DAPK1在下咽鳞状细胞癌中的异常甲基化及表达

发布时间：2018-02-01 19:01

本文关键词： DAPk1 下咽鳞状细胞癌甲基化表达　出处：《山东大学》2014年博士论文　论文类型：学位论文

【摘要】：研究背景下咽部的恶性肿瘤是头颈部恶性程度最高的肿瘤之一,而鳞状细胞癌是最常见的恶性肿瘤类型,下咽鳞状细胞癌占头颈肿瘤的5%左右。下咽癌的发生、发展是一个多阶段的过程,其中包含了多个基因水平的改变。由于其解剖部位的特殊性,下咽癌的早期症状不明显,且大多数分化程度较差,局部呈侵袭性生长并易沿黏膜下浸润和扩散,在病程早期就容易出现颈部淋巴结的转移。现如今,多学科联合综合治疗是下咽鳞状细胞癌主要的治疗方案,包括手术、放射疗法及化疗,近年来,尽管治疗方法得到了很大的改进,但是下咽癌的预后仍不尽如人意,5年生存率仅在40-50%左右。死亡相关蛋白激酶-1(DAPk1,或简称为DAPk)是近年来发现的一种抑癌基因,属于DAPk蛋白家族,后者包括DAPk1, DAPk2/DRP-1, DAPk3/ZIPk/Dlk, DRAK1和DRAK2。DAPk1定位于人染色体9q34.1,编码的蛋白分子量为160-kD,由1430个氨基酸组成。它由酶催化区、钙调蛋白调节区、8个锚蛋白重复序列、ROC-COR域,死亡结构域和富含丝氨酸的C-末端组成。DAPk1是一种凋亡的正性调节因子,且作为一种丝氨酸/苏氨酸调节激酶,DAPk1参与到多种信号通路调节凋亡、自噬、半胱天冬酶依赖的细胞死亡、细胞粘附和迁移。因此,它不仅起到在炎症反应中起到一定的作用,还发挥着抗肿瘤与抑制转移的功能。现如今,越来越多关于DAPk1的抗肿瘤功能被陆续发掘,包括抑制整合素信号、推动失巢凋亡、抑制细胞活动、肿瘤代谢的调节等。作为一个抑癌基因,DAPk1在许多种类的肿瘤中都呈现表达下调,而DNA启动子区CpG岛的甲基化被认为是一个主要的机制,它能够影响基因转录活性,却不涉及DNA的序列改变。DNA甲基化是表观遗传学的重要机制,与细胞生长、分化及转化密切相关,在肿瘤的发生、发展中也起到重要的作用。抑癌基因的DNA启动子区CpG岛异常甲基化在许多肿瘤中均频繁发生,DNA启动子区的高甲基化状态可通过基因失活、删失及突变等方式导致基因沉默。DAPk1的异常甲基化在许多肿瘤中均有报道,包括喉癌、结直肠癌、乳腺癌等。目前尚未有关于DAPk1在下咽癌中的甲基化及表达情况的类似报道,因此,在本次研究中,我们拟通过检测DAPk1在下咽癌中的甲基化及表达情况,探讨其与临床病理特征和预后的关系。目的我们收集了53对下咽鳞状细胞癌肿瘤组织和癌旁非肿瘤黏膜组织样本,拟检测下咽鳞状细胞癌中DAPk1的甲基化及表达情况,分析其与临床病理特征的关系,并评价DAPk1对下咽癌患者预后的影响。方法 1.53对肿瘤组织及癌旁非肿瘤黏膜组织样本来源于2010年山东大学齐鲁医院耳鼻咽喉科的下咽鳞状细胞癌住院手术患者。肿瘤组织取自肿瘤的中心,而相应的癌旁非肿瘤黏膜组织取自距离肿瘤边缘至少2cm的形态学正常的黏膜组织。 2.我们采用甲基化特异性PCR(MSP)来检测样本中DAPk1的甲基化状态,并利用实时定量RT-PCR、免疫组织化学染色和蛋白质印迹分析分别从mRNA水平和蛋白水平检测DAPk1的表达情况。分析DAPk1的甲基化及表达情况之间的关系,以及其与患者的临床病理特征和预后的关系。结果 1. DAPk1甲基化检测结果显示在下咽鳞状细胞癌肿瘤组织中甲基化率为60.38%(32/53),癌旁非肿瘤黏膜组织中甲基化率为26.42%(14/53),下咽癌中肿瘤组织的甲基化率明显高于癌旁组织,两者之间的差异具有统计学意义(P=0.001)。 2.实时定量RT-PCR中肿瘤组织的DAPk1mRNA相对表达量(0.863+0.095)较癌旁非肿瘤黏膜组织中(1.000)明显下调(P＝0.002)；蛋白质印迹分析肿瘤组织中DAPk1的蛋白表达水平(0.459±0.036)较癌旁非肿瘤黏膜组织中(0.666+0.037)明显降低(P0.001)；免疫组织化学染色中,DAPk1蛋白主要在胞浆表达,DAPk1在肿瘤组织中的蛋白表达阳性率为32.08%(17/53),低于癌旁非肿瘤黏膜组织中的58.49%(31/53)(P=0.006)。以上结果显示在下咽鳞状细胞癌肿瘤组织中DAPk1的mRNA及蛋白水平较癌旁非肿瘤黏膜组织均明显降低,差异具有统计学意义。 3.下咽癌肿瘤组织中甲基化组的DAPk1mRNA相对表达量为0.633+0.095,显著低于非甲基化组的DAPk1mRNA相对表达量(1.212+0.167),两者之间的差异具有统计学意义,DAPk1甲基化与mRNA的相对表达之间的关系呈负相关(P=0.002,r=-0.521)。存在区域淋巴结转移的病例中,其DAPk1甲基化程度增高(P=0.001),且mRNA表达降低(0.516±0.069v.s.1.128±0.142,P=0.001)；同样地,在高等级的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表达降低(0.698±0.096v.s.1.156±0.188,P=0.019)。肿瘤组织中DAPk1甲基化、mRNA表达与年龄、原发肿瘤分期、组织病理学分级无关。 4.生存分析结果显示在53例下咽鳞状细胞癌患者中,DAPk1未甲基化组患者的生存率显著高于甲基化组患者(P=0.031), DAPk1的mRNA表达上调组的患者生存率也显著高于表达下调组(P=0.045)。在多因素Cox比例风险回归分析中,区域淋巴结转移是影响预后的独立危险因素(P=0.043,HR5.387,95%CI1.052-27.600)。而DAPk1在肿瘤组织中的甲基化、mRNA表达,原发肿瘤分期、临床TNM分期均不能作为独立危险因素,它们协同发挥作用从而影响下咽鳞状细胞癌患者的预后。结论 DAPk1在下咽鳞状细胞癌肿瘤组织中较癌旁非肿瘤黏膜组织中的甲基化率明显增高,且mRNA及蛋白水平均表达降低,DAPk1的甲基化状态和mRNA表达情况两者之间呈负相关。DAPk1的高甲基化及低表达与下咽癌的区域淋巴结转移、高等级临床分期及预后不良密切相关,区域淋巴结转移是影响下咽癌患者预后的独立危险因素。
[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.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.6

【共引文献】