【作者】 吴芳；

【导师】 卢放根； 胡春宏；

【作者基本信息】 中南大学 ， 肿瘤内科学， 2009， 博士

【摘要】 研究背景肺癌是当今发病率和肿瘤相关死亡最高的恶性肿瘤,其中80%～85%为非小细胞肺癌（non-small cell lung cancer,NSCLC）。以铂类为基础的联合化疗是NSCLC的标准化疗方案,对铂类药物的耐受是导致化疗失败,肿瘤复发和转移的主要原因,也是NSCLC治疗中亟待解决的难题。随着表遗传学研究的深入,最近有研究显示,hMLH1启动子甲基化可能介导了卵巢癌和结直肠癌的顺铂耐药,去甲基化干预可以部分逆转肿瘤的顺铂耐药。那么hMLH1启动子甲基化是否也导致了NSCLC的顺铂耐药,是否能作为NSCLC耐药逆转的靶点,目前国内外尚无系统研究。本研究细胞试验部分通过检测NSCLC顺铂敏感细胞株A549和耐顺铂细胞株A549/DDP的hMLH1启动子甲基化状态,探讨hMLH1启动子甲基化是否与NSCLC的顺铂耐药有关,并观察去甲基化药物（5-Aza-dc）能否去除该基因启动子甲基化,逆转肿瘤耐药。在临床试验部分,检测了83例NSCLC患者hMLH1启动子甲基化状态,结合患者的临床病理和随访资料,以探讨hMLH1启动子甲基化是否与NSCLC临床特征相关,是否能作为一项有效的分子标志用于预测术后顺铂辅助化疗后患者的生存和复发转移。第一章hMLH1基因启动子甲基化与非小细胞肺癌细胞铂耐药的相关研究目的探讨hMLH1基因启动子甲基化是否与非小细胞肺癌细胞A549/DDP顺铂耐药相关;去甲基化药物5-Aza-dc对A549/DDP细胞hMLH1基因启动子甲基化状态、基因表达和顺铂耐受性的影响。方法MSP法检测非小细胞肺癌顺铂耐药细胞株A549/DDP和其亲本株A549的hMLH1启动子甲基化状态;MTT法和流式细胞术测定A549/DDP对顺铂的耐药指数、5-Aza-dc对A549/DDP细胞的细胞毒性作用和不同浓度5-Aza-dc处理48小时后A549/DDP细胞对顺铂的IC₅₀和耐药逆转指数;MSP法、荧光定量RT-PCR（Real-time RT-PCR）和Western blot检测5-Aza-dc对A549/DDP细胞hMLH1基因启动子甲基化状态、mRNA和蛋白表达的影响。结果A549和A549/DDP细胞对顺铂的IC₅₀分别为5.20±0.14μM和30.15±0.76μM,A549/DDP的耐药指数为5.80,确定为耐顺铂的非小细胞肺癌细胞株;MSP检测A549为hMLH1基因启动子非甲基化细胞株,A549/DDP为hMLH1基因启动子部分甲基化细胞株;MTT及流式细胞术检测作用48小时时,20μM 5-Aza-dc为对A549/DDP细胞的无毒剂量,40μM为低毒剂量;以无毒和低毒剂量5-Aza-dc（20μM、40μM）处理A549/DDP后,顺铂对A549/DDP细胞的IC₅₀分别降低到16.54±0.35μM和6.82±0.16μM;耐药逆转指数分别为1.82和4.42;MSP、Real-time RT-PCR及Western blot检测发现无毒和低毒剂量5-Aza-dc均能很好地去除hMLH1启动子甲基化,hMLH1mRNA和蛋白表达增高。结论hMLH1基因启动子甲基化可能参与了非小细胞肺癌细胞A549/DDP的顺铂耐药;小剂量5-Aza-dc对A549/DDP细胞无明显的增殖抑制和促凋亡作用,但可能通过去除hMLH1启动子甲基化,上调hMLH1基因表达而部分逆转A549/DDP对顺铂的化疗耐药。第二章hMLH1基因启动子甲基化在非小细胞肺癌中的表达和临床意义目的检测手术切除并接受顺铂为基础辅助化疗后的非小细胞肺癌患者肿瘤组织中hMLH1基因启动子甲基化状态,探讨hMLH1基因启动子甲基化与患者临床病理特征及预后的关系。方法采用改良的酚/氯仿手工法提取83例接受顺铂为基础辅助化疗的NSCLC患者的福尔马林固定-石蜡包埋（Formalin-fixed andParrffin-Embedded,FFPE）肿瘤组织DNA;巢式MSP（nested-MSP）法检测hMLH1基因启动子甲基化状态;结合临床病理及随访资料,x²检验分析hMLH1基因启动子甲基化与其临床病理特征的关系;单因素Kaplan-Meier法（Log-rank检验）和多因素Cox回归对其启动子甲基化与患者总体生存进行统计学分析;多因素Logistic回归对其启动子甲基化与患者无病生存进行分析。结果83例NSCLC肿瘤样本中80例成功进行了巢式MSP扩增,其中33例发生hMLH1基因启动子甲基化（41.25%）。hMLH1启动子甲基化与患者年龄、性别、吸烟状态、肿瘤T分期、病理类型、分化程度、三年总体生存无关,与淋巴结转移分期（N）、三年无病生存相关。总体生存的单因素分析和多因素Cox分析均显示仅肿瘤N分期与总体生存有关,hMLH1启动子甲基化状态与总体生存无统计学相关,肿瘤复发和转移的多因素Logistic分析结果显示hMLH1启动子甲基化状态和肿瘤N分期与患者无病生存相关,甲基化阳性的患者更容易发生淋巴结转移和术后顺铂辅助化疗后的肿瘤复发和转移。结论hMLH1启动子甲基化是NSCLC的普遍事件,与NSCLC患者的N分期和无病生存（DFS）相关,与总体生存（OS）无关;并有望作为预测顺铂辅助化疗后的NSCLC复发和转移的分子标志。更多还原

【Abstract】 Background Lung cancer is one kind of malignant tumors with the highest incidence and mortality,while non-small cell lung cancer（NSCLC） is the most common type which accounts for 80%-85%of the lung cancer. Platinum-based chemotherapy is the standard chemotherapy for both early and advanced NSCLC,so platinum-resistance is the main reason for chemotherapy failure which causes tumor recurrence and metastasis,and has been a problem in the clinical practice.With in-depth study of epigenetics,the latest research showed that hMLH1 gene promoter methylation probably mediated cisplatin-resistance in ovarian cancer and colorectal cancer,demethylation intervention could well reverse the chemoresistance.However,there is no relevant research on hMLH1 methylation in NSCLC,and whether it could be used as a target for resistance reversal.This study first reported the hMLH1 methylation status in NSCLC cell line A549 and cisplatin -resistant cell line A549/DDP,in order to clear whether or not it correlate with platinum-resistance of NSCLC,then we observed whether demethylation intervention（5-Aza-dc） could remove the hMLH1 gene methylation and reverse platinum-resistance in A549/DDP cell line.In the next clinical study,we reported the hMLH1 methylation status of 83 NSCLC patients,to explore whether hMLH1 methylation was associated with the clinicopathological features of NSCLC,and if it could be used as an effective biomarker for the prediction of survival,recurrence and metastasis of NSCLC patients who accepted postoperative adjuvant cisplatin-based chemotherapy.ChapterⅠStudy on the role of hMLH1 gene promoter methylation in the platinum-resistance of NSCLC cell lineObjective To explore whether the hMLH1 promoter methylation correlate with the cisplatin-resistance in NSCLC A549/DDP cell line;the impact of demethylation drug 5-Aza-dc on hMLH1 gene methylation,gene expression and cisplatin-resistance of A549/DDP cell line.Methods MSP for detection of hMLH1 methylation status in cisplatin-resistant NSCLC cell line A549/DDP and its parental cell line A549.MTT method and flow cytometry（FCM） for test of cisplatin resistance index in A549/DDP cell,the cytotoxicity of 5-Aza-dc on A549/DDP,and the IC₅₀ and cisplatin resistance index of A540/DDP cells 48 hours after different concentration of 5-Aza-dc（0μM,20μM,40μM） treatment.MSP,Fluorescence quantitative PCR（Real-time RT-PCR） and western blot for detection of hMLH1 methylation status,mRNA and protein expression.Results The IC₅₀ values of cisplatin in A549 and A549/DDP cells were 5.20±0.14μM and 30.15±0.76μM,the resistance index of A549/DDP was 5.80,A549/DDP cells was identified as cisplatin-resistant NSCLC cell line.MSP showed that A549 was hMLH1 unmethylated cell,A549/DDP was hMLH1 partly methylated cell.MTT and FCM results demonstrated that during 48 hours’ 5-Aza-dc treatment,20μM was non-toxic dose,40μM was low-toxic dose,non-toxic dose and low-toxic dose of 5-Aza-dc（20μM, 40μM） reduced IC₅₀ values of cisplatin to 16.54±0.35μM（RI=1.82） and 6.82±0.16μM（RI=4.42）.MSP,Fluorescence quantitative PCR（Real-time RT-PCR） and western blot showed that non-toxic and low-toxic 5-Aza-dc could well remove the hMLH1 methylation,hMLH1 mRNA and protein expression increased.Conclusion hMLH1 promoter methylation may be involved in the cisplatin-resistance of NSCLC.Low dose 5-Aza-dc cause no significant growth inhibition and apoptosis to cisplatin-resistant NSCLC cells,but it can partly reverse the chemoresistance through removal of hMLH1 methylation and increased expression of hMLH1 gene. ChapterⅡThe clinical significance of hMLHl gene promoter methylation in NSCLCObjective To detect the hMLHl methylation status in the surgical resection specimens of NSCLC patients who accepted postoperative adjuvant cisplatin-based chemotherapy,and value its clinical significance, such as its relation to clinicopathological features and prognosis.Methods we used modified saturated phenol/chloroform method to extract FFPE（Formalin-fixed and Parrffin-Embedded） tumor tissue DNA from 83 NSCLC patients who accepted postoperative adjuvant cisplatin-based chemotherapy,nested MSP（nMSP） to detect the hMLH1 methylation status.x² test to analysis the relationship between hMLH1 methylation and clinicopathological features,univariate Kaplan-Meier （Log-rank test） and multivariate Cox regression for the hMLH1 methylation and overall survival（OS） analysis,Logistic regression for the hMLHl methylation and disease-free survival（DFS） analysis.Results In 80 patients with successful amplification of nested MSP, 33 were hMLH1 methylation cases（41.25%）.hMLH1 methylation was not associated with age,sex,smoking status,T stage,phathologic type, differentiation and 3-year survival,but correlated with N status and 3-year disease-free survival.Univariate Kaplan-Meier and multivariate Cox analysis of survival revealed that N status significantly affect the patients’ survival,but hMLH1 methylation was not associated with the overall survival.Logistic regression analysis showed that hMLH1 methylation and N staging were both risk factors of tumor recurrence and metastasis.Conclusion hMLH1 gene promoter methylation is a common event in NSCLC,it correlats with N status and disease-free survival of NSCLC, but is not associated with overall survival.hMLH1 hypermethylation may become a recurrence and metastasis prediction of NSCLC patients who accepted postoperative adjuvant cisplatin-based chemotherapy,and is expected to become a biomarker for NSCLC patients.更多还原