was detected with RT-PCR in breast cancer cell lines and tissues.Methylation specific PCR（MSP）was used to check whether it was methylated in the promoter and exon 1 CpG island of WWOX in MDA-MB-231,MCF-7 cell lines and tissues.Furthermore, the effect of hydralazine on demetlrylation of WWOX gene regulatory region CpG islands was evaluated,and 5-Aza-dcA was used as positive control.Part 1 Expression of WWOX protein in breast cancer and cell linesObjective To determine the expression of WWOX protein in breast cancer tissues and cells,and evaluate the association between WWOX expression and clinicopathological features.Methods Expression of WWOX was determined by immunohistochemical（IHC） staining on 155 tissues which was comprised of specimens of 56 normal breast tissurs,12 ductal carcinoma in situ（DCIS）and 87 primary invasive carcinoma. Furthermore,expression of WWOX protein was evaluated by IHC on breast cancer cell lines,MDA-MB-231 and MCF-7 cells.Results Decreased or lost WWOX expression was observed more frequently in invasive tumor（62.1%）than control（28.6%）（P=0.001）.82.1%of ER negative tumors and 52.5%of ER positive tumors showed decreased or lost WWOX expression（P=0.026）.22.4%of premenopausal tumors were completely negative for WWOX expression versus 51.8%for postmenopausal breast carcinomas（P=0.024）. Furthermore,23.1%of StageⅠ（6/26）,28.6%of StageⅡ（10/35）and 46.2%of StageⅢ（12/26）tumors showed negative WWOX expression（P=0.001）. Immunohistochemical analysis showed a highly reduced WWOX staining in MDA-MB-231 cells and a moderately reduced WWOX reaction in MCF-7 cells.Conclusions Lost WWOX expression is a common event in breast cancer.The associations of WWOX expression with ER status,menopausal status and clinical stage of breast cancer respectively support the hypothesis that WWOX play a role in carcinogenesis and development of breast cancer in a pathway or pathways involved sex steroid metabolism.Part 2 Methylation of the WWOX promoter and exon 1 CpG islands in breast cancer tissue and breast cancer cellsObjective To evaluate CpG islands methylation status of WWOX gene in breast cancer and anlysis the association between methylation and WWOX mRNA expression.Methods The expression of WWOX mRNA was detected with RT-PCR in breast cancer cell lines and tissues.Methylation specific PCR（MSP）was used to check whether it was methylated in the promoter and exon 1 CpG island of WWOX in MDA-MB-231,MCF-7 cell lines and tissues,and normal breast tissues were used as control.Results The breast cancer cell lines MDA-MB-231 and specimens of breast cancers were found methylated in WWOX DNA promoter and exon 1 CpG island.In breast cancer specimens,methylation rate of promoter and exon 1 CpG island of WWOX gene was 55%and 45%respectively.Whereas,WWOX CpG islands of normal mammary tissues were completely unmethylated in all cases.CpG islands of WWOX promoter and exon 1 were methylated in MDA-MB-231 cell line but not MCF-7 cell.The methylated cell lines and breast cancer tissues expressed decreassed WWOX mRNA.DNA CpG islands methylation contributes to the tumor-specific silencing of WWOX mRNA expression.The expression of WWOX mRNA was decreased significantly in MDA-MB-231 cells and partly due to the methylation of WWOX CpG islands.Conclusions The disfigurement of WWOX gene plays an important role in the carcinogenesis of breast cancer,and this alteration is partially caused by methylation of WWOX DNA regulatery region CpG islands. Part 3 Effect of hydralazine on demethylation of WWOX gene promoter and exon 1 in breast cancer cellsObjective To evaluate the effect of hydralazien on demethylation of WWOX gene promoter and exon 1 in breast cancer cellsMethods The demethylating agent,hydralazine,was used to treat methylated breast cancer cells and then,breast cancer cell growth was measured in vivo,and 5-Aza-CdR was used as positive control.The expression of WWOX mRNA was detected with RT-PCR in breast cancer cell lines before and after demethylating treatment,and WWOX protein expression was evaluated by immunohistochemical staining. Furthermore,to study the effects of restoration of WWOX expression in breast cancer cells,cell proliferation was evaluated.Results After hydralazine treatmentm WWOX CpG islands of MDA-MB-231 cells were unmethylated.Hydralazine could reactivate the expression of WWOX mRNA in methylated MDA-MB-231 cells.Restoration of WWOX expression led to suppression of growth of Wwox-deficient breast cancer-derived cells,through activation of the intrinsic caspase pathway,but did not affect growth of Wwox-sufficient MCF7 cells.Conclusions Hydralazine could effectively cause demethylation and inhibit the growth of MDA-MB-231 cells by reactivation the gene transcription silenced by aberrant hypermethylation.There are no significant differences in the demethylation of methylated MDA-MB-231 cells,restoration of WWOX mRNA expression,and suppression of the growth of cell lines for 1.0μmol/L,2.0μmol/L,4.0μmol/L, 6.0μmol/L 5-Aza-CdR.Summary WWOX gene is a candidate tumor suppressor gene located on chromosome 16q23.3-24.1.Abnormalities of WWOX at genomic and/or expression levels have been reported in numerous neoplasias including breast cancer.Lost WWOX expression is a common event in breast cancer.The associations of WWOX expression with ER status,menopausal status and clinical stage of breast cancer respectively supported the hypothesis that WWOX played a role in carcinogenesis and development of breast cancer in a pathway or pathways involved sex steroid metabolism.The disfigurement of WWOX gene plays an important role in the development of ovarian cancer,and this alteration is partially caused by methylation of WWOX DNA.MDA-MB-231 was found methylated by MSP in WWOX DNA promoter and exon 1 CpG island and methylation rate of the promoter and exon 1 CpG islands was 55%and 45%in breast cancer specimens respectively.The methylated cell lines and breast cancer tissues expressed decreased WWOX mRNA. The expression of WWOX mRNA was decreased significantly in MDA-MB-231breast cancer cells.The WWOX mRNA decrease was partly due to the methylation of WWOX CpG islands.Hydralazine and 5-Aza-CdR could reactivate the expression of WWOX mRNA in methylated MDA-MB-231 cells.Restoration of WWOX expression led to suppression of growth of WWOX-deficient breast cancer-derived cells,through activation of the intrinsic caspase pathway,but did not affect growth of WWOX-sufficient MCF7 cells.The finding that the WWOX regulatory region was methylated in MDA-MB-231 cells suggested that WWOX might be restored in breast cancer cells by hydralazine treatment.The cardiovascular drugs have a promising tumor suppressor-reactivating action and could pontentially be used in clinic as an anticancer treatment,mostly likely to increase the efficacy of current biological or chemotherapeutic treatments.更多还原