融合基因在脑胶质瘤中的发现和功能研究

发布时间：2018-06-14 10:50

本文选题：PTEN-COL17融合基因 + EGFR-RP11-745C15.2融合基因　；参考：《首都医科大学》2017年博士论文

【摘要】：【背景】脑胶质瘤是最常见的脑肿瘤,致死率多年来居高不下。融合基因由两个染色体基因重排形成。在血液系统肿瘤和实体肿瘤中已经发现了许多促进肿瘤发生发展的融合基因。转录本测序目前已成为发现和检验肿瘤中融合基因的非常重要的工具。已经有许多融合基因都是通过转录本测序这一工具被发现。第一个通过转录本测序发现并鉴定出来的脑胶质瘤融合基因是FGFR3-TACC3融合基因。FGFR3-TACC3融合基因定位于细胞有丝分裂纺锤体极,有激酶活性,延迟细胞的有丝分裂和染色体分离过程,同时促进非整倍体的形成。EGFR-PSPH14融合基因能促进胶质瘤细胞的更新和生长,活化STAT3通路,并对EGFR的抑制剂敏感。应用转录本测序的方法检测CGGA数据库中部分胶质瘤样本,目前已经发现了不少融合基因,其中PTPRZ1-MET融合基因在继发胶质母细胞瘤中发现并证实。同时CGGA转录本测序还发现了2例PTEN-COL17A1融合基因和4例EGFR-RP11-745C15.2融合基因的病例,尚未经行验证研究。【方法】PCR方法扩增待测样本的融合位点序列,阳性PCR产物进行焦磷酸测序,用PCR和测序方法验证CGGA转录本测序发现的PTEN-COL17A1融合基因和EGFR-RP11-745C15.2阳性样本,同样的方法在另外40例独立样本中验证。用Western blot方法检测PTEN-COL17A1融合基因对蛋白表达影响情况,发现转录本测序PTEN-COL17A1融合基因阳性的样本中野生型Collagen XVII表达升高。体外实验,应用si RNA干扰方法降低U251细胞的COL17A1表达,在H4和U87中过表达COL17A1。通过CCK8增殖和克隆形成实验观察敲低和过表达COL17A1后细胞的增殖能力,通过迁移和侵袭实验观察敲低和过表达COL17A1后细胞的迁移和侵袭能力。通过Overlap PCR方法克隆EGFR-RP11-745C15.2融合基因全长,构建质粒,在U87细胞系中过表达EGFR-RP11-745C15.2,进行功能研究。【结果】用PCR和焦磷酸测序方法证实了CGGA转录本测序发现的1例PTEN-COL17A1和2例EGFR-RP11-745C15.2融合基因真实存在。同时发现并证实了4例新的EGFR-RP11-745C15.2融合基因病例,且都在较高级别胶质瘤中。发现了转录本测序PTEN-COL17A1融合基因阳性的样本中野生型Collagen XVII表达升高。应用si RNA干扰方法降低U251细胞中COL17A1表达,随着Collagen XVII表达降低MMP9蛋白表达也随之减低,细胞侵袭能力明显降低。在H4和U87细胞中过表达COL17A1,Collagen XVII升高后MMP9蛋白表达也随之升高,细胞侵袭能力明显增强。Overlap PCR方法成功克隆R-RP11-745C15.2融合基因全长,成功构建质粒,在U87细胞系中观察到了EGFR-RP11-745C15.2融合基因过表达以后抑制肿瘤的迁移和增殖,促进凋亡。【结论】本研究证实了PTEN-COL17A1和EGFR-RP11-745C15.2融合基因真实存在,同时发现4例新的EGFR-RP11-745C15.2融合基因病例,且都在高级别胶质瘤中。EGFR-RP11-745C15.2融合基因可能与高级别胶质瘤尤其是WHOⅣ胶质母细胞瘤密切相关。发现了转录本测序PTEN-COL17A1融合基因阳性的样本中野生型Collagen XVII表达升高。证实了COL17A1通过调节MMP9增加胶质瘤细胞的侵袭能力。EGFR-RP11-745C15.2融合基因可能抑制肿瘤的迁移和增殖,促进凋亡。
[Abstract]:[background] glioma is the most common brain tumor. Death rate has been high for many years. Fusion gene is rearranged by two chromosomal genes. Many fusion genes have been found in blood system tumors and solid tumors. Transcriptional sequencing has now become a fusion gene for detection and detection of tumors. Very important tools. Many fusion genes have been found by the transcriptional sequencing tool. The first glioma fusion gene, identified and identified through transcriptional sequencing, is the FGFR3-TACC3 fusion gene.FGFR3-TACC3 fusion gene located in cell mitotic spindle pole, kinase activity, and delayed cell The process of mitosis and chromosome segregation, and promoting the formation of aneuploidy.EGFR-PSPH14 fusion gene can promote the regeneration and growth of glioma cells, activate the STAT3 pathway, and be sensitive to the inhibitors of EGFR. The method of sequencing the transcriptional sequencing is used to detect some of the glioma samples in the CGGA database. Many fusion genes have been found. The PTPRZ1-MET fusion gene was found and confirmed in secondary glioblastoma. At the same time, 2 cases of PTEN-COL17A1 fusion gene and 4 EGFR-RP11-745C15.2 fusion gene were also found, which have not been verified. [method] PCR method was used to amplify the fusion site sequence of the samples to be measured and the positive PCR products were used for focal phosphorus. Acid sequencing, PCR and sequencing methods were used to verify the PTEN-COL17A1 fusion gene and EGFR-RP11-745C15.2 positive samples found in the CGGA transcriptional sequence. The same method was verified in 40 other independent samples. The Western blot method was used to detect the influence of the PTEN-COL17A1 fusion gene on the protein expression, and the transcriptional transcriptional sequence PTEN-COL17A1 fusion gene was found. In the positive samples, the expression of wild type Collagen XVII was increased. In vitro, the COL17A1 expression of U251 cells was reduced by Si RNA interference. The overexpression of COL17A1. in H4 and U87 was observed by the proliferation and cloning of CCK8. The proliferation ability of the cells was observed after the knockout and overexpressed COL17A1, and the knockout and overexpressed C were observed through the migration and invasion experiments. The cell migration and invasion ability after OL17A1. Overlap PCR method was used to clone the full length of EGFR-RP11-745C15.2 fusion gene, construct plasmid, express EGFR-RP11-745C15.2 in U87 cell line and perform functional study. [results] 1 cases of PTEN-COL17A1 and 2 EGFR-RP11-745 of CGGA transcriptional sequencing were confirmed by PCR and pyrosequencing method. The C15.2 fusion gene was true. At the same time, 4 new EGFR-RP11-745C15.2 fusion genes were found and found in high grade gliomas. The expression of wild type Collagen XVII in the samples of the transcriptional PTEN-COL17A1 fusion gene positive was found. The COL17A1 expression in U251 cells was reduced with the Si RNA interference method, with the decrease of COL17A1 expression in U251 cells. The expression of Collagen XVII decreased and the expression of MMP9 protein decreased and the cell invasiveness decreased. COL17A1 was overexpressed in H4 and U87 cells, the expression of MMP9 protein increased after Collagen XVII increased, and the invasion ability of the cells was obviously enhanced by.Overlap PCR method, and the full length of the R-RP11-745C15.2 fusion gene was successfully cloned and the plasmid was successfully constructed. In the cell line, EGFR-RP11-745C15.2 fusion gene was observed to inhibit the migration and proliferation of tumor and promote apoptosis. [Conclusion] this study confirmed the true existence of PTEN-COL17A1 and EGFR-RP11-745C15.2 fusion genes, and 4 new EGFR-RP11-745C15.2 fusion genes were found, and all of them were.EGFR-RP1 in high grade gliomas. The 1-745C15.2 fusion gene may be closely related to the high grade glioma, especially the WHO IV glioblastoma. The expression of wild type Collagen XVII in the samples of the transcriptional PTEN-COL17A1 fusion gene positive is found to be elevated. It is confirmed that COL17A1 can increase the invasion ability of the glioma cells by regulating MMP9 to increase the invasion ability of.EGFR-RP11-745C15.2 fusion gene. It can inhibit the migration and proliferation of tumor and promote apoptosis.
【学位授予单位】：首都医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.41

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