口腔鳞状细胞癌中PRSS8及PTPN22的表达情况及甲基化状态的研究

发布时间：2018-05-27 03:16

本文选题：口腔鳞状细胞癌 + PRSS8　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)是世界范围内最常见的口腔颌面部恶性肿瘤之一。近年来大样本的流行病学研究表明,在中国,OSCC发病率呈逐年上升趋势,且发病年龄日趋年轻化,发病率在3.6/10-8.0/10万之间。因其具有恶性程度高,易发生淋巴结转移,预后差等特点,导致OSCC的5年生存率在50%左右。目前对于OSCC早期诊断及预后的研究较缺乏,当前的研究认为在OSCC发生发展中的基因变化因素主要包括基因突变与表观遗传修饰异常。DNA甲基化的研究可以使我们更好的了解OSCC发生机制,为OSCC的早期诊断及预后提供新的理论基础。现阶段研究表明,基因突变与表观遗传修饰异常是影响OSCC发生的重要因素,表观遗传修饰是一种可遗传、可逆转的生物学行为,主要包括DNA核苷酸胞嘧啶的甲基化修饰、组蛋白修饰、非编码RNA调控等。其中,DNA甲基化的研究成为表观遗传学的研究热点。PRSS8基因又称为人类的前列腺蛋白(Prostasin)或CAP-1,位于染色体16p11.2上,不仅能维持机体的正常生理功能,而且参与生长因子的调节、细胞的迁移多种炎症、表皮生长、肿瘤的发生等过程。近年来研究发现食管鳞癌、结直肠癌等癌组织中存在PRSS8基因甲基化状态及PRSS8mRNA表达异常,PRSS8基因的异常改变有可能参与了癌症的发生。蛋白酪氨酸磷酸酶非受体型22(PTPN22)基因位于染色体1p13.3-13.1上并参与上皮粘附。TCGA数据库显示PTPN22甲基化水平在肿瘤组织与正常组织之间有显著差异。然而,尽管PTPN22基因在癌症发生中有潜在重要性,但是关于它与肿瘤的报道很少。目前尚未发现PRSS8及PTPN22在OSCC中的表观遗传学变化相关报道。本研究以52例OSCC术后标本(河北医科大学第四医院口腔科)为研究对象,通过分析OSCC组织和相应正常黏膜组织中PRSS8及PTPN22基因的启动子区甲基化状态及其mRNA的表达水平,并结合患者的临床病理特征进行统计分析,旨在揭示OSCC的发病机制,为OSCC的早期发现与预后的评估提供实验理论依据。方法:1应用甲基化特异性聚合酶链反应(Methylation Specific PCR,MSP)检测52例经术后病理诊断为OSCC的癌组织及相应正常黏膜组织中PRSS8及PTPN22基因启动子区甲基化状态。2应用逆转录-聚合酶链反应(Reverse Transcriptase-PCR,RT-PCR)技术检测52例术后经病理诊断为OSCC的标本的癌组织及相应正常黏膜组织中PRSS8及PTPN22的mRNA相对表达量。3应用统计软件SPSS21.0对实验结果进行统计学分析。结果:1 OSCC和正常组织中PRSS8及PTPN22基因的甲基化状态52例OSCC组织中PRSS8及PTPN22基因启动子甲基化率分别为57.7%(30/52),51.9%(27/52),52例正常组织甲基化率为34.6%(18/52),30.8%(16/52),经统计学分析,OSCC组织PRSS8及PTPN22基因启动子甲基化率均明显高于相应正常组织,其差异都具有统计学意义(χ~2=5.571,P=0.018;χ~2=4.798,P=0.029)。(Table 1,2)(Fig.1,2)2 OSCC和正常组织中PRSS8及PTPN22基因mRNA的表达情况52例标本中OSCC组织和正常组织PRSS8mRNA的表达量均符合正态分析,PRSS8mRNA的相对表达量为0.44±0.12,相应正常组织的表达量为0.71±0.13,采用配对t检验,PRSS8基因mRNA的相对表达量在OSCC组织中明显低于相应正常组织,其差异具有统计学意义(t=-10.620,P=0.000)。(Table 3)(Fig.3)PTPN22 mRNA的相对表达量为0.44±0.20,相应正常组织的表达量为0.63±0.20,PTPN22基因mRNA的相对表达量在OSCC组织中明显低于相应正常组织,其差异具有统计学意义(t=-4.647,P=0.000)。(Table 4)(Fig.4)3 PRSS8及PTPN22基因甲基化组与非甲基化组中mRNA相对表达量的比较在52例OSCC组织中,甲基化组PRSS8mRNA的相对表达量为0.43±0.12,非甲基化组为0.52±0.11,利用独立样本t检验进行分析,结果显示甲基化组与非甲基化组PRSS8mRNA相对表达量之间的差异具有统计学意义(t=-2.880,P=0.006)。(Table 5)PTPN22mRNA在甲基化组相对表达量为0.47±0.18,在非甲基化组相对表达量为0.56±0.08,甲基化组与非甲基化组中PTPN22mRNA相对表达量之间的差异有统计学意义(t=-2.171,P=0.035)。(Table6)4 PRSS8和PTPN22基因启动子甲基化率与患者临床参数之间的关系男性组PRSS8和PTPN22基因启动子甲基化率分别为53.3%(16/30),46.7%(14/30),女性组甲基化率分别为63.6%(14/22),59.1%(13/22),其差异均无统计学意义(χ~2=0.552,P=0.458),(χ~2=0.785,P=0.376);年龄≥60岁组甲基化率分别为62.5%(20/32),56.3%(18/32),年龄60岁组患者甲基化率分别为50.0%(10/20),45.0%(9/20),其差异均无统计学意义(χ~2=0.788,P=0.375),(χ~2=0.624,P=0.430);饮酒组甲基化率分别为59.3%(16/27),51.9%(14/27),不饮酒组甲基化率分别为56.0%(14/25),52.0%(13/25),其差异均无统计学意义(χ~2=0.056,P=0.812);(χ~2=0.000,P=0.991);吸烟组甲基化率分别为51.7%(15/29),48.3%(14/29),不吸烟组甲基化率分别为65.2%(15/23),56.5%(13/23),其差异均无统计学意义(χ~2=0.957,P=0.328),(χ~2=0.349,P=0.554);临床Ⅰ期+Ⅱ期组甲基化率分别为38.1%(8/21),28.6%(6/21),临床Ⅲ期+Ⅳ期组甲基化率分别为71.0%(22/31),67.7%(21/31),其差异均有统计学意义(χ~2=5.543,P=0.019),(χ~2=66.589,P=0.000);淋巴结转移组甲基化率分别为73.9%(17/23),65.2%(13/23),无淋巴结转移组甲基化率分别为44.8%(13/29),41.4%(12/29),差异均有统计学意义(χ~2=4.446,P=0.035),(χ~2=2.920,P=0.047)。高中分化组甲基化率分别为47.1%(16/34),41.2%(14/34),低分化组甲基化率分别为77.8%(14/18),72.2%(13/18),差异均具有统计学意义(χ~2=4.550,P=0.033),(χ~2=4.544,P=0.033)。(Table 7,8)5 OSCC中PRSS8和PTPN22基因mRNA相对表达水平及与临床参数之间的关系男性组患者PRSS8和PTPN22mRNA的相对表达量分别为0.44±0.13,0.41±0.19,女性组的相对表达量分别为0.46±0.11,0.49±0.21,差异均无统计学意义(t=-0.586,P=0.560),(t=-1.481,P=0.145);年龄≥60岁组的相对表达量分别为0.45±0.13,0.44±0.20,年龄60岁组相对表达量分别为0.43±0.10,0.46±0.20,差异均无统计学意义(t=-0.481,P=0.633),(t=-0.451,P=0.654);饮酒组的相对表达量分别为0.43±0.11,0.47±0.22,不饮酒组的相对表达量分别为0.47±0.13,0.42±0.17,差异均无统计学意义(t=-1.197,P=0.237),(t=1.016,P=0.315);吸烟组相对表达量分别为0.44±0.11,0.48±0.22,不吸烟组相对表达量分别为0.45±0.13,0.40±0.18,差异均无统计学意义(t=-475,P=0.637),(t=1.323,P=0.192);根据TNM分期,进行临床分期,其中Ⅰ+Ⅱ期相对表达量分别为0.49±0.11,0.56±0.12,Ⅲ+Ⅳ期分别为0.41±0.12,0.46±0.20,均具有显著差异,差异有统计学意义(t=2.335,P=0.024),(t=2.076,P=0.043)。淋巴结转移组分别为0.42±0.11,0.44±0.17,无淋巴结转移组分别为0.49±0.13,0.55±0.11,差异有统计学意义(t=-2.119,P=0.039),(t=-2.688,P=0.010)。高中分化组相对表达量分别为0.48±0.12,0.57±0.11,低分化组相对表达量分别为0.39±0.11,0.49±0.12,具有统计学意义(t=2.736,P=0.009),(t=2.188,P=0.033)。(Table9,10)结论:1 PRSS8和PTPN22基因在OSCC组织中的mRNA相对表达量明显低于其相应的正常黏膜组织,提示PRSS8和PTPN22基因在OSCC中可能主要起抑癌基因的作用。2 OSCC组织中PRSS8和PTPN22基因启动子区甲基化率明显高于其相应的正常黏膜组织,提示PRSS8和PTPN22基因甲基化可能是OSCC发生的机制之一。3 PRSS8和PTPN22启动子区甲基化率与患者性别、年龄等因素无关,与分化程度、淋巴结转移、病理分级等有关,提示PRSS8和PTPN22基因启动子区甲基化可能与OSCC的预后有关。
[Abstract]:Objective: oral squamous cell carcinoma (OSCC) is one of the most common oral and maxillofacial malignant tumors in the world. In recent years, the epidemiological study of large samples shows that the incidence of OSCC is increasing year by year in China, and the age of the disease is becoming younger and younger. The incidence of OSCC is between 3.6/10-8.0/10 million. High degree of sex, easy to occur lymph node metastasis, poor prognosis and so on, resulting in the 5 year survival rate of about 50% of OSCC. At present, the research on early diagnosis and prognosis of OSCC is lack. The current research suggests that the gene change factors in the development of OSCC mainly include gene mutation and epigenetic modification of abnormal.DNA methylation. We have a better understanding of the OSCC mechanism and provide a new theoretical basis for the early diagnosis and prognosis of OSCC. Current studies have shown that gene mutation and epigenetic modification are an important factor affecting the occurrence of OSCC. Epigenetic modification is a genetic, reversible biological behavior, mainly including the methylation of the DNA nucleotides cytosine. Chemical modification, histone modification, non coded RNA regulation, among which, DNA methylation has become the focus of epigenetic research. The.PRSS8 gene, known as human prostate protein (Prostasin) or CAP-1, is located on chromosome 16p11.2. It not only maintains normal physiological functions of the body, but also participates in the regulation of growth factors and the migration of cells. In recent years, it has been found that the methylation status of PRSS8 gene and the abnormal PRSS8mRNA expression in the cancer tissues of the esophageal squamous cell carcinoma and colorectal cancer have been found, and the abnormal changes of the PRSS8 gene may be involved in the occurrence of cancer. The protein tyrosine phosphatase non receptor 22 (PTPN22) gene is located in the chromosome 1p13.3 -13.1 and involved in the epithelial adhesion.TCGA database show that there is a significant difference between the level of PTPN22 methylation in the tumor tissue and the normal tissue. However, although the PTPN22 gene is of potential importance in the occurrence of cancer, there are few reports about it and the tumor. The epigenetic changes associated with PRSS8 and PTPN22 in OSCC have not been found. In this study, 52 specimens of OSCC (Department of Stomatology, Hebei Medical University, Fourth Hospital of Hebei Medical University) were studied. The methylation status of the promoter region of the PRSS8 and PTPN22 genes in the OSCC tissues and the corresponding normal mucosa tissues and the expression level of mRNA were analyzed, and the clinicopathological features of the patients were statistically analyzed in order to reveal the hair of OSCC. The mechanism of the disease provides experimental theoretical basis for the assessment of early detection and prognosis of OSCC. Methods: 1 the methylation specific polymerase chain reaction (Methylation Specific PCR, MSP) was used to detect the reversal of the methylation status of PRSS8 and PTPN22 gene promoter regions in the cancerous tissues of OSCC and in the corresponding normal mucosal tissues after the pathological diagnosis of OSCC. Reverse Transcriptase-PCR (RT-PCR) technique was used to detect the mRNA relative expression of PRSS8 and PTPN22 in the specimens of 52 patients with OSCC after surgery and.3 application statistics software SPSS21.0 to analyze the results of the experimental data. Results: 1 OSCC and PRSS8 and PTPN22 in normal tissues. The methylation status of genes in 52 OSCC tissues was 57.7% (30/52), 51.9% (27/52) and 34.6% (18/52) and 30.8% (16/52) in 52 normal tissues, respectively. The methylation rates of PRSS8 and PTPN22 genes in OSCC tissues were significantly higher than those of the corresponding normal tissues, and the differences were all of the differences. Statistical significance (x ~2=5.571, P=0.018, X ~2=4.798, P=0.029). (Table 1,2) (Fig.1,2) 2 OSCC and the expression of PRSS8 and PTPN22 gene mRNA in normal tissues. The expression of OSCC tissue and normal tissues in 52 specimens were both 0.44 + 0.12, and the expression of the corresponding normal tissues was 0.71 The relative expression of PRSS8 gene mRNA was significantly lower than that of the corresponding normal tissue in OSCC tissue by paired t test. The difference was statistically significant (t=-10.620, P=0.000). (Table 3), the relative expression of PTPN22 mRNA was 0.44 + 0.20, the expression of the corresponding normal fabric was 0.63 + 0.20, and the relative expression of PTPN22 gene mRNA The difference in CC tissue was significantly lower than that of the corresponding normal tissue (t=-4.647, P=0.000). (Table 4) (Fig.4) the relative expression of mRNA in the methylation group and the non methylation group was compared with the non methylation group in 52 cases of OSCC tissues, the relative expression of PRSS8mRNA in the methylation group was 0.43 + 0.12, and the non methylation group was 0.52 + 0.11. Analysis by independent sample t test showed that the difference between the relative expression of PRSS8mRNA in methylation group and non methylation group was statistically significant (t=-2.880, P=0.006). (Table 5) PTPN22mRNA in methylation group was 0.47 + 0.18, the expression amount was 0.56 + 0.08 in non methylation group, methylation group and non methylation group. The differences in the relative expression of PTPN22mRNA were statistically significant (t=-2.171, P=0.035). (Table6) the relationship between the methylation rate of 4 PRSS8 and PTPN22 gene promoters and the clinical parameters of the patients was 53.3% (16/30), 46.7% (14/30), and 63.6% (14/22) and 59.1% (1) in the female group, respectively. 3/22), the difference was not statistically significant (x ~2=0.552, P=0.458), (x ~2=0.785, P=0.376), the methylation rates of the age group were 62.5% (20/32), 56.3% (18/32) and 50% (10/20) and 45% (9/20) in the 60 year old group, respectively, and the difference was not statistically significant (x ~2=0.788, P=0.375), and the methylation of the drinking group. The rates of methylation were 59.3% (16/27) and 51.9% (14/27). The methylation rates of non drinking groups were 56% (14/25) and 52% (13/25). The differences were not statistically significant (x ~2=0.056, P=0.812), and the methylation rates of smoking group were 51.7% (15/29), 48.3% (14/29), 65.2% (15/23) and 56.5% (13/23), respectively, and the differences were not all Statistical significance (x ~2=0.957, P=0.328), (x ~2=0.349, P=0.554); the methylation rates in phase I + stage group were 38.1% (8/21), 28.6% (6/21), and 71% (22/31) and 67.7% (21/31) in phase III + IV group, respectively, with statistical significance (x ~2=5.543, P=0.019), (chi ~2=66.589, P=0.000), and lymph node metastasis group methylation rates. The methylation rates of 73.9% (17/23), 65.2% (13/23), no lymph node metastasis group were 44.8% (13/29) and 41.4% (12/29). The difference was statistically significant (x ~2=2.920, P=0.047). The methylation rates of the high school group were 47.1% (16/ 34), 41.2% (14/34), and 41.2% (14/18), 72.2% (13/18), respectively, 72.2% (13/18), and 72.2% (13/18), respectively. Statistical significance (x ~2=4.550, P=0.033), (x ~2=4.544, P=0.033). (Table 7,8) the relative expression level of PRSS8 and PTPN22 gene mRNA in 5 OSCC and the relationship with the clinical parameters, the relative expression of PRSS8 and PTPN22mRNA in the male group was 0.44 + 0.19, and the relative expression of the female group was 0.46 + 0.21, respectively. The difference was not statistically significant (t=-0.586, P=0.560), (t=-1.481, P=0.145), the relative expression of age group was 0.45 + 0.13,0.44 0.20, and the relative expression of age 60 year old group was 0.43 + 0.10,0.46 + 0.20 respectively, the difference was not statistically significant (t=-0.481, P= 0.633), (t=-0.451, P=0.654), and the relative expression of alcohol drinking group was 0.43 + 0, respectively. The relative expression of.11,0.47 + 0.22 was 0.47 + 0.13,0.42 + 0.17, respectively, and the difference was not statistically significant (t=-1.197, P=0.237), (t=1.016, P=0.315), and the relative expression of smoking group was 0.44 + 0.11,0.48 + 0.22 respectively, and the relative expression of non smoking group was 0.45 + 0.13,0.40 + 0.18 respectively, and the difference was not statistically significant (T, P=0.637). (T, P=0.637) =1.323, P=0.192), according to TNM staging, clinical staging was carried out, in which the relative expression of stage I + II was 0.49 + 0.11,0.56 + 0.12 and 0.41 + 0.12,0.46 + 0.20 respectively. The difference was significant (t=2.335, P=0.024), (t=2.076, P=0.043). The lymph node metastasis group was 0.42 + 0.11,0.44 + 0.17, no lymph node. The transfer group was 0.49 + 0.13,0.55 + 0.11 respectively (t=-2.119, P=0.039), (t=-2.688, P=0.010). The relative expression of the high school division group was 0.48 + 0.12,0.57 + 0.11 respectively, and the relative expression of the low differentiation group was 0.39 + 0.11,0.49 + 0.12 respectively, which had statistical significance (t=2.736, P=0.009), (t=2.188, P=0.033). (Table9,10) conclusion: 1 The relative expression of the RSS8 and PTPN22 genes in the OSCC tissues was significantly lower than that of the corresponding normal mucosa, suggesting that the PRSS8 and PTPN22 genes may play a role in the tumor suppressor gene in OSCC. The methylation rate of the PRSS8 and PTPN22 gene promoter regions in.2 OSCC tissues is significantly higher than that of the corresponding normal mucosal tissue, suggesting PRSS8 and bases. Methylation may be one of the mechanisms of OSCC, one of the mechanisms of.3 PRSS8 and PTPN22 promoter methylation is related to the degree of differentiation, lymph node metastasis, and pathological classification. It suggests that the methylation of the promoter region of the PRSS8 and PTPN22 genes may be related to the preimplantation of OSCC.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.8

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