食管鳞癌中AJUBA突变的分子机制及临床意义

发布时间：2018-08-06 16:15

【摘要】：实验背景：食管癌是世界第八大肿瘤,占据肿瘤导致死亡的第六位,其中绝大部分食管癌的病理类型为鳞癌,但是食管鳞癌的五年生存率只有不足15%,近年来总生存期也没有明显的延长,这跟目前缺少有效的治疗有关。随着高通量测序技术的发展,在多个食管鳞癌队列当中发现AJUBA基因突变,而相关研究是匮乏的,探索AJUBA突变在食管鳞癌发生发展中的分子机制及临床意义,可能改善患者的预后,提供新的治疗靶点。实验方法：本论文收集了五个食管鳞癌的队列,共545例患者,都有临床信息及全外显子组测序数据,通过统计学分析AJUBA突变与患者TNM分期的相关性,分别绘制AJUBA突变型与野生型患者的Kaplan-Meier曲线,通过Log-Rank比较两者是否有显著差异,以获得可能的AJUBA突变的临床意义,指导进一步的机制探索及临床应用。同时,运用293T细胞包装慢病毒,慢病毒感染目的细胞构建可供研究AJUBA突变的稳转细胞系,这包括稳定敲降的细胞系,过表达野生型AJUBA的细胞系及过表达突变型的细胞系,通过流式荧光分选、潮霉素/嘌呤霉素进行筛选,RT-PCR及蛋白印迹进行验证。在构建细胞系基础上,进行CCK-8、Transwell细胞功能实验,体外验证AJUBA突变与细胞增殖、迁移及侵袭的关系。实验结果：在545例患者当中,AJUBA突变型及野生型的TNM分期没有显著差异(Fisher确切概率p=1.000),AJUBA突变型患者Kaplan-Meier曲线有生存时间更长的趋势(Log-Rank检验p=0.13)。构建稳转敲降细胞株,感染后10日通过潮霉素/流式荧光分选进行筛选,RT-PCR及蛋白印迹验证有一定的敲降效率,但是敲降效率不高。构建过表达野生型及突变型细胞株,通过嘌呤霉素进行筛选,运用蛋白印迹技术验证,可见野生型AJUBA条带及截短的突变型AJUBA条带,构建稳转过表达细胞系成功。通过CCK-8比较过表达细胞系与野生型细胞系的增殖能力,发现三个过表达细胞系的增殖能力都强于野生型细胞系(t检验48h:p0.001,p0.001, p0.001; 72h:p=0.008, p0.001, p0.001)。Transwell实验比较过表达细胞系与野生型细胞系的迁移及侵袭能力,三个过表达细胞系的迁移能力强于野生型细胞系(t检验：p0.001,p0.001,p0.001),过表达野生型AJUBA及突变1的侵袭能力强于野生型细胞系(t检验：p0.001,p=0.035,p=0.576),其中过表达野生型AJUBA细胞系的迁移及侵袭能力增强最为显著。结论：AJUBA突变型的食管鳞癌患者预后相对于AJUBA野生型的患者有更好的趋势。构建稳转敲降细胞系可作为进一步研究的对照,并为构建敲降后过表达突变型AJUBA的细胞系打下基础。过表达突变型细胞系可供研究AJUBA突变,作为进一步体外体内实验的基础。过表达野生型及突变型AJUBA的KYSE450细胞系具有更强的增殖能力。过表达野生型及突变型AJUBA的KYSE450细胞系具有更强的迁移和侵袭能力(除了突变2的侵袭能力),其中以过表达野生型AJUBA的细胞系增强的更为明显。
[Abstract]:Background: esophageal cancer is the eighth largest tumor in the world, occupying the sixth place in the death caused by tumor. The majority of esophageal cancer is squamous cell carcinoma. However, the 5-year survival rate of esophageal squamous cell carcinoma is less than 15%, and the total survival time has not been significantly prolonged in recent years, which is related to the lack of effective treatment. With the development of high-throughput sequencing technique, AJUBA gene mutations were found in many esophageal squamous cell carcinoma cohorts, but the related studies were scarce. The molecular mechanism and clinical significance of AJUBA mutation in the carcinogenesis and development of esophageal squamous cell carcinoma were explored. It may improve the prognosis of patients and provide new therapeutic targets. Methods: five cohorts of esophageal squamous cell carcinoma were collected in this paper. All 545 patients had clinical information and total exon sequence data. The correlation between AJUBA mutation and TNM staging was analyzed statistically. The Kaplan-Meier curves of AJUBA mutation and wild-type patients were drawn, and the differences between the two were compared by Log-Rank, in order to obtain the clinical significance of the possible AJUBA mutation and to guide the further exploration of the mechanism and clinical application. At the same time, using 293T cells to package lentivirus, lentivirus infected target cells to construct stable cell lines for studying AJUBA mutation, including stable knockdown cell line, overexpression of wild-type AJUBA cell line and over-expression mutant cell line. By flow cytometry, hygromycin / purine mycin was screened by RT-PCR and Western blotting. On the basis of the constructed cell line, CCK-8 transwell cell function test was carried out to verify the relationship between AJUBA mutation and cell proliferation, migration and invasion in vitro. Results: there was no significant difference in the TNM stages of AJUBA mutation and wild type (Fisher exact probability p1.000) among 545 patients with AJUBA mutation, the Kaplan-Meier curve of AJUBA mutation had a longer survival time (Log-Rank test p0.13). The stable knockdown cell line was constructed and screened by hygromycin / flow fluorescence sorting on 10 days after infection. RT-PCR and Western blotting showed that the knockdown efficiency was not high but the knockdown efficiency was not high. Overexpression wild-type and mutant cell lines were constructed and screened by purine mycin. The results showed that the wild-type AJUBA bands and truncated mutant AJUBA bands were successfully constructed. The proliferative ability of overexpression cell line and wild type cell line was compared by CCK-8. It was found that the proliferation ability of the three overexpression cell lines was stronger than that of wild type cell lines (t test 48h: p0.001p0.001, p0.001; 72h: p0.008, p0.001, p0.001) .Transwell experiment was used to compare the migration and invasion ability between overexpression cell lines and wild type cell lines. The migration ability of three overexpression cell lines was stronger than that of wild type cell lines (t test: p0.001 + p0.001p0.001p0.001), and the invasion ability of overexpression of wild type AJUBA and mutant 1 was stronger than that of wild type cell lines (t test: 0. 001 p0. 001 p0. 035 p0. 576), among them, overexpression of wild type AJUBA cell line was stronger than that of wild type AJUBA cell line. And the most significant enhancement of invasion ability. Conclusion the prognosis of the esophageal squamous cell carcinoma with the mutation of: AJUBA is better than that of the wild type of AJUBA. The construction of stable knockdown cell line could be used as a control for further study and lay a foundation for the construction of mutant AJUBA cell line after knockdown. Overexpression mutant cell line can be used to study AJUBA mutation as a basis for further in vitro experiments. Overexpression of wild-type and mutant AJUBA KYSE450 cell lines have stronger proliferative ability. Overexpression of wild-type and mutant AJUBA in KYSE450 cell lines had stronger migration and invasion ability (except for the invasion ability of mutant 2), especially in the over-expression of wild-type AJUBA cell lines.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.1

【相似文献】