哈族食管鳞癌组织中PLCE1基因启动子甲基化状态及其临床意义

发布时间：2018-04-04 04:50

本文选题：哈萨克族　切入点：食管鳞癌　出处：《石河子大学》2017年硕士论文

【摘要】：目的:探讨PLCE1基因启动子甲基化程度与PLCE1蛋白表达的关系及其与临床病理特征的关系;检测4株不同食管鳞癌细胞系Eca109,TE-1,EC9706,Kyse150中PLCE1基因启动子甲基化程度与PLCE1蛋白表达的关系并探讨甲基转移酶抑制剂5-aza-d C对细胞PLCE1甲基化程度与PLCE1蛋白表达的影响;应用PLC抑制剂U73122后对食管鳞癌细胞生长、凋亡及侵袭转移的影响。方法:收集51例新疆哈萨克族食管鳞癌组织及对应的51例癌旁正常组织,用免疫组化Envision法检测PLCE1的蛋白表达,并提取这51对癌与癌旁组织的DNA,应用甲基化特异性PCR(MSP)技术检测PLCE1基因启动子甲基化状态,分析PLCE1基因启动子甲基化程度与PLCE1蛋白表达的相关性,及其与患者性别、年龄、肿瘤最大直径、淋巴结和远处转移、TNM分期及预后的相关性;用Western blot检测4株食管癌细胞系在5-aza-d C作用前后的PLCE1蛋白表达,并用MSP检测它们的PLCE1基因启动子甲基化状态;将PLC抑制剂U73122作用于食管癌细胞系Eca109、EC9706,应用Western blot检测抑制效率,并在作用48小时后,用MTT和平板克隆检测细胞生长情况,流式细胞术和TUNEL染色检测细胞凋亡情况,transwell侵袭实验检测细胞侵袭能力,并用Western blot检测凋亡及上皮间叶转化的相关蛋白表达情况。结果:(1)新疆哈族食管鳞癌组织中的PLCE1蛋白表达明显高于癌旁正常组织;食管鳞癌组织PLCE1基因启动子甲基化率明显低于正常组织,两组比较差异有统计学意义(P0.001);新疆哈族食管鳞癌组织中,PLCE1蛋白高表达的组织其PLCE1基因启动子甲基化水平低于PLCE1蛋白低表达的组织,两组比较差异有统计学意义(P=0.028)。在这51例食管鳞癌组织中PLCE1基因启动子甲基化程度与患者性别、年龄、肿瘤大小及患者术后生存时间无关,与患者淋巴结及远处转移(χ2=7.242,P=0.027)和TNM分期(χ2=7.883,P=0.019)相关。(2)MSP检测四株食管癌细胞系Eca109,TE-1,EC9706,Kyse150中PLCE1基因启动子甲基化程度与PLCE1蛋白表达成反比,甲基转移酶抑制剂5-aza-d C抑制了TE-1和Kyse150的PLCE1甲基化程度、增高了其蛋白表达水平。(3)U73122在浓度为10u M时作用于食管鳞癌细胞48h后对PLCE1的抑制效果最好,当U73122作用后,MTT和平板克隆技术证明细胞生长能力降低,流式细胞术和TUNEL法证明细胞凋亡增多,促凋亡分子Bax、Cleaved-PARP、Caspase3、Caspase7表达均增高,而抗凋亡分子Bcl-2表达降低;transwell实验证明细胞侵袭能力减弱,上皮标记物E-cadherin表达升高,间叶标记物Vimentin降低。结论:PLCE1的去甲基化导致其在食管鳞癌细胞中高表达从而促进食管癌细胞的恶性生物学行为。
[Abstract]:Objective: To investigate the relationship between methylation of PLCE1 gene promoter and expression of PLCE1 protein and its relationship with clinicopathological features; detection of 4 different strains of esophageal squamous cell carcinoma cell lines Eca109, TE-1, EC9706, Kyse150 in PLCE1 gene promoter methylation and expression of PLCE1 protein and explore the methyltransferase inhibitor 5-aza-d C on cells the degree of PLCE1 methylation and expression of PLCE1 protein on the growth of esophageal squamous cell carcinoma; application of PLC inhibitor U73122, apoptosis and metastasis. Methods: 51 cases were collected in Xinjiang Kazakh esophageal squamous cell carcinoma and 51 cases of cancer adjacent normal tissues, the expression of PLCE1 by immunohistochemical Envision method to detect and extract the protein. 51 of the carcinoma and adjacent tissue DNA by methylation specific PCR (MSP) technique to detect the methylation status of PLCE1 gene promoter and analysis of PLCE1 gene promoter methylation degree and PLCE1 protein The correlation between the expression, and age and gender, and the maximum diameter of tumor, lymph node and distant metastasis, the correlation between TNM staging and prognosis; 4 strains of human esophageal cancer cell line Western to detect the blot expression of 5-aza-d before and after C in the role of the PLCE1 protein, and MSP detection of PLCE1 gene promoter methylation status of their will; PLC inhibitor U73122 on esophageal cancer cell lines Eca109, EC9706, blot application of Western detection and suppression efficiency, and in 48 hours after using MTT and clone detection cell growth, flow cytometry and TUNEL staining were used to detect cell apoptosis, Transwell to detect the invasive ability of cells, and the expression of related protein in leaves the transformation of epithelial apoptosis and Western blot detection between. Results: (1) the expression of Xinjiang Kazakh esophageal squamous cell carcinoma PLCE1 protein was significantly higher than that of adjacent normal tissue; PLCE1 gene in esophageal squamous cell carcinoma. The promoter methylation rate was significantly lower than that in normal tissues, there was significant difference between two groups (P0.001); Xinjiang Kazakh esophageal squamous cell carcinoma, promoter methylation level is lower than the low expression of PLCE1 protein in the tissue of high expression of PLCE1 protein in the tissue of PLCE1 gene, there was significant difference between two groups (P=0.028) in the PLCE1. In 51 cases of esophageal carcinoma gene promoter methylation in patients with gender, age, tumor size and survival time. Patients with lymph node and distant metastasis (2=7.242, P=0.027) and TNM stage (2= 7.883, P=0.019). (2) MSP was detected in four strains of esophageal carcinoma cell line Eca109, TE-1, EC9706, Kyse150 in PLCE1 gene promoter methylation and PLCE1 protein expression is inversely proportional to the methyltransferase inhibitor 5-aza-d inhibited the C methylation level of PLCE1 TE-1 and Kyse150, increased the expression of U73122 (3). At the concentration of 10u M in esophageal squamous cell carcinoma cell line 48h after the best inhibitory effect on PLCE1, when U73122, MTT and plate cloning technology that reduces cell growth ability, that the number of apoptosis cells by flow cytometry and TUNEL assay, apoptosis molecule Bax, Cleaved-PARP, Caspase3, Caspase7 expression was significantly increased, while the anti the decreased expression of apoptosis Bcl-2; Transwell experiments proved that the weakened cell invasion, epithelial marker E-cadherin increased expression of mesenchymal marker Vimentin decreased. Conclusion: PLCE1 demethylation leads to its high expression in esophageal squamous cell carcinoma and promote the malignant biological behavior of esophageal carcinoma cells.

【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

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