【作者】 胡汛；

【导师】 倪一鸣；

【作者基本信息】 浙江大学 ， 外科学， 2014， 硕士

【摘要】 背景：肺癌居死亡原因世界范围内第一位,其发病率占男性癌症患者的18.4%,占女性的21.9%。相关数据显示,超过90万例患者每年死于肺癌。到目前为止,还没有发现这种致命的疾病的有效诊断和预测风险的生物标记物。在过去十年的科学研究进展中,越来越多的研究指向microRNAs (miRNAs or miRs)在参与各种类型的肺部恶性肿瘤中的重要作用,发挥的角色包括促发因子、调节因子、衡量危险分层的分子、作为诊断的生化标志物等。目前已有研究证实了miR126,let-7, miR-372, miR-182*,以及miR-220这5个miRNA与总体生存率显著相关,而且miR126是保护性的miRNA因子,细胞周期蛋白cyclinD的表达水平受miR126的调控。临床治疗方面,CT引导下肿瘤射频消融术(CT-PRFA)是一种用于治疗原发性及继发性肿瘤的微创技术,但目前尚未有研究探究肺癌射频消融术术前以及术后的miRNA的表达水平的变化,在非小细胞肺癌和肺转移性肝癌患者中miRNA表达水平的差别,及其与细胞周期蛋白cyclinD表达水平的关系,以及相关miRNA的表达水平与患者预后的关系。目的：本课题的研究目的是1.探讨CT-射频消融术治疗肺癌对主要研究终点的功效和对中期生存的评价的影响。2.CT-射频治疗肺癌前后miRNA表达水平的变化。3.miR-126与miR200c在非小细胞肺癌以及肺转移性肝癌中的表达的比较。4.miR-126与miR200c的表达水平在非小细胞肺癌以及肺转移性肝癌患者中对生存率的影响。5.miR-126与miR200c的表达水平在非小细胞肺癌以及肺转移性肝癌病灶组织中与细胞周期蛋白cyclin D的关系。6.cox比例风险回归模型分析非小细胞肺癌以及肺转移性肝癌病的预后因子。方法：本研究入选了从2009年1月到2013年10月,根据WHO诊断标准,经过病理显微镜镜检诊断为非小细胞肺癌,或者肺转移性肝癌,且纳入CT-PRFA治疗的患者26名。进行CT引导下肿瘤射频消融术,定量实时PCR (QRT-PCR)方法测定术前以及术后microRNA表达水平,实时PCR系统进行定量分析。采用细胞免疫组化的方法测定肿瘤组织中细胞周期蛋白cyclinD的表达。随访截止时间为2014年6月。使用两种独立样本t检验或单因素方差分析评估miRNA的表达水平与临床参数之间的可能关系。我们采用kaplan-Meier法比较生存率,风险比(HR)和95%可信区间(CI)研究患者射频消融术后生存率。生存功能比较通过使用Log Rank对数秩检验进行,P<0.05为有显著差异。用Cox比例风险模型进行多变量分析预测非小细胞肺癌与肺转移性肝癌的预后因子。结果：14例经病理确诊的原发性非小细胞肺癌(NSCLC)和12例肝细胞癌肺转移患者入选本研究。所有14例的原发性非小细胞肺癌患者只有一个单独的病灶,9例肝细胞癌肺转移患者在肺部只有一个单独病灶,而其他三例则有两处肿瘤病灶。中位随访时间为15个月(3-34个月的范围),没有患者失访。在随访过程中只有一位患者接受了全身系统化疗,用以辅助射频消融术治疗,而其他患者则没有接受任何抗肿瘤的治疗。患者总体2年生存率为80.8%(95%CI：35%,95%)。结果显示以miRNA126分两组,在继发性肺癌患者中kaplan meier生存分析显示,低ACt组与高ACt组的生存率没有显著差异(p=0.497)；而在原发性肺癌中miRNA126高ACt组的生存率明显高于低ACt组(p=0.033)。以miRNA200c分两组,在继发性肺癌患者低ACt组的生存率明显高于高ACt组(p--0.037)而在原发性肺癌中miRNA200c,低ACt组与高ACt组的生存率没有显著差异,(p=0.09)。CT-PRFA是一种有潜在价值的有效的治疗方案,既可应用于高选择性的孤立原发非小细胞肺癌,也可以用来治疗肝癌继发肺部肿瘤。CT-PRFA的功效可能与miRNA的表达上调或下降有关,miR-126的表达水平与非小细胞肺癌患者的生存率显著相关,且与细胞周期蛋白cyclin D的表达成反比关系,cox风险比例回归模型分析得出miR126的表达水平是非小细胞肺癌患者的预后因子。结论：CT-PRFA是一种有潜在价值的有效的治疗方案,既可应用于高选择性的孤立原发非小细胞肺癌,也可以用来治疗肝癌继发肺部肿瘤。CT-PRFA的功效可能与miRNA的表达上调或下降有关,miR-126的表达水平与非小细胞肺癌患者的生存率显著相关,且与细胞周期蛋白cyclin D的表达成反比关系。更多还原

【Abstract】 Background:Lung cancer ranks first in the cause of death worldwide, the incidence rate of male cancer patients is18.4%, and21.9%of women. Data showed that more than900,000cases of patients die each year from lung cancer. So far, scientists have not found effective biomarkers for diagnosing and predicting the risk of this deadly disease. Over the past decade, more and more research points to microRNAs (miRNAs or miRs), which play an important role in participating in various types of lung cancers, and the roles include precipitating factor, adjustment factor, a measurement of risk molecules stratification, and also as a diagnostic and other biochemical markers. Recently, study confirmed the miR126, let-7, miR-372, miR-182*, and miR-220are miRNA which were significantly correlated with the overall survival rate, and miR126miRNA is one of the protective factors, cell cycle proteins cyclinD regulated according miR126expression level.As to the clinical treatment, CT-guided radiofrequency tumor ablation is a method for the treatment of both primary and secondary tumors with minimally invasive, as to our best knowledge, there is not a study to explore miRNA expression levels before radiofrequency tumor ablation of lung cancer as well as postoperative in non-small cell lung cancer and lung metastatic liver cancer patients, its relationship with the expression levels of cyclin cyclinD, how the expression levels of miRNA associated with patient’s prognosis, and the predictor for overall survival of lung cancer patients.Objectives:The purposes of this project are:l.to investigate CT-radiofrequency ablation efficacy of the primary endpoint of lung cancer and its impact on the evaluation of the medium-term survival.2. Comparison of miRNA expression before and after CT-radiofrequency ablation3. miR-126and miR200c’s expression difference between non small cell lung cancer and metastatic liver cancer in the lung.4, The relationship of the expression level of miR-126and miR200c in non-small cell and metastatic liver lung cancer patients and their overall survival.5, the expression level of miR-126and miR200c relationship with cyclin D in non-small cell lung cancer and metastatic liver lesions in lung tissue.6, cox proportional hazards regression model analysis prognostic factors of non-small cell lung cancer and lung metastatic liver disease.Methods:This study began from January2009to October2013, according to the WHO diagnostic criteria, after pathological microscopic examining, all cases were diagnosed as non-small cell lung cancer, or lung metastatic liver cancer,26patients were enrolled who also finished lung cancer treatment with CT-guided radiofrequency tumor ablation(CT-PRFA), quantitative real-time PCR (QRT-PCR) method for the determination preoperative and postoperative microRNA expression levels, real-time PCR system for quantitative analysis. Cell immunohistochemical method was adapt to detect the expression of cell cycle proteins cyclinD in tumor tissue. Patients were followed up untill June2014. Using two independent samples t-test or ANOVA assess the possible relationship between miRNA expression levels and clinical parameters. We used kaplan-Meier method to compare survival hazard ratio (HR) and95%confidence intervals (CI) in patients with radiofrequency ablation. Comparison of survival functions carried out by using the Log Rank log-rank test. Cox proportional hazards model with multivariate analysis to detect prognostic factors of non-small cell lung cancer and pulmonary metastatic liver cancer. P<0.05was considered statistically significant.Results:14patients with pathologically diagnosed as primary non-small cell lung cancer (NSCLC) and12cases of hepatocellular carcinoma and lung metastases. All14cases of primary non-small cell lung cancer patients had only a single lesion,9cases of hepatocellular carcinoma in patients with lung metastases had only a single lesion in the lungs, while the other three cases had two tumor lesions. The median follow-up time was15months (range3-34months), no patients lost to follow. During follow-up, only one patient received systemic chemotherapy, which were used to assist radiofrequency ablation, and other patients did not receive any anti-tumor therapy. The overall two-year survival rate for all patients is80.8%(95%CI:35%,95%). Patients were divided into two groups according to miRNA126expression level (median ACt), kaplan meier survival analysis showed no significant difference (p=0.497)for patients who suffered from secondary lung cancer between high and low△Ct group; while in primary lung cancer, survival rate of miRNA126low ACt group was significantly higher than that of high△Ct (p=0.033). Patients were also divided into two groups according to miRNA200c expression level (median△Ct),the survival rate of patients with secondary cancer was significantly higher in the high△Ct group than that of the low△Ct group (p=0.037), but there was no significant difference in primary lung cancer patients (p=0.09). CT-PRFA is a potential value of effective treatment options, it can be applied to highly selective isolation of the non-small cell lung cancer, and can also be used to treat secondary lung tumors. CT-PRFA efficacy may be related to the expression of some miRNA, miR-126expression levels was inversely correlated with non small cell lung cance patients, and had a signigicantly relationship with expression of cyclin D, cox proportional hazards regression model derived that expression levels of miR126is a prognostic factor in non-small cell lung cancer patients.Conclusion:CT-PRFA is a potentially valuable and effective treatment option, can be applied to the highly selective isolation node of the major non-small cell lung cancer, as well as secondary lung cancer. CT-PRFA efficacy may be related to the expression of some miRNA, miR-126expression levels was inversely correlated with non small cell lung cance patients, and had a signigicantly relationship with expression of cyclin D, cox proportional hazards regression model derived that expression levels of miR126is a prognostic factor in non-small cell lung cancer patients.更多还原