【作者】 李春艳；

【导师】 高文信； 周延民；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2009， 博士

【摘要】 口腔鳞状细胞癌(Oral Squamous Cell Carcinoma, OSCC)是口腔颌面部最常见的恶性肿瘤,其发病机理目前尚不明确。本研究在吉林省科技厅基金资助下(项目编号200705349)对凋亡蛋白酶活化因子1(Apaf-1)和死亡相关蛋白激酶(DAPK)在OSCC发生发展过程中的作用机制及去甲基化药物5-氮杂-2’-脱氧胞苷(5-Aza-deoxycytidine, 5-Aza-CdR)对舌鳞癌Tca8113细胞生物学行为的影响进行探讨。目前国内外罕有见到对Apaf-1基因、DAPK基因在OSCC组织及细胞系中表达及甲基化情况系统研究的报道。本研究应用免疫组织化学方法和半定量RT-PCR方法检测Apaf-1和DAPK蛋白及mRNA表达,结果显示正常口腔黏膜组织Apaf-1、DAPK蛋白及mRNA表达无减少或缺失。OSCC组织中Apaf-1、DAPK蛋白表达及mRNA表达均显著低于正常口腔黏膜。应用甲基化特异性PCR检测基因启动子区甲基化情况,结果显示正常口腔黏膜组织中Apaf-1基因和DAPK基因启动子区无甲基化。OSCC组织中Apaf-1基因、DAPK基因启动子区甲基化率均显著高于正常口腔黏膜,基因启动子区甲基化与其在肿瘤组织中的蛋白表达及mRNA表达下调呈正相关。Apaf-1基因和DAPK基因的蛋白表达、mRNA表达及基因启动子区甲基化均与肿瘤组织的病理分级、患者的年龄及性别无关。应用去甲基化药物5-Aza-CdR处理舌鳞癌Tca8113细胞,研究Apaf-1基因、DAPK基因去甲基化的转录调节作用,结果显示经5-Aza-CdR处理,舌鳞癌Tca8113细胞的存活率明显降低,凋亡率明显增高,Apaf-1基因、DAPK基因发生去甲基化及mRNA表达上调,且呈药物剂量依赖性。通过本研究,得出以下结论:1. OSCC的发生发展与Apaf-1和DAPK的mRNA及蛋白表达明显降低密切相关。2.口腔鳞状细胞癌组织中,Apaf-1基因和DAPK基因启动子区发生甲基化,是导致Apaf-1和DAPK蛋白表达、mRNA表达下调的重要原因之一。3. Apaf-1基因、DAPK基因去甲基化后,Apaf-1基因、DAPK基因表达上调,促进肿瘤细胞凋亡,因此Apaf-1基因、DAPK基因可作为口腔鳞状细胞癌治疗的潜在靶点。4. Apaf-1和DAPK基因的表达及甲基化均与OSCC的病理分级、患者的年龄及性别无关。更多还原

【Abstract】 Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors in oral and maxillofacial region. Its occurrence and development is a multi-stage process. In this process a variety of gene level changes, including activation of oncogenes and inactivation of tumor suppressor genes. Apoptosis is the mechanism responsible for the physiological deletion of cells and appears to be intrinsically programmed in certain physiological or pathological conditions. Apoptosis-related inactivation of tumor suppressor genes leads to abnormal cell proliferation, and eventually results in tumorigenesis.Apoptosis protease activating factor-1(Apaf-1), as the really core of apoptosome complex, is an important pro-apoptotic factor in mitochondrial apoptosis pathway. Apaf-1 plays a role in activation of Caspase-3, simultaneously needing the participation of Apaf-2 (Cyto C) and Apaf-3 (Caspase-9). After Apaf-1 / Cyto C complex binding with ATP / dATP, they can form the apoptosome complex which activate Caspase-3 and start Caspase cascade reaction of apoptosis through Apaf-1’s CARD domain convening Caspase-9. Because of the important role of Apaf-1 in apoptosis pathway, the abnormality of Apaf-1 leads to the disorder of apoptosis process. Apaf-1 is also directly or indirectly associated with many diseases, such as: cancer or the nervous system diseases and so on.Death-associated protein kinase (DAPK), which participates many transduction pathway mediated by the P53, TNF-α, Fas, IFN-γ, TNF-βand other apoptosis factors, is a positive regulator of apoptosis. DAPK was considered as a tumor suppressor gene and closely related to the occurrence, development, and metastasis of tumor. At the early stage of tumorigenesis, the abnormal expression of DAPK inhibits apoptosis so that leads to normal cells continually division, proliferation and finally tumor formation; while in the case of tumor metastasis, DAPK inhibits cell detachment from the extracellular matrix, which induce tumor cells tolerance to various apoptosis stimuli.The relationship between DAPK gene and apaf-1 gene in p53-dependent apoptotic pathway:DAPK can activate p19ARF/p53-dependent apoptotic pathway through phosphorylation of p19ARF. Then p53 triggers mitochondrial apoptotic pathway so that lead to DNA fragmentation and apoptosis by means of inducing the expression of some specific apoptotic genes including Bax, PUMA, Noxa, Apaf-1, et al. DAPK gene and Apaf-1 gene, which play an important role in p53-dependent apoptotic pathway, are respectively located in the upstream and downstream of p53. The importance of p53 in starting and early stage of apoptosis has been widely confirmed. The research on these two genes’ expression in the OSCC will contribute to explore anti-apoptotic pathway of tumor cells and provide guidance for future treatment. Epigenetics is a study of heritable changes occurring in gene function without changes in the sequence of nuclear DNA. In recent years, there has been rapid progress in understanding epigenetic mechanisms which include DNA methylation, changes in chromatin structure, as well as the changes in genomes of cytoplasmic elements (for instance: chloroplasts and mitochondria). Other mechanisms have also been proposed. These changes will induce the phenomena such as gene silencing, imprinting, para-mutation and RNA interference; therefore, the abnormal modification may lead to tumorigenesis. Despite molecular pathogenesis of the tumor remains unclear, the close relationship between the tumor occurrence and the abnormal methylation of tumor suppressor genes has been recognized by the majority of scholars. The abnormal methylation of genes mainly concentrated in the CpG island in their 5’-ending promoter regions.Scholars have found that Apaf-1 is a kind of tumor suppressor gene which rarely mutate. The mechanism underlying Apaf-1 gene’s loss of function includes loss of heterozygosity (LOH) and promoter methylation. The methylation-induced gene silencing is the main reason for the down-regulation of gene expression. Also, as tumor suppressor gene, DAPK gene has been concerned by scholars because of its expression down-regulation in variety kinds of tumors. Studies have shown that the down-regulation of DAPK gene expression is relevant with the promoter hypermethylation.Transcriptional silencing of tumor suppressor genes led by methylation of CpG island in promoter regions is a reversible epigenetic modification proces,sand this kind of reversal may resume the function of the tumor suppressor genes, suggesting demethylation of DNA as a novel gene therapeutics. The biological activity of 5-Aza-deoxycytidine as a methylation inhibitor is associated with its incorporation into cellular DNA and/or RNA, with subsequent sequestration of DNA methyl transferases (DNMT) via covalent bond formation between C6 of 5-Aza- deoxycytidine and cysteine thiolate of DNMTs. Under physiologic conditions, this enzyme-DNA/RNA adduct is formed irreversibly, thereby depleting the cells of DNMT activity and causing demethylation of cellular DNA. The 5-Aza-deoxycytidine’s role of demethylation can reactivate a variety of tumor suppressor genes to restore their function of inducing apoptosis, as well as inhibiting tumor cell growth and providing therapeutics for chemotherapy-resistance tumors. Therefore, it may have broad clinical application prospects.DAPK and Apaf-1 are apoptosis-related tumor suppressor genes. Their expression and methylation level in tissues or cells of OSCC have rarely been reported. In this research we detected expression and methylation of DAPK gene and Apaf-1 gene in OSCC tissues and tongue squamous cell carcinoma Tca8113 cell lines. Meanwhile, we observed demethylation in transcriptional regulation of DAPK gene and Apaf-1 gene in Tca8113 cell lines. Specific research contents and results are as follow:Ⅰ. Study on expression of Apaf-1 gene and DAPK gene in OSCC The protein expression and mRNA expression of Apaf-1 gene and DAPK gene was detected by immunohistochemistry and semiquantitative RT-PCR in 23 cases of normal oral mucosa and 53 cases of OSCC tissues.Results are as follow:1. We detected the protein expression and mRNA expression of Apaf-1 gene and DAPK gene in all 23 cases of normal oral mucosa. The positive rate was 100%. There was no gene reduction or deletion.2. Apaf-1 protein were positively stained in 23 cases out of 53 OSCC cases (43.4%), The expression of Apaf-1 protein was significantly decreased in OSCC tissues compared with in normal oral mucosa(P<0.01).3. DAPK protein were positively stained in 17 cases out of 53 OSCC cases (32.1%), The expression of DAPK protein was significantly decreased in OSCC tissues compared with in normal oral mucosa(P<0.01).4. Only in two OSCC tissues of the 53 cases (3.77%), the mRNA index of Apaf-1 gene was 50% greater than that in normal tissues. While in the other 51 cases (96.23%), the mRNA index of Apaf-1 gene showed decreased expression. The expression of Apaf-1 gene was significantly decreased in OSCC tissues compared with in normal oral mucosa(P<0.01).5. Only in three OSCC tissues of 53 cases (5.66%), the mRNA index of DAPK gene was 50% greater than that in normal tissues. While in the other 50 cases (94.34%), the mRNA index of DAPK gene showed decreased expression. The expression of DAPK gene was significantly decreased in OSCC tissues compared with in normal oral mucosa(P<0.01).6. Protein expression and mRNA expression of Apaf-1 gene and DAPK gene was not significantly correlated with the pathological grade, age and gender of patients (P> 0.05).7. The Pearson correlation analysis between Apaf-1 gene and DAPK gene: In statistics, the Pearson correlation coefficient was 0.950(P<0.01),which manifested the expression of these two genes in OSCC tissues had correlation.Ⅱ. Study on methylation of Apaf-1 gene and DAPK gene in OSCC The methylation in promoter region of Apaf-1gene and DAPK gene were detected by methylation specific PCR in 23 cases of normal oral mucosa and 53 cases of OSCC tissues.Results are as follow:1. There was no methylation in promoter region of Apaf-1 gene and DAPK gene in normal oral mucosa.2. We detected exhaustive methylation in promoter region of Apaf-1 gene in 41 cases(77.36%)and partial methylation in 5 cases (9.43%)in OSCC. The total methylation rate was 86.79%(46/53) in OSCC, which has significant differences compared with normal oral mucosa(P<0.01). 3. We detected exhaustive methylation in promoter region of DAPK gene in 30 cases(56.60%)and partial methylation in 8 cases( 15.09%)in OSCC. The total methylation rate was 71.69%(38/53) in OSCC, which has significant difference compared with normal oral mucosa(P<0.01).4. The methylation of Apaf-1 gene and DAPK gene was not significantly correlated with pathological grade, age and gender of patients(P>0.05).5. Promoter methylation of Apaf-1 gene and DAPK gene tended to correlate positively with their decreased protein expression and mRNA expression in tumor tissues.Ⅲ. Study on demethylation treatment of Apaf-1 gene and DAPK gene in human tongue squamous cell carcinoma cell line Tca8113To study the effect of demethylated Apaf-1 gene and DAPK gene on Tca8113 cells, we detected Tca8113 cells treated by 5-Aza-deoxycytidine by means of MTT, Annexin V-PI dual stain, TUNEL, methylation specific PCR and semiquantitive RT-PCR.Results are as follow:1. In tongue Tca8113 cells treated by 5-Aza-deoxycytidine, the cell viability rate was significantly decreased, which has significant differences compared with that of control group(P<0.05). The cell viability appears a dose-dependent effect.2. The apoptosis of Tca8113 cells were induced by 5-Aza-deoxycytidine and has significant difference compared with that of control group(P<0.05). The cell apoptosis appears a dose-dependent effect.3. After treated by 5-Aza-deoxycytidine, Apaf-1 gene and DAPK gene were demethylated and the most obvious effect was observed in 5μmol/L 5-Aza group.4. After treated by 5-Aza-deoxycytidine, the expression Apaf-1 mRNA and DAPK mRNA in Tca8113 cells was increased, which has significant difference compared with control group(P<0.05). The mRNA expression appears a dose-dependent effect.5. The mRNA expression of Apaf-1 gene and DAPK gene were upregulated after gene demethylation, which lead to gene exerting their pro-apoptotic role. They can be potential targets for the therapy of oral squamous cell carcinoma. On the base of the results above, we can conclude as follow: 1. The occurrence and development of OSCC are closely related to significantly decrease of the mRNA and protein expression of Apaf-1 gene and DAPK gene.2. In OSCC tissues, the promoter region of Apaf-1 gene and DAPK gene are usually methylated. Gene methylation is one of the main reasons that lead to the decreasing expression of the mRNA and protein of the Apaf-1 gene and DAPK gene.3. The expression of Apaf-1 gene and DAPK gene were both increased after demethylation, which promoted apoptosis of the tumor cells. Therefore, Apaf-1 gene and DAPK gene can be used as the potential target for oral squamous cell carcinoma treatment.4. The expression and methylation of Apaf-1 gene and DAPK gene were not significantly correlated with the pathological grade, age and gender of patients.更多还原