本文關(guān)鍵詞： DAPk1 下咽鱗狀細(xì)胞癌 甲基化 表達(dá)　出處：《山東大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景 下咽部的惡性腫瘤是頭頸部惡性程度最高的腫瘤之一,而鱗狀細(xì)胞癌是最常見的惡性腫瘤類型,下咽鱗狀細(xì)胞癌占頭頸腫瘤的5%左右。下咽癌的發(fā)生、發(fā)展是一個(gè)多階段的過(guò)程,其中包含了多個(gè)基因水平的改變。由于其解剖部位的特殊性,下咽癌的早期癥狀不明顯,且大多數(shù)分化程度較差,局部呈侵襲性生長(zhǎng)并易沿黏膜下浸潤(rùn)和擴(kuò)散,在病程早期就容易出現(xiàn)頸部淋巴結(jié)的轉(zhuǎn)移。現(xiàn)如今,多學(xué)科聯(lián)合綜合治療是下咽鱗狀細(xì)胞癌主要的治療方案,包括手術(shù)、放射療法及化療,近年來(lái),盡管治療方法得到了很大的改進(jìn),但是下咽癌的預(yù)后仍不盡如人意,5年生存率僅在40-50%左右。 死亡相關(guān)蛋白激酶-1(DAPk1,或簡(jiǎn)稱為DAPk)是近年來(lái)發(fā)現(xiàn)的一種抑癌基因,屬于DAPk蛋白家族,后者包括DAPk1, DAPk2/DRP-1, DAPk3/ZIPk/Dlk, DRAK1和DRAK2。DAPk1定位于人染色體9q34.1,編碼的蛋白分子量為160-kD,由1430個(gè)氨基酸組成。它由酶催化區(qū)、鈣調(diào)蛋白調(diào)節(jié)區(qū)、8個(gè)錨蛋白重復(fù)序列、ROC-COR域,死亡結(jié)構(gòu)域和富含絲氨酸的C-末端組成。DAPk1是一種凋亡的正性調(diào)節(jié)因子,且作為一種絲氨酸/蘇氨酸調(diào)節(jié)激酶,DAPk1參與到多種信號(hào)通路調(diào)節(jié)凋亡、自噬、半胱天冬酶依賴的細(xì)胞死亡、細(xì)胞粘附和遷移。因此,它不僅起到在炎癥反應(yīng)中起到一定的作用,還發(fā)揮著抗腫瘤與抑制轉(zhuǎn)移的功能。現(xiàn)如今,越來(lái)越多關(guān)于DAPk1的抗腫瘤功能被陸續(xù)發(fā)掘,包括抑制整合素信號(hào)、推動(dòng)失巢凋亡、抑制細(xì)胞活動(dòng)、腫瘤代謝的調(diào)節(jié)等。 作為一個(gè)抑癌基因,DAPk1在許多種類的腫瘤中都呈現(xiàn)表達(dá)下調(diào),而DNA啟動(dòng)子區(qū)CpG島的甲基化被認(rèn)為是一個(gè)主要的機(jī)制,它能夠影響基因轉(zhuǎn)錄活性,卻不涉及DNA的序列改變。DNA甲基化是表觀遺傳學(xué)的重要機(jī)制,與細(xì)胞生長(zhǎng)、分化及轉(zhuǎn)化密切相關(guān),在腫瘤的發(fā)生、發(fā)展中也起到重要的作用。抑癌基因的DNA啟動(dòng)子區(qū)CpG島異常甲基化在許多腫瘤中均頻繁發(fā)生,DNA啟動(dòng)子區(qū)的高甲基化狀態(tài)可通過(guò)基因失活、刪失及突變等方式導(dǎo)致基因沉默。DAPk1的異常甲基化在許多腫瘤中均有報(bào)道,包括喉癌、結(jié)直腸癌、乳腺癌等。目前尚未有關(guān)于DAPk1在下咽癌中的甲基化及表達(dá)情況的類似報(bào)道,因此,在本次研究中,我們擬通過(guò)檢測(cè)DAPk1在下咽癌中的甲基化及表達(dá)情況,探討其與臨床病理特征和預(yù)后的關(guān)系。 目的 我們收集了53對(duì)下咽鱗狀細(xì)胞癌腫瘤組織和癌旁非腫瘤黏膜組織樣本,擬檢測(cè)下咽鱗狀細(xì)胞癌中DAPk1的甲基化及表達(dá)情況,分析其與臨床病理特征的關(guān)系,并評(píng)價(jià)DAPk1對(duì)下咽癌患者預(yù)后的影響。 方法 1.53對(duì)腫瘤組織及癌旁非腫瘤黏膜組織樣本來(lái)源于2010年山東大學(xué)齊魯醫(yī)院耳鼻咽喉科的下咽鱗狀細(xì)胞癌住院手術(shù)患者。腫瘤組織取自腫瘤的中心,而相應(yīng)的癌旁非腫瘤黏膜組織取自距離腫瘤邊緣至少2cm的形態(tài)學(xué)正常的黏膜組織。 2.我們采用甲基化特異性PCR(MSP)來(lái)檢測(cè)樣本中DAPk1的甲基化狀態(tài),并利用實(shí)時(shí)定量RT-PCR、免疫組織化學(xué)染色和蛋白質(zhì)印跡分析分別從mRNA水平和蛋白水平檢測(cè)DAPk1的表達(dá)情況。分析DAPk1的甲基化及表達(dá)情況之間的關(guān)系,以及其與患者的臨床病理特征和預(yù)后的關(guān)系。 結(jié)果 1. DAPk1甲基化檢測(cè)結(jié)果顯示在下咽鱗狀細(xì)胞癌腫瘤組織中甲基化率為60.38%(32/53),癌旁非腫瘤黏膜組織中甲基化率為26.42%(14/53),下咽癌中腫瘤組織的甲基化率明顯高于癌旁組織,兩者之間的差異具有統(tǒng)計(jì)學(xué)意義(P=0.001)。 2.實(shí)時(shí)定量RT-PCR中腫瘤組織的DAPk1mRNA相對(duì)表達(dá)量(0.863+0.095)較癌旁非腫瘤黏膜組織中(1.000)明顯下調(diào)(P＝0.002)；蛋白質(zhì)印跡分析腫瘤組織中DAPk1的蛋白表達(dá)水平(0.459±0.036)較癌旁非腫瘤黏膜組織中(0.666+0.037)明顯降低(P0.001)；免疫組織化學(xué)染色中,DAPk1蛋白主要在胞漿表達(dá),DAPk1在腫瘤組織中的蛋白表達(dá)陽(yáng)性率為32.08%(17/53),低于癌旁非腫瘤黏膜組織中的58.49%(31/53)(P=0.006)。以上結(jié)果顯示在下咽鱗狀細(xì)胞癌腫瘤組織中DAPk1的mRNA及蛋白水平較癌旁非腫瘤黏膜組織均明顯降低,差異具有統(tǒng)計(jì)學(xué)意義。 3.下咽癌腫瘤組織中甲基化組的DAPk1mRNA相對(duì)表達(dá)量為0.633+0.095,顯著低于非甲基化組的DAPk1mRNA相對(duì)表達(dá)量(1.212+0.167),兩者之間的差異具有統(tǒng)計(jì)學(xué)意義,DAPk1甲基化與mRNA的相對(duì)表達(dá)之間的關(guān)系呈負(fù)相關(guān)(P=0.002,r=-0.521)。存在區(qū)域淋巴結(jié)轉(zhuǎn)移的病例中,其DAPk1甲基化程度增高(P=0.001),且mRNA表達(dá)降低(0.516±0.069v.s.1.128±0.142,P=0.001)；同樣地,在高等級(jí)的TNM分期中,即Ⅲ期和Ⅳ期的病例中,DAPk1甲基化程度增高(P=0.009),且mRNA表達(dá)降低(0.698±0.096v.s.1.156±0.188,P=0.019)。腫瘤組織中DAPk1甲基化、mRNA表達(dá)與年齡、原發(fā)腫瘤分期、組織病理學(xué)分級(jí)無(wú)關(guān)。 4.生存分析結(jié)果顯示在53例下咽鱗狀細(xì)胞癌患者中,DAPk1未甲基化組患者的生存率顯著高于甲基化組患者(P=0.031), DAPk1的mRNA表達(dá)上調(diào)組的患者生存率也顯著高于表達(dá)下調(diào)組(P=0.045)。在多因素Cox比例風(fēng)險(xiǎn)回歸分析中,區(qū)域淋巴結(jié)轉(zhuǎn)移是影響預(yù)后的獨(dú)立危險(xiǎn)因素(P=0.043,HR5.387,95%CI1.052-27.600)。而DAPk1在腫瘤組織中的甲基化、mRNA表達(dá),原發(fā)腫瘤分期、臨床TNM分期均不能作為獨(dú)立危險(xiǎn)因素,它們協(xié)同發(fā)揮作用從而影響下咽鱗狀細(xì)胞癌患者的預(yù)后。 結(jié)論 DAPk1在下咽鱗狀細(xì)胞癌腫瘤組織中較癌旁非腫瘤黏膜組織中的甲基化率明顯增高,且mRNA及蛋白水平均表達(dá)降低,DAPk1的甲基化狀態(tài)和mRNA表達(dá)情況兩者之間呈負(fù)相關(guān)。DAPk1的高甲基化及低表達(dá)與下咽癌的區(qū)域淋巴結(jié)轉(zhuǎn)移、高等級(jí)臨床分期及預(yù)后不良密切相關(guān),區(qū)域淋巴結(jié)轉(zhuǎn)移是影響下咽癌患者預(yù)后的獨(dú)立危險(xiǎn)因素。
[Abstract]:Research background
Hypopharyngeal cancer is one of the most malignant tumor of head and neck squamous cell carcinoma, and malignant tumor is the most common type of hypopharyngeal squamous cell carcinoma of head and neck cancer accounted for about 5%. Under the incidence of pharyngeal cancer, development is a multi-stage process, which includes a plurality of base level changes. Because of its special anatomic site, hypopharyngeal cancer early symptoms are not obvious, and most poorly differentiated, a local invasive growth and easy invasion and spread along the submucosal, early in the course of the disease prone to metastasis of cervical lymph node. Now, a multidisciplinary comprehensive treatment is to swallow treatment. Squamous cell carcinoma include surgery, radiotherapy and chemotherapy, in recent years, although the treatment has been greatly improved, but the prognosis of pharynx cancer is still not satisfactory, the 5 year survival rate of only about 40-50%.
Death associated protein kinase -1 (DAPk1 or DAPk) is a tumor suppressor gene discovered in recent years, which belongs to the family of DAPk proteins, including DAPk1, DAPk2/DRP-1, DAPk3/ZIPk/Dlk, DRAK1 and DRAK2.DAPk1 located on chromosome 9q34.1, encoding a protein of 160-kD, consisting of 1430 amino acids. It is catalyzed by enzymes District, Calmodulin regulatory region, 8 ankyrin repeats, ROC-COR domain, C- at the end of the death domain and a serine rich composition of.DAPk1 is a positive regulator of apoptosis, and as a serine / threonine kinase regulating, DAPk1 involved in multiple signaling pathways regulating autophagy, apoptosis, caspase dependent cell death, cell adhesion and migration. Therefore, it is not only to play a role in inflammation, also exerts antitumor and anti metastasis function. Nowadays, more and more anti swelling on the DAPk1 The tumor function has been excavated in succession, including inhibiting the integrin signal, promoting anoikis, inhibiting cell activity and regulating tumor metabolism.
As a tumor suppressor gene, DAPk1 is down regulated in many kinds of tumors, and DNA methylation of CpG islands in the promoter region is considered to be a major mechanism, it can influence gene transcription activity, but does not involve changes in DNA sequence of.DNA methylation is an important epigenetic mechanisms, and cell growth, differentiation and transformation is closely related in tumorigenesis, development also plays an important role. The tumor suppressor gene DNA promoter CpG island methylation in many tumors are frequent, DNA promoter hypermethylation of promoter region can be obtained by gene inactivation and mutation, censored such as lead to abnormal methylation.DAPk1 gene silence has reported in many cancers including laryngeal cancer, colorectal cancer, breast cancer and so on. Yet there are about DAPk1 in similar reports, hypopharyngeal cancer methylation and expression. Therefore, in this study, I We intend to examine the relationship between the methylation and expression of DAPk1 in hypopharyngeal carcinoma and its clinicopathological features and prognosis.
objective
We collected 53 pairs of hypopharyngeal squamous cell carcinoma tissues and adjacent non tumor mucosal tissue samples. We detected the methylation and expression of DAPk1 in hypopharyngeal squamous cell carcinoma, analyzed their relationship with clinicopathological characteristics, and evaluated the effect of DAPk1 on the prognosis of hypopharyngeal carcinoma.
Method
1.53 pairs of tumor tissues and cancer adjacent non tumor mucosa samples from 2010 in Qilu Hospital of Shandong University Department of Otorhinolaryngology of hypopharyngeal squamous cell carcinoma in hospitalized patients. The tumor tissue from the center of the tumor, and the corresponding adjacent non tumor tissues from morphology from the tumor edge at least 2cm normal mucosa.
2. we use methylation specific PCR (MSP) to detect the methylation status of DAPk1 in the sample, and using real-time quantitative RT-PCR, immunohistochemistry and Western blot analysis respectively from the expression of mRNA and protein level. DAPk1 detection and expression analysis of DAPk1 methylation relationship between, and its relationship with the clinical pathological characteristics and prognosis of the patient.
Result
1. DAPk1 methylation detection results in hypopharyngeal squamous cell carcinoma tumor tissue methylation rate was 60.38% (32/53), adjacent non tumor tissues of the methylation rate was 26.42% (14/53), hypopharyngeal carcinoma tumor tissue methylation rate was significantly higher than that of adjacent tissues, the difference was statistically significant between the two (P=0.001).
The relative expression of 2. tumor tissues by quantitative real-time RT-PCR DAPk1mRNA (0.863+0.095) than in adjacent non tumor tissues (1) was significantly reduced (P = 0.002); analysis of tumor tissue DAPk1 protein was expression level (0.459 + 0.036) than in adjacent non tumor mucosa tissue (0.666+0.037) decreased significantly (P0.001); in immunohistochemical staining, DAPk1 protein was mainly expressed in the cytoplasm, the positive expression of DAPk1 protein in tumor tissues was 32.08% (17/53), lower than that in the adjacent non tumor tissues 58.49% (31/53) (P= 0.006). These results show that mRNA and DAPk1 protein levels in pharyngeal squamous cell carcinoma in tumor tissue than in adjacent non tumor tissues were significantly lower in the lower, the difference was statistically significant.
Methylation group 3. hypopharyngeal carcinoma tumor tissue DAPk1mRNA relative expression of 0.633+0.095 was significantly lower than that of the relative expression of non methylation group DAPk1mRNA (1.212+0.167), the difference was statistically significant between the two, DAPk1 methylation and mRNA relative to the relationship between the expression of negative correlation (P=0.002, r=-0.521). There are regional lymph nodes node metastasis cases, the methylation level of DAPk1 increased (P=0.001), and the decreased expression of mRNA (0.516 + 0.069v.s.1.128 + 0.142, P=0.001); similarly, at a high level of TNM in the staging of the disease, patients with stage III and IV in the methylation level of DAPk1 increased (P=0.009), and the expression of mRNA reduce (0.698 + 0.096v.s.1.156 + 0.188, P=0.019). DAPk1 methylation in tumor tissues, mRNA expression and age, tumor stage, histological grade.
4. survival analysis showed that in 53 cases of patients with hypopharyngeal squamous cell carcinoma, the survival rate of DAPk1 methyl group were significantly higher than that of methyl groups (P=0.031, DAPk1) mRNA expression group was significantly higher than the survival rate of patients with down regulated expression group (P=0.045). In the multivariate Cox proportional hazard regression analysis and regional lymph node metastasis were independent prognostic factors (P=0.043, HR5.387,95%CI1.052-27.600) and DAPk1 methylation in tumor tissues, the expression of mRNA, tumor staging, clinical TNM stage were not the independent risk factors, their synergistic effect which affects the prognosis of hypopharyngeal squamous cell carcinoma.
conclusion
DAPk1 in hypopharyngeal squamous cell carcinoma tissues than in non methylation rate of the tumor tissues was significantly increased, and the mRNA and protein levels were decreased expression between methylation status and mRNA expression of DAPk1 showed hypermethylation and low expression of.DAPk1 is negatively related to regional lymph node and hypopharyngeal carcinoma transfer of high grade, clinical staging and prognosis is closely related to regional lymph node metastasis is the independent risk factors influencing the prognosis of hypopharyngeal cancer patients.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.6

【共引文獻(xiàn)】

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