联合血清SOX1和VIM基因甲基化对肝细胞癌诊断价值的研究

发布时间：2018-04-09 12:07

  本文选题：甲基化　切入点：SOX1　出处：《山东大学》2017年硕士论文

【摘要】：背景:肝细胞癌(HCC)是全世界发病率最高的恶性肿瘤之一,其死亡率在恶性肿瘤中高居第三位。肝癌起病隐匿,60-70%的患者由于得不到及时的诊断在初次发现时便已发展至肝癌晚期,失去了手术切除等根治性治疗的机会。因此,肝癌的早期诊断十分重要。目前,对于肝癌的诊断和预后判断的主要依据是临床病理和影像学检查。临床病理学检查虽然是诊断肝癌的金标准,但是其需要手术切除的标本或者肝穿刺标本,有创性大大限制了它的临床应用,故一般不作为首选检查方法。各种影像学技术包括超声,计算机断层扫描(CT)和磁共振成像(MRI)等也被广泛用于临床检查。然而,这些影像学技术都存在某些方面的局限性。此外,甲胎蛋白(AFP)等多种血清蛋白作为肝癌的辅助性诊断指标在临床上被广泛应用。但其存在假阳性和假阴性的不足,且灵敏度仅为22-60%,远远不能满足临床需求。因此,寻找肝癌特异性标记物用于肝癌早期诊断迫在眉睫。各种生物标记物被广泛用于诊断和评估癌症预后。循环肿瘤DNA(ctDNA)是指肿瘤细胞释放到外周血中的基因组小片段,它可以作为肿瘤生物标记物,为诊断肿瘤提供一种简单、无创的方法。表观遗传学在人类肿瘤的发生和发展过程中起重要作用。在肿瘤中基因启动子区域异常甲基化所导致的抑癌基因沉默是最常见的表观遗传学改变。许多研究已经证实DNA异常甲基化可作为生物标记物用于HCC等癌症的早期诊断。目的:本研究旨在探究肝细胞癌病人血清SRY(sex determining region Y)-box 1(SOX1)和波形蛋白(VIM)基因启动子甲基化状态,以及血清SOX1和VIM基因启动子甲基化对HCC的诊断价值。方法:本研究包括360名受试者,其中240名HCC患者,29名肝硬化(LC)患者,66名慢性乙型肝炎(CHB)患者和25名健康对照(HC)。HCC的诊断标准为美国肝病研究学会(AASLD)2010年发布的《肝细胞癌临床指南》,CHB和LC的诊断标准为AASLD 2009年发布的《慢性乙型肝炎实践指南》。所有血液样本均为清晨采取的空腹静脉血。经血清分离,通过试剂盒法提取血清中DNA,具体操作步骤严格按照说明书进行。经重亚硫酸盐进行甲基化修饰处理,应用甲基化特异性聚合酶链反应(MSP)检测HCC,LC,CHB患者和健康对照者血清中SOX1和VIM基因启动子甲基化状态。结果:1.HCC组血清SOX1基因启动子甲基化率(72.08%)显著高于LC组(17.24%,p0.001),CHB 组(15.15%,p0.001)和 HC 组(4.00%,p0.001)。HCC组血清VIM基因启动子甲基化率(61.67%)明显高于LC组(24.14%,p0.001),CHB 组(13.64%,p0.001)和 HC 组(12.00%p0.001)。然而,LC组和CHB组,CHB组和HC组或LC组和HC组之间的SOX1和VIM基因启动子甲基化率没有显著差异。2.HCC患者血清SOX1基因启动子甲基化状态与肿瘤数量(χ2=6.107,p =0.013),肿瘤大小(χ2= 7.986,p=0.018)和 TNM 分期(χ2= 12.458,p = 0.001)相关。HCC患者血清VIM基因启动子甲基化状态与门静脉癌栓形成(χ=7.528,p= 0.006),肿瘤数目(X2= 9.997,p = 0.002),肿瘤大小(χ2= 19.451,p=0.001),TNM 分期(χ2= 8.291,p = 0.004)和血管转移(χ2= 8.832,p = 0.003)相关。单变量逻辑回归分析提示HCC患者血清SOX1基因启动子甲基化与多个肿瘤结节(OR = 0.208,p0.001)和 TNM 分期 Ⅲ-Ⅳ)(OR = 4.987,p0.001)相关;VIM基因启动子甲基化与门静脉癌栓(OR = 0.006,0.001),TNM分期(Ⅲ-Ⅳ)(OR = 3.978,p0.001),肿瘤大小(5cm)(OR=2.340,p = 0.037)和血管转移(OR = 33.681,p0.001)相关。3.当用于诊断HCC与LC和CHB患者时,SOX1基因启动子甲基化的灵敏度为72.08%,VIM基因启动子甲基化的灵敏度为61.67%,均高于AFP(56.67%,χ2= 12.436,0.001;χ2= 1.242,p = 0.265)。联合 SOX1 和 VIM 基因启动子甲基化用于诊断HCC的灵敏度为82.50%,特异度为78.95%。当AFP大于20ng/ml或SOX1和VIM中至少一个基因发生甲基化时,其诊断HCC的灵敏度、特异度、阳性预测值和阴性预测值分别为88.33%、72.63%、89.08%和71.13%。当AFP大于20ng/ml且SOX1和VIM基因均发生甲基化时,其诊断HCC的灵敏度、特异度、阳性预测值和阴性预测值分别为48.33%、97.89%、98.31%和42.86%。结论:1.本研究首次发现HCC患者血清中存在SOX1和VIM基因启动子区域异常甲基化且甲基化率高于LC和CHB患者。2.SOX1和VIM基因启动子甲基化与HCC患者临床病理指标间具有相关性,提示SOX1和VIM启动子甲基化与HCC的进展相关。3.AFP联合SOX1和VIM基因启动子甲基化可明显提高HCC诊断的灵敏度。提示血清SOX1和VIM基因甲基化可作为诊断HCC和预测HCC进展的生物标记物。
[Abstract]:Background: hepatocellular carcinoma (HCC) is the world's highest incidence of malignant tumors, the mortality rate ranks third in malignant tumors. The insidious onset of liver cancer 60-70% patients due to the lack of timely diagnosis in first discovered when it has developed to the advanced liver cancer, and lost the chance of surgical resection and radical treatment. Therefore, early diagnosis is very important. At present, the main basis for the diagnosis and prognosis of hepatocellular carcinoma is the clinical pathological and radiological examination. The clinical pathological examination is the gold standard for the diagnosis of liver cancer, but the need of surgical resection or liver biopsy specimens, noninvasive greatly limits the clinical application it's generally not as the preferred method of examination. Ultrasound technology includes various imaging, computed tomography (CT) and magnetic resonance imaging (MRI) have been widely used in clinical examination. However, these imaging technologies There are certain limitations. In addition, alpha fetoprotein (AFP) and other serum proteins as auxiliary diagnostic index of liver cancer is widely used in clinical practice. But the problems of false positive and false negative, and the sensitivity is only 22-60%, far can not meet the clinical needs. Because of this, looking for hepatoma specific markers for early diagnosis of hepatocellular carcinoma. All imminent biomarkers are widely used for diagnosis and evaluation of prognosis of cancer. Circulating tumor DNA (ctDNA) refers to the release of tumor cells into the peripheral blood of the small fragment, it can be used as a tumor biomarker for the diagnosis of cancer to provide a simple, noninvasive method. Epigenetics plays an important role in the occurrence and development of human tumors. Tumor suppressor gene silencing in tumor gene promoter region methylation which is the most common epigenetic changes in many research. We have confirmed that DNA methylation can be used as a biomarker for early diagnosis of HCC cancer. Objective: This study aimed to explore the levels of serum SRY in patients with hepatocellular carcinoma (sex determining region Y) -box 1 (SOX1) and vimentin (VIM) gene promoter methylation status, and serum SOX1 and VIM gene promoter methylation of the diagnostic value of HCC. Methods: This study consisted of 360 subjects, including 240 HCC patients, 29 liver cirrhosis (LC) patients, 66 chronic hepatitis B (CHB) patients and 25 healthy controls (HC).HCC diagnostic criteria for the study of liver disease in the United States (AASLD) of liver cell carcinoma clinical guidelines published in 2010 < >, the criteria for the diagnosis of CHB and LC for the fasting blood AASLD released in 2009 "chronic hepatitis B practice guidelines. All blood samples were taken in the morning. The serum separated by Kit Method to extract DNA in serum, the specific steps In strict accordance with the instructions. The methylation modification treated by bisulfite, using methylation specific polymerase chain reaction (MSP) detection of HCC, LC, CHB patients and healthy controls were SOX1 and VIM in serum of gene promoter Zi Jiaji state. Results: the serum 1.HCC of SOX1 gene promoter methylation rate (72.08%) was significantly higher than that of group LC (17.24%, p0.001), group CHB (15.15%, p0.001) and group HC (4%, p0.001) group.HCC serum VIM gene promoter methylation rate (61.67%) was significantly higher than that of group LC (24.14%, p0.001), group CHB (13.64%, p0.001) and HC group (12.00%p0.001). However, LC group and the CHB group, CHB group and HC group or LC group and HC group between SOX1 and VIM gene promoter methylation rate did not start significant difference of serum SOX1 in patients with.2.HCC gene methylation and tumor promoter number (x 2=6.107, P =0.013), tumor size (2= 7.986, p=0.018) and TNM stage (x 2= 12.458, P = 0 .001)鐩稿叧.HCC鎮ｈ，

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