發(fā)布時間：2018-04-09 12:07

  本文選題：甲基化　切入點：SOX1　出處：《山東大學》2017年碩士論文

【摘要】：背景:肝細胞癌(HCC)是全世界發(fā)病率最高的惡性腫瘤之一,其死亡率在惡性腫瘤中高居第三位。肝癌起病隱匿,60-70%的患者由于得不到及時的診斷在初次發(fā)現(xiàn)時便已發(fā)展至肝癌晚期,失去了手術切除等根治性治療的機會。因此,肝癌的早期診斷十分重要。目前,對于肝癌的診斷和預后判斷的主要依據(jù)是臨床病理和影像學檢查。臨床病理學檢查雖然是診斷肝癌的金標準,但是其需要手術切除的標本或者肝穿刺標本,有創(chuàng)性大大限制了它的臨床應用,故一般不作為首選檢查方法。各種影像學技術包括超聲,計算機斷層掃描(CT)和磁共振成像(MRI)等也被廣泛用于臨床檢查。然而,這些影像學技術都存在某些方面的局限性。此外,甲胎蛋白(AFP)等多種血清蛋白作為肝癌的輔助性診斷指標在臨床上被廣泛應用。但其存在假陽性和假陰性的不足,且靈敏度僅為22-60%,遠遠不能滿足臨床需求。因此,尋找肝癌特異性標記物用于肝癌早期診斷迫在眉睫。各種生物標記物被廣泛用于診斷和評估癌癥預后。循環(huán)腫瘤DNA(ctDNA)是指腫瘤細胞釋放到外周血中的基因組小片段,它可以作為腫瘤生物標記物,為診斷腫瘤提供一種簡單、無創(chuàng)的方法。表觀遺傳學在人類腫瘤的發(fā)生和發(fā)展過程中起重要作用。在腫瘤中基因啟動子區(qū)域異常甲基化所導致的抑癌基因沉默是最常見的表觀遺傳學改變。許多研究已經(jīng)證實DNA異常甲基化可作為生物標記物用于HCC等癌癥的早期診斷。目的:本研究旨在探究肝細胞癌病人血清SRY(sex determining region Y)-box 1(SOX1)和波形蛋白(VIM)基因啟動子甲基化狀態(tài),以及血清SOX1和VIM基因啟動子甲基化對HCC的診斷價值。方法:本研究包括360名受試者,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名健康對照(HC)。HCC的診斷標準為美國肝病研究學會(AASLD)2010年發(fā)布的《肝細胞癌臨床指南》,CHB和LC的診斷標準為AASLD 2009年發(fā)布的《慢性乙型肝炎實踐指南》。所有血液樣本均為清晨采取的空腹靜脈血。經(jīng)血清分離,通過試劑盒法提取血清中DNA,具體操作步驟嚴格按照說明書進行。經(jīng)重亞硫酸鹽進行甲基化修飾處理,應用甲基化特異性聚合酶鏈反應(MSP)檢測HCC,LC,CHB患者和健康對照者血清中SOX1和VIM基因啟動子甲基化狀態(tài)。結(jié)果:1.HCC組血清SOX1基因啟動子甲基化率(72.08%)顯著高于LC組(17.24%,p0.001),CHB 組(15.15%,p0.001)和 HC 組(4.00%,p0.001)。HCC組血清VIM基因啟動子甲基化率(61.67%)明顯高于LC組(24.14%,p0.001),CHB 組(13.64%,p0.001)和 HC 組(12.00%p0.001)。然而,LC組和CHB組,CHB組和HC組或LC組和HC組之間的SOX1和VIM基因啟動子甲基化率沒有顯著差異。2.HCC患者血清SOX1基因啟動子甲基化狀態(tài)與腫瘤數(shù)量(χ2=6.107,p =0.013),腫瘤大小(χ2= 7.986,p=0.018)和 TNM 分期(χ2= 12.458,p = 0.001)相關。HCC患者血清VIM基因啟動子甲基化狀態(tài)與門靜脈癌栓形成(χ=7.528,p= 0.006),腫瘤數(shù)目(X2= 9.997,p = 0.002),腫瘤大小(χ2= 19.451,p=0.001),TNM 分期(χ2= 8.291,p = 0.004)和血管轉(zhuǎn)移(χ2= 8.832,p = 0.003)相關。單變量邏輯回歸分析提示HCC患者血清SOX1基因啟動子甲基化與多個腫瘤結(jié)節(jié)(OR = 0.208,p0.001)和 TNM 分期 Ⅲ-Ⅳ)(OR = 4.987,p0.001)相關;VIM基因啟動子甲基化與門靜脈癌栓(OR = 0.006,0.001),TNM分期(Ⅲ-Ⅳ)(OR = 3.978,p0.001),腫瘤大小(5cm)(OR=2.340,p = 0.037)和血管轉(zhuǎn)移(OR = 33.681,p0.001)相關。3.當用于診斷HCC與LC和CHB患者時,SOX1基因啟動子甲基化的靈敏度為72.08%,VIM基因啟動子甲基化的靈敏度為61.67%,均高于AFP(56.67%,χ2= 12.436,0.001;χ2= 1.242,p = 0.265)。聯(lián)合 SOX1 和 VIM 基因啟動子甲基化用于診斷HCC的靈敏度為82.50%,特異度為78.95%。當AFP大于20ng/ml或SOX1和VIM中至少一個基因發(fā)生甲基化時,其診斷HCC的靈敏度、特異度、陽性預測值和陰性預測值分別為88.33%、72.63%、89.08%和71.13%。當AFP大于20ng/ml且SOX1和VIM基因均發(fā)生甲基化時,其診斷HCC的靈敏度、特異度、陽性預測值和陰性預測值分別為48.33%、97.89%、98.31%和42.86%。結(jié)論:1.本研究首次發(fā)現(xiàn)HCC患者血清中存在SOX1和VIM基因啟動子區(qū)域異常甲基化且甲基化率高于LC和CHB患者。2.SOX1和VIM基因啟動子甲基化與HCC患者臨床病理指標間具有相關性,提示SOX1和VIM啟動子甲基化與HCC的進展相關。3.AFP聯(lián)合SOX1和VIM基因啟動子甲基化可明顯提高HCC診斷的靈敏度。提示血清SOX1和VIM基因甲基化可作為診斷HCC和預測HCC進展的生物標記物。
[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(zhèn)ｈ，

本文編號：1726371