本文選題：PTEN-COL17融合基因 + EGFR-RP11-745C15.2融合基因��； 參考：《首都醫(yī)科大學(xué)》2017年博士論文

【摘要】：【背景】腦膠質(zhì)瘤是最常見的腦腫瘤,致死率多年來居高不下。融合基因由兩個染色體基因重排形成。在血液系統(tǒng)腫瘤和實(shí)體腫瘤中已經(jīng)發(fā)現(xiàn)了許多促進(jìn)腫瘤發(fā)生發(fā)展的融合基因。轉(zhuǎn)錄本測序目前已成為發(fā)現(xiàn)和檢驗?zāi)[瘤中融合基因的非常重要的工具。已經(jīng)有許多融合基因都是通過轉(zhuǎn)錄本測序這一工具被發(fā)現(xiàn)。第一個通過轉(zhuǎn)錄本測序發(fā)現(xiàn)并鑒定出來的腦膠質(zhì)瘤融合基因是FGFR3-TACC3融合基因。FGFR3-TACC3融合基因定位于細(xì)胞有絲分裂紡錘體極,有激酶活性,延遲細(xì)胞的有絲分裂和染色體分離過程,同時促進(jìn)非整倍體的形成。EGFR-PSPH14融合基因能促進(jìn)膠質(zhì)瘤細(xì)胞的更新和生長,活化STAT3通路,并對EGFR的抑制劑敏感。應(yīng)用轉(zhuǎn)錄本測序的方法檢測CGGA數(shù)據(jù)庫中部分膠質(zhì)瘤樣本,目前已經(jīng)發(fā)現(xiàn)了不少融合基因,其中PTPRZ1-MET融合基因在繼發(fā)膠質(zhì)母細(xì)胞瘤中發(fā)現(xiàn)并證實(shí)。同時CGGA轉(zhuǎn)錄本測序還發(fā)現(xiàn)了2例PTEN-COL17A1融合基因和4例EGFR-RP11-745C15.2融合基因的病例,尚未經(jīng)行驗證研究�！痉椒ā縋CR方法擴(kuò)增待測樣本的融合位點(diǎn)序列,陽性PCR產(chǎn)物進(jìn)行焦磷酸測序,用PCR和測序方法驗證CGGA轉(zhuǎn)錄本測序發(fā)現(xiàn)的PTEN-COL17A1融合基因和EGFR-RP11-745C15.2陽性樣本,同樣的方法在另外40例獨(dú)立樣本中驗證。用Western blot方法檢測PTEN-COL17A1融合基因?qū)Φ鞍妆磉_(dá)影響情況,發(fā)現(xiàn)轉(zhuǎn)錄本測序PTEN-COL17A1融合基因陽性的樣本中野生型Collagen XVII表達(dá)升高。體外實(shí)驗,應(yīng)用si RNA干擾方法降低U251細(xì)胞的COL17A1表達(dá),在H4和U87中過表達(dá)COL17A1。通過CCK8增殖和克隆形成實(shí)驗觀察敲低和過表達(dá)COL17A1后細(xì)胞的增殖能力,通過遷移和侵襲實(shí)驗觀察敲低和過表達(dá)COL17A1后細(xì)胞的遷移和侵襲能力。通過Overlap PCR方法克隆EGFR-RP11-745C15.2融合基因全長,構(gòu)建質(zhì)粒,在U87細(xì)胞系中過表達(dá)EGFR-RP11-745C15.2,進(jìn)行功能研究�！窘Y(jié)果】用PCR和焦磷酸測序方法證實(shí)了CGGA轉(zhuǎn)錄本測序發(fā)現(xiàn)的1例PTEN-COL17A1和2例EGFR-RP11-745C15.2融合基因真實(shí)存在。同時發(fā)現(xiàn)并證實(shí)了4例新的EGFR-RP11-745C15.2融合基因病例,且都在較高級別膠質(zhì)瘤中。發(fā)現(xiàn)了轉(zhuǎn)錄本測序PTEN-COL17A1融合基因陽性的樣本中野生型Collagen XVII表達(dá)升高。應(yīng)用si RNA干擾方法降低U251細(xì)胞中COL17A1表達(dá),隨著Collagen XVII表達(dá)降低MMP9蛋白表達(dá)也隨之減低,細(xì)胞侵襲能力明顯降低。在H4和U87細(xì)胞中過表達(dá)COL17A1,Collagen XVII升高后MMP9蛋白表達(dá)也隨之升高,細(xì)胞侵襲能力明顯增強(qiáng)。Overlap PCR方法成功克隆R-RP11-745C15.2融合基因全長,成功構(gòu)建質(zhì)粒,在U87細(xì)胞系中觀察到了EGFR-RP11-745C15.2融合基因過表達(dá)以后抑制腫瘤的遷移和增殖,促進(jìn)凋亡�！窘Y(jié)論】本研究證實(shí)了PTEN-COL17A1和EGFR-RP11-745C15.2融合基因真實(shí)存在,同時發(fā)現(xiàn)4例新的EGFR-RP11-745C15.2融合基因病例,且都在高級別膠質(zhì)瘤中。EGFR-RP11-745C15.2融合基因可能與高級別膠質(zhì)瘤尤其是WHOⅣ膠質(zhì)母細(xì)胞瘤密切相關(guān)。發(fā)現(xiàn)了轉(zhuǎn)錄本測序PTEN-COL17A1融合基因陽性的樣本中野生型Collagen XVII表達(dá)升高。證實(shí)了COL17A1通過調(diào)節(jié)MMP9增加膠質(zhì)瘤細(xì)胞的侵襲能力。EGFR-RP11-745C15.2融合基因可能抑制腫瘤的遷移和增殖,促進(jìn)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.
【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R739.41

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