【作者】 贺黉裕；

【导师】 秦新裕；

【作者基本信息】 复旦大学 ， 外科学， 2010， 博士

【摘要】 背景：基因组印记是指来自父方和母方的一对等位基因,其中一方发生DNA修饰导致单等位基因表达,发生修饰的基因不表达,未修饰的基因表达。印记基因相当于功能上的单倍体,仅一次突变就使该基因失活,从而导致肿瘤。SLC22A18是位于人类染色体1 1p15.5的父源印记基因,影响细胞生长、胚胎发育。印记基因发生突变或印记异常后使抑癌印记基因活性丧失,促癌印记基因双等位基因表达,基因产物成倍增加,细胞增殖过度促使肿瘤形成,促进肿瘤发展。SLC22A18是成人肺的肿瘤抑制基因和胎儿肾印记性肿瘤抑制基因。本试验的目的是研究SLC22A18基因与乳腺癌生物学行为的相关性,测定SLC22A18在乳腺良性疾病及乳腺癌组织中的表达情况,探讨其与乳腺癌预后的关系。第一部分不同转移力乳腺癌细胞株的SLC22A18表达差异及侵袭能力差异目的：研究SLC22A18在不同转移力的乳腺癌细胞株MDA-MB-231、MCF-7中表达差异,评估此两种细胞株的侵袭能力差异。方法：应用荧光定量逆转录聚合酶链反应方法和蛋白质印记方法检测MDA-MB-231细胞株和MCF-7细胞株的RNA和蛋白质的表达。用transwell方法评估此两种细胞株的侵袭能力。结果：MDA-MB-231细胞和MCF-7细胞,SLC22A18 RNA和蛋白质表达均有差异。高转移的乳腺癌细胞株MDA-MB-231 SLC22A18表达少,穿过膜的细胞数多,侵袭能力强；低转移的乳腺癌细胞株MCF-7 SLC22A18表达多,穿过膜的细胞数较少,侵袭能力弱。t值为21.0,P=0.004。小结：印记基因SLC22A18可能作为一个抑癌基因影响乳腺癌的侵袭。第二部分SLC22A18过表达稳转细胞株的鉴定及SLC22A18过表达后对细胞侵袭能力的影响目的：建立SLC22A18过表达稳转细胞株(U-231,U-7),并对其进行鉴定。比较稳转株与未处理细胞株侵袭能力(MDA-MB-231, MCF-7)的差异性。方法：SLC22A18过表达稳转细胞株为复旦大学法医学实验室提供。应用蛋白质印记方法鉴定稳转株蛋白质表达情况。用transwell方法评估此稳转细胞株和未处理细胞株侵袭能力的变化。结果：U-231稳转株与对照组MDA-MB-231相比,其SLC22A18蛋白表达明显上调,表达有差异。U-7稳转株与对照组MCF-7相比,其SLC22A18蛋白表达明显上调,表达有差异。高转移力乳腺癌细胞株在SLC22A18表达上调(U-231)后与未处理的MDA-MB-231相比,穿过膜的细胞数减少,分别为25.33个和59.33个,t值为12.424,P值为0.000,差异有统计学意义。小结：U-231,U-7稳转株构建成功,SLC22A18表达上调后,乳腺癌细胞的侵袭能力减弱。在两种不同转移力的细胞株上都有体现。SLC22A18作为一个抑癌基因,上调其表达后,抑癌功能增强,减弱了肿瘤的侵袭能力。第三部分乳腺肿瘤组织中SLC22A18表达水平与临床病理学特征相关性目的：分析乳腺癌的临床病理学特征与SLC22A18蛋白表达量的关系。方法：收集复旦大学附属中山医院普外科2005年1月-2008年3月间住院的194例乳腺肿瘤石蜡标本,其中良性肿瘤38例,乳腺癌156例。用免疫组化法检测SLC22A18蛋白的表达,用计算机图像分析软件检测计算肿瘤标本中SLC22A18蛋白表达的相对量。根据患者病理学特征进行分组。采用软件SPSS17.0对数据进行统计分析,评价SLC22A18表达与乳腺癌生物病理学特征的相关性。结果：1)乳腺良性肿瘤中SLC22A18表达水平明显高于乳腺癌组织。2)SLC22A18表达水平与乳腺癌患者的患病年龄无关。3)SLC22A18表达水平与乳腺癌肿瘤大小呈负相关。肿瘤直径小于2cm组的SLC22A18表达水平高于肿瘤直径2-5cm组和大于5cm组,肿瘤直径2-5cm组的SLC22A18表达水平高于大于5cm组,差异有统计学意义。4)无脉管内癌栓的癌组织中SLC22A18表达水平高于有脉管内癌栓组。5)SLC22A18在有淋巴结转移的乳腺癌中表达水平较低。以乳腺癌淋巴结转移个数(pN区域淋巴结病理分期)分组,无淋巴结转移组的SLC22A18表达水平高于淋巴结转移1-3个组和10个以上组,差异有统计学意义,有淋巴结转移的各组间肿瘤组织的SLC22A18表达水平差异无统计学意义。6)以乳腺癌患者的雌孕激素受体和HER-2的免疫组化指标为分组指标,ER,PR及HER-2各自的分组中的SLC22A18表达水平差异无统计学意义。7)乳腺癌SLC22A18表达水平与肿瘤分化程度、肿瘤类型无明显关系。8)SLC22A18表达水平随临床TNM分期的增加而递减。结论：乳腺良性肿瘤组织中SLC22A18表达水平明显高于乳腺癌,且随着乳腺癌的临床分期和恶性程度的增高,SLC22A18的表达明显下降,并且在癌发生淋巴转移后SLC22A18表达下降更明显。第四部分SLC22A18表达水平在乳腺癌预后价值的研究目的：研究印记基因SLC22A18是否与乳腺癌患者术后复发转移相关,鉴定SLC22A18是否能作为一个独立的预后因子预测乳腺癌患者的复发转移。方法：对所有石蜡标本患者进行术后随访,记录首次出现复发转移的时间、无病生存时间和总生存时间。以免疫组化所测得的SLC22A18表达的相对量的中位数将所有病例分组。采用软件SPSS17.0统计分析。用Kaplan-Meier方法分析患者复发转移的情况；用log-rank检验比较患者无病生存分布。用寿命表法估计各分组患者的1年、2年的无病生存率。COX模型进行多因素回归分析,辨认预测预后的因素。结果：生存分析Kaplan-Meier累积生存函数图和log-rank检验表明SLC22A18低表达组的术后无病生存时间少于高表达组。Cox Regression风险比例模型分析SLC22A18低表达组和高表达组的术后复发转移的相对危险度为2.624：1。COX模型多因素回归分析结果提示SLC22A18分组和组织学分级与乳腺癌复发转移相关,SLC22A18低表达组复发风险是高表达组的2.624倍,组织学分级高组复发风险是组织学分级低组的2.065倍。小结：乳腺癌原发灶的SLC22A18表达水平与患者的预后相关,SLC22A18高表达的乳腺癌患者术后的复发转移风险较小。SLC22A18作为抑癌基因参与乳腺癌的发病过程,检测SLC22A18的表达有助于乳腺癌预后的判定。更多还原

【Abstract】 Background SLC22A18, also known as/IMPT1/BWR1A/TSSC5, is located in the region of human chromosome 11p15.5. SLC22A18 encodes an efflux transporter-like protein with 10 transmembrane domains, whose regulation may affect drug sensitivity, cellular metabolism and growth. Human chromosome 11 p15.5 is of interest because it is frequently lost in a wide variety of tumors, including Wilms’tumor, lung cancer, hepatocarcinoma cells and breast cancer, suggesting that one or more tumor suppressor gene map to this region. The aims of this study were to evaluate the relationship of SLC22A18 expression with clinicopathologic features and investigate the prognostic value of SLC22A18 expression in breast cancer after surgery.Part 1 Differential SLC22A18 expression and invasion abilities in 2 breast cancer cell lines MDA-MB-231 and MCF-7Purpose:To investigate the differential SLC22A18 expression in 2 breast cancer cell lines MDA-MB-231 and MCF-7 and to evaluate their invasion abilities.Methods:Real-time quantitative reverse transcriptase-polymerase chain reaction(Realtime RT-PCR) and western blot was applied on 2 breast cancer cell lines. Transwell was employed to evaluate the differential invasion abilities of 2 breast cancer cell lines.Results:Distinctive expression of SLC22A18 was observed in 2 breast cancer cell lines MDA-MB-231 and MCF-7. The difference was significant. Lower expression of SLC22A18 was detected in high metastasis breast cancer cell line MDA-MB-231 while higher expression of SLC22A18 was detected in low metastasis breast cancer cell line MCF-7. Transwell showed that comparing with MCF-7, breast cancer cell lines MDA-MB-231 had a much more cells that across the matrigel membrane. The distinction was notable. P=0.004.Conclusions:High metastasis breast cancer cell line MDA-MB-231 had strong invasion ability and low level of SLC22A18 expression. Imprinted gene SLC22A18 might be a tumor suppressor gene that effect metastasis of breast cancer.Part 2 Up-regulation of SLC22A18 expression on breast cancer cell linesPurpose:To up-regulate the expression of SLC22A18 on breast caner cell lines (U-231, U-7), and to identify the over-expression of SLC22A18. Compare the varied invasion abilities of up-regulation cell lines U-231,U-7 and breast cancer cell lines MDA-MB-231.MCF-7.Methods:U-231, U-7 was provided by Department of Forensic Medicine, Shanghai Medical College, Fudan University. Western Blot was applied to identify the expression of SLC22A18 protein in U-231,U-7. Transwell was employed to evaluate the differential invasion abilities of up-regulation cell lines and breast cancer cell lines.Results:Compared with MDA-MB-231, higher expression of SLC22A18 protein was detected in U-231. The difference is significant. The result remained the same when comparing MCF-7 with U-7. Transwell showed that up-regulation cell lines had lower cells that across the matrigel membrane. The distinctive was notable.Conclusions:Up-regulation cell lines were successfully constructed. Over expression of SLC22A18 may reduce the invasion abilities of breast cancer cells. SLC22A18,serve as tumor suppressor gene, may restrain the growth of tumor and weakened the invasion abilities of caner cells.Part 3 Expression of SLC22A18 in breast tissues and its relationship with clinicopathologic parameters and clinical outcomesPurpose:To detect the expression of SLC22A18 in benign breast tissues and breast cancer tissues. To analyze the correlation between SLC22A18 expression and clinicopathologic parameters in those breast cancer patients.Methods:From Jan.2004-Mar.2008,156 cases with breast cancer and 38 cases with benign breast diseases were randomly retrieved from a prospectively collected database of a total 574 cases that treated at the general surgery department, Zhongshan hospital, Fudan University, Shanghai, China. Immunohistochemistry was used to evaluate the expression of SLC22A18 protein in those breast tissues. Quantification of immunostaining was performed through digital image analysis. Statistical comparisons were done by SPSS 17.0 software to evaluate the relationship of SLC22A18 expression with clinicopathologic parameters and clinical outcomes.Results:1) We found that expression of SLC22A18 was down regulated in breast cancer and one way ANOVA analysis revealed expression of SLC22A18 was significantly higher in benign than in malignant tumors.2) The patients’age did not contribute to any significant difference in SLC22A18 expression levels.3) Breast tumor size reversely correlated with SLC22A18 expression levels. The larger size of breast cancer is, the lower expression of SLC22A18 appears.4) The expression of SLC22A18 is associated with extensive lymphovascular invasion. Lower expression was viewed in ones with extensive lymphovascular invasion than ones without extensive lymphovascular invasion.5) The expression level of SLC22A18 decreased gradually with the increasing of metastatic lymph nodes. Higher expression of SLC22A18 showed in lymph nodes positive groups than that in lymph node negative groups. However the expression level showed no significant relationship with numbers of metastatic lymph nodes.6) Estrogen receptor, progesterone receptor and HER-2 status contributed nothing to any significant difference in SLC22A18expression levels.7) The grade of tumor and the type of tumor was not related to SLC22A18 expression level.8) The expression level of SLC22A18 decreased gradually with the increasing of TNM staging.Conclusions:The expression of SLC22A18 in benign tissue was significant higher than that in malignant tumor tissue. The more advanced malignancy of breast cancer, the lower expression of SLC22A18. The trend appeared clearer as the emerging of metastatic events.Part 4 Prognostic roles of imprinted gene SLC22A18 in breast cancerPurpose:To investigate the prognostic value of SLC22A18 expression in breast cancer after surgery.Methods:We followed up all breast cancer patients through phone calling and clinical visits. We recorded the first time when recurrence or metastases occurred. The relapse-free interval (RFI) of the patients was calculated. Survival analysis was done using the Kaplan-Meier method and the Cox proportional hazards model to estimate variable with a significant independent prognostic role by means of a backward stepwise elimination procedure. P<0.05(two-sided) was judged statistically significant.Results:Kaplan-Meier analysis identified SLC22A18 expression was associated with relapse-free survival (RFS) of breast cancer. Higher expression SLC22A18 group had longer cum survival compared to the group with low expression. The difference was significant (p=0.003, log-rank test). Cox’s regression analysis identified tumor size, lymph nodes metastasis, nuclear stage, extensive lymphovascular invasion, SLC22A18 expression as prognostic factor for RFS. Nuclear stage and SLC22A18 expression were the most meaning histopathologic parameter in predicting tumor recurrence. Comparing with the group showing higher expression of SLC22A18, group with lower expression had more chance to relapse. The HR is 2.624 (p=0.035).Conclusions:Less risk of relapse and metastasis could be observed in breast cancer patients with higher expression level of SLC22A18. The result find SLC22A18, a probably tumor suppressor genes, participate the development of breast cancer. Testing expression of SLC22A18 could be helpful for diagnosis and prognosis in breast cancer.更多还原