LATS1基因在食管鳞癌中的功能及甲基化状态研究

发布时间：2018-05-26 01:33

本文选题：LATS1 + 食管鳞状细胞癌　；参考：《重庆医科大学》2017年硕士论文

【摘要】：背景:大肿瘤抑制基因1(LATS1)是Hippo信号通路中的一个关键抑癌基因。有研究表明在多种肿瘤中LATS1基因存在启动子甲基化,并且证实与其发生发展相关联,但是在食管鳞状细胞癌中LATS1的功能及与甲基化状态的关系尚未有研究。目的:了解LATS1基因表达与LATS1甲基化状态的联系,及LATS1在食管鳞状细胞癌中的功能,并探讨LATS1作为食管鳞癌基因靶向治疗的可能性。方法:1.通过qRT-PCR、免疫组化检测LATS1蛋白及mRNA在食管鳞癌及配对癌旁组织中的表达水平差异,并探讨与临床病理特点及分期的关系。2.甲基化特异性PCR(MSP)和亚硫酸氢盐测序法(BGS)检测食管鳞癌组织与细胞中LATS1基因启动子Cp G岛是否发生了甲基化。3.慢病毒转染食管鳞癌细胞株TE-10和EC109,稳定表达LATS1基因。4.CCK-8和克隆形成实验检测LATS1基因对TE-10和EC109细胞增殖的影响。5.采用流式细胞术检测LATS1基因对TE-10和EC109细胞周期及细胞凋亡的影响。6.通过transwell实验检测LATS1基因对TE-10和EC109细胞迁移和侵袭能力的影响。7.使用SPSS23.0对研究所得数据进行统计学分析。结果:1.LATS1基因在两种组织中表达水平有明显差异,癌组织中普遍低表达,而癌旁组织中则广泛表达。2.在食管鳞癌细胞TE-10及EC109中过表达LATS1基因能抑制细胞增殖。3.在TE-10及EC109细胞中过表达LATS1基因能阻滞细胞周期于G2/M期,同时能诱导细胞凋亡。4.在TE-10及EC109细胞中过表达LATS1基因能减少细胞的迁移和侵袭力。5.在食管鳞状细胞癌组织及细胞中,LAST1基因启动子CpG岛未发现甲基化位点。结论:我们的实验结果表明LATS1在食管鳞癌中起到抑癌基因的作用,并且与肿瘤的临床分期相关,而其下调与CpG岛的高甲基化无关。在食管鳞癌细胞中过表达LATS1明显下调YAP,抑制细胞增殖、细胞迁移和侵袭,同时能够促进细胞凋亡。因此,LATS1基因值得进一步探索作为食管鳞癌生物标志物的可能用途和未来分子诊断和治疗的目标。
[Abstract]:Background: large tumor suppressor gene 1 (LATS1) is a key tumor suppressor gene in Hippo signaling pathway. Some studies have shown that there is promoter methylation of LATS1 gene in many kinds of tumors and has been proved to be associated with its occurrence and progression. However, the function of LATS1 and its relationship with methylation status have not been studied in esophageal squamous cell carcinoma. Objective: to investigate the relationship between LATS1 gene expression and LATS1 methylation, and the function of LATS1 in esophageal squamous cell carcinoma, and to explore the possibility of LATS1 as a target therapy for esophageal squamous cell carcinoma. Method 1: 1. The expression of LATS1 protein and mRNA in esophageal squamous cell carcinoma and paracancerous tissues were detected by using qRT-PCR, and the relationship between the expression of mRNA and clinicopathological features and staging was discussed. Methylation specific LATS1 gene promoter CpG island was detected in esophageal squamous cell carcinoma tissues and cells by methylation specific PCR and bisulfite sequencing. Lentivirus was transfected into esophageal squamous cell carcinoma cell lines TE-10 and EC109, and LATS1 gene was stably expressed. 4. CCK-8 and clone formation assay were used to detect the effect of LATS1 gene on the proliferation of TE-10 and EC109 cells. The effect of LATS1 gene on TE-10 and EC109 cell cycle and apoptosis was detected by flow cytometry. The effect of LATS1 gene on the migration and invasion of TE-10 and EC109 cells was detected by transwell assay. Use SPSS23.0 to carry on statistical analysis to the data obtained from the study. Results: 1. The expression level of LATS1 gene was significantly different between the two tissues. The expression of LATS1 gene was generally low in cancer tissues, but widely expressed in paracancerous tissues. Overexpression of LATS1 gene in esophageal squamous cell TE-10 and EC109 can inhibit cell proliferation. 3. Overexpression of LATS1 gene in TE-10 and EC109 cells could block cell cycle in G 2 / M phase and induce apoptosis. Overexpression of LATS1 gene in TE-10 and EC109 cells can reduce cell migration and invasiveness. No methylation site was found in CpG island of LAST1 gene promoter in esophageal squamous cell carcinoma tissues and cells. Conclusion: our results suggest that LATS1 plays a role as a tumor suppressor gene in esophageal squamous cell carcinoma and is related to the clinical stage of the tumor, but its down-regulation is not associated with hypermethylation of CpG island. Overexpression of LATS1 in esophageal squamous cell carcinoma cells significantly down-regulated Yap, inhibited cell proliferation, cell migration and invasion, and promoted apoptosis. Therefore, the LATS1 gene is worthy of further exploration as a possible biomarker for esophageal squamous cell carcinoma and a target for molecular diagnosis and treatment in the future.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

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