本文選題：NSCLC + NGS��； 參考：《中國人民解放軍軍事醫(yī)學科學院》2017年碩士論文

【摘要】：【研究背景】肺癌又稱原發(fā)性支氣管肺癌,是具有高發(fā)病率及高死亡率的肺部惡性腫瘤。國家癌癥中心計算,我國2015年新發(fā)肺癌患者73.3萬人,死亡61萬人。肺癌是男性腫瘤發(fā)病率和死亡率的第一位,是女性腫瘤發(fā)病率的第二位和死亡率的第一位。在世界范圍內,肺癌是男性腫瘤發(fā)病率和死亡率的第一位,是女性腫瘤發(fā)病率的第三位和死亡率的第二位。按病理類型可將肺癌分為腺癌、鱗癌、大細胞癌、小細胞癌,其中腺癌、鱗癌、大細胞癌又統(tǒng)稱為非小細胞肺癌(non-small cell lung cancer,NSCLC)。由于肺癌早期沒有明顯癥狀,絕大部分病人就診時已經(jīng)是局部晚期或已發(fā)生轉移,失去了手術根治的機會。骨骼是其常見的血行轉移部位之一,一半的肺癌骨轉移患者并發(fā)骨相關事件。一旦發(fā)生骨相關事件,患者生存時間可縮短一半。NSCLC約占整個肺癌的75%-80%。NSCLC預后總體較差,5年生存率約15%-16%。肺癌的標準治療包括手術、放療、化療等綜合治療,目前化療已發(fā)展至瓶頸期。隨著分子靶向藥物及免疫檢查點抑制劑的引入,肺癌的治療策略發(fā)生了巨大的變化,迎來了新的曙光。肺癌的發(fā)生發(fā)展是個復雜的過程,多個驅動基因異常參與其中,目前主要的可用于非小細胞肺癌治療的靶向基因包括表皮生長因子受體(epidermal growth factor receptor,EGFR)敏感突變、間變性淋巴瘤激酶(anaplasticlymphoma kinase,ALK)融合、c-ros肉瘤致癌基因1(c-ros oncogene 1,ROS1)融合,其他的還包括人表皮生長因子受體2(human epidermal growth factor receptor 2,HER2)突變、BRAF V600E突變、高水平MET擴增或MET14外顯子跳躍突變、RET重排等。然而仍然還有許多未知的驅動基因參與其中。尋找出新的驅動基因或治療靶點是目前肺癌研究的熱點。腫瘤基因組圖譜數(shù)據(jù)庫(the cancer genome atlas,TCGA)收集了大量人類腫瘤特別是肺癌的測序結果,旨在通過系統(tǒng)分析,繪制出人類腫瘤的基因變異圖譜,為癌癥的診斷、治療提供幫助,同時以期尋找新的治療腫瘤的方法和策略。我們對TCGA數(shù)據(jù)庫進行分析,篩選出了483個腫瘤相關的基因,希望通過二代測序技術(next generation sequencing,NGS)在臨床NSCLC患者組織樣本中對這些基因進行測序,檢測基因的變異情況,以期發(fā)現(xiàn)新的與肺癌特別是肺癌骨轉移相關的突變基因;同時,對TCGA中肺腺癌核糖核酸測序結果分析顯示,GDP甘露糖-4,6-脫水酶(GDP-mannose-4,6-dehydratase,GMDS)是其中差異性最顯著的基因之一。GMDS是巖藻糖基化過程中的關鍵酶之一,與GDP-4-酮-6-脫氧甘露糖-3,5異構酶-4-還原酶(GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase,FX)共同參與GDP巖藻糖的合成。在肺癌、結直腸癌、乳腺癌、卵巢癌、肝癌、胰腺癌等多個腫瘤中存在巖藻糖基化異常,巖藻糖基化異常與腫瘤的發(fā)生發(fā)展密切相關。GMDS位于6號染色體,編碼372氨基酸,其功能缺失導致白細胞粘附缺陷癥的發(fā)生。結直腸癌及其轉移灶中均存在GMDS突變,而轉移灶GMDS突變明顯高于原發(fā)灶,其可能與疾病的進展相關。結直腸腫瘤細胞系HCT116中GMDS外顯子缺失導致功能缺陷,從而使其逃脫自然殺傷細胞的免疫監(jiān)視。同時巖藻糖基化中的其他關鍵酶也與腫瘤的發(fā)生發(fā)展密切相關,與原發(fā)性結直腸腫瘤細胞系sw480相比,轉移性結直腸腫瘤細胞系sw620高表達FX,增強了腫瘤-內皮細胞粘附作用,肺腺癌細胞系CL1-5高表達FUT8,增加了腫瘤增殖、侵襲、轉移能力。GMDS在NSCLC中的表達情況及其在腫瘤發(fā)生發(fā)展中的作用目前還未見報道,因此,我們希望通過免疫組化檢測臨床NSCLC患者中GMDS表達情況,同時在細胞水平研究GMDS對NSCLC發(fā)生發(fā)展的作用及其可能的機制,以探討GMDS可否作為NSCLC治療的新靶點�！狙芯磕康摹繌腡CGA數(shù)據(jù)庫中篩選出了483個與腫瘤相關的基因,檢測其在NSCLC合并骨轉移患者中的變異情況,以期尋找與肺癌特別是骨轉移密切相關的基因。觀察NSCLC患者癌組織中GMDS表達與癌旁是否存在差異,NSCLC細胞系中GMDS表達情況,同時采用慢病毒介導的RNAi沉默GMDS,觀察抑制GMDS表達對細胞增殖、凋亡的影響�！狙芯糠椒ā坎捎肏iseq2000_PE75二代測序平臺,檢測8例NSCLC同時合并骨轉移患者中483個腫瘤相關基因的變異情況,以期尋找與NSCLC特別是與骨轉移相關的基因,尋找新的潛在治療靶點。應用免疫組織化學(immunohistochemistry,IHC)方法檢測10例肺腺癌患者癌組織及癌旁組織GMDS表達情況,利用聚合酶鏈式反應(polymerase chain reaction,PCR)、實時聚合酶鏈式反應(real time polymerase chain reaction,RT-PCR)及蛋白印記(western-blot,WB)檢測NSCLC細胞系中GMDS表達情況。慢病毒介導的RNA干擾(RNA interference,RNAi)技術沉默GMDS后,使用RT-PCR檢測干擾效率。GMDS表達被抑制后,MTT檢測細胞活力,流式細胞儀檢測細胞周期及細胞凋亡。【結果】第一部分:8例NSCLC合并骨轉移患者中,共檢測出3620個基因突變,多個與肺癌相關的信號通路基因均存在突變;同時存在16個突變一致性基因,6例(75%)患者存在HNF1α1394_1395ins8突變,5例(62.5%)患者存在APC A818G突變,4例(50%)患者存在CD22 G1703A突變。對HNF1α及CD22行Sanger測序,結果顯示與NGS測序結果一致。第二部分:10例肺腺癌患者癌組織與癌旁組織GMDS IHC檢測,癌組織GMDS表達為3.597±1.908,癌旁組織GMDS表達為0.453±1.119,癌組織GMDS表達明顯高于癌旁組織(P值0.01)。RNA干擾后,A549細胞干擾組GMDS表達量為0.28±0.017,明顯低于對照組的1.03±0.097(P值0.01);H1299細胞干擾組GMDS表達量為0.19±0.022,明顯低于對照組的1.00±0.004(P值0.01)。細胞活力方面,GMDS-Si RNA組H1299細胞在96,120小時OD值分別為0.464±0.019,0.453±0.038,低于對照組細胞的0.815±0.051,0.969±0.012(P值0.01);GMDS-Si RNA組A549細胞在96,120小時的OD值為0.744±0.005,0.858±0.041,低于對照組細胞的1.105±0.026,1.441±0.042(P值0.01)。細胞周期分布方面,GMDS-Si RNA組H1299細胞在G1期的比例為46.15±1.20%,高于對照組的27.96±0.93%(P值0.01);在S期的比例為50.80±0.82%,低于對照組的59.40±0.75%(P值0.01);在G2期的比例為3.05±0.41%,低于對照組的12.64±1.21%(P值0.01)。GMDS-Si RNA組A549細胞在G1期的比例58.2±1.32%,高于對照組的54.83±0.41%(P值0.05);在G2期的比例10.62±0.80,低于對照組的14.42±0.42%(P值0.01)。凋亡方面,GMDS-Si RNA組H1299細胞中凋亡細胞占到14.90±0.39%,明顯高于對照組的3.83±0.23%(P值0.01)。GMDS-Si RNA組A549細胞中凋亡細胞占到23.01±0.45%,明顯高于對照組的3.59±0.13%(P值0.01)�！窘Y論】NGS結果顯示多個肺癌相關信號通路基因突變參與了NSCLC的發(fā)生發(fā)展,同時HNF1α(1394_1395ins8)、CD22(G1703A)、APC(A818G)等高頻一致性突變可能與NSCLC骨轉移相關。GMDS在肺腺癌組織中高表達,沉默NSCLC細胞系GMDS表達后,明顯抑制了細胞增殖,促進了細胞凋亡,GMDS表達可能可以作為NSCLC增殖、凋亡的預測指標,GMDS也許可以作為NSCLC治療的潛在靶標。
[Abstract]:[background] lung cancer, also known as primary bronchogenic lung cancer, is a lung malignant tumor with high morbidity and mortality. The National Cancer Center has calculated 733 thousand new lung cancer patients in 2015 and 610 thousand deaths. Lung cancer is the first of the male tumor incidence and mortality, and the second and mortality rate of the female tumor incidence. In the world, lung cancer is the first of the male tumor incidence and death rate, the third and second mortality rate of the female tumor, which can be divided into adenocarcinoma, squamous cell carcinoma, large cell carcinoma, small cell carcinoma, including adenocarcinoma, squamous cell carcinoma, and large cell carcinoma (non-small cell). Lung cancer, NSCLC). Due to no obvious symptoms in the early stage of lung cancer, most patients have been locally advanced or metastasize, losing the chance of radical operation. Bone is one of the common blood transfer sites, and half of the patients with lung cancer are associated with bone related events. Once bone related events occur, the patient's survival time The overall prognosis of 75%-80%.NSCLC for lung cancer is less than half.NSCLC, and the 5 year survival rate is about 15%-16%. lung cancer, including surgery, radiotherapy, chemotherapy and so on. The chemotherapy has developed to the bottleneck. With the introduction of the molecular targeting drug and immunologic checkpoint inhibitors, the treatment strategy of lung cancer has changed greatly. The development of lung cancer is a complex process, with many abnormal driving genes involved, and the main target genes for the treatment of non-small cell lung cancer include epidermal growth factor receptor (EGFR) sensitivity mutation, and anaplasticlymphoma kina Se, ALK) fusion, fusion of c-ros sarcoma oncogene 1 (c-ros oncogene 1, ROS1), and other human epidermal growth factor receptor 2 (human epidermal growth factor receptor 2, HER2) mutation, high level amplification or exons jumping, rearrangement, etc. However, there are still many unknown driving genes involved. The Cancer Genome Atlas (TCGA), which has collected a large number of human tumors, especially lung cancer, has collected the results of a large number of human tumors, especially lung cancer. The purpose of this study is to draw the genetic mutation map of human tumor by systematic analysis to provide the diagnosis and treatment of cancer. Help, while looking for new methods and strategies for the treatment of tumors. We analyzed the TCGA database and screened 483 tumor related genes. We hope to sequence the genes in the tissue samples of the clinical NSCLC patients through the two generation sequencing technology (next generation sequencing, NGS) to detect the mutation of the gene in order to develop the gene. A new mutant gene associated with bone metastasis of lung cancer, especially lung cancer, and the analysis of nucleotide sequencing results of lung adenocarcinoma in TCGA shows that GDP mannose -4,6- dehydrase (GDP-mannose-4,6-dehydratase, GMDS) is one of the most distinct genes in which.GMDS is one of the key enzymes in the process of fucoidylation and GDP-4- ketone -6- The oxygen mannose -3,5 isomerase -4- reductase (GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase, FX) participates in the synthesis of GDP fucose together. There are abnormal algonglycylation in many tumors, such as lung cancer, colorectal cancer, breast cancer, ovarian cancer, liver cancer, and pancreatic cancer, and the abnormal algonglycylation is closely related to the development and development of the tumor, which is closely related to the occurrence and development of the tumor, and is closely related to the.GMDS position. On chromosome 6, encoding 372 amino acids, the loss of function leads to the occurrence of leukocyte adhesion deficiency. There is a GMDS mutation in both colorectal cancer and its metastasis, and the GMDS mutation in the metastases is significantly higher than that of the primary lesion. It may be related to the progression of the disease. The deletion of exon GMDS in the colorectal tumor cell line HCT116 causes functional defects, thus making The other key enzymes in the fucosylation are also closely related to the occurrence and development of the tumor. Compared with the primary colorectal tumor cell line SW480, the metastatic colorectal tumor cell line SW620 highly expresses FX, enhancing the adhesion of the tumor to the endothelial cell, and the high expression of FUT in the lung adenocarcinoma cell line CL1-5. 8, the expression of tumor proliferation, invasion and metastasis of.GMDS in NSCLC and its role in the development of tumor have not yet been reported. Therefore, we hope to detect the expression of GMDS in clinical NSCLC patients by immunohistochemistry, and study the role of GMDS on the development of NSCLC and the possible mechanism at the level of cell at the same time. To explore the possibility of GMDS as a new target for NSCLC treatment. [Objective] to screen 483 genes related to tumor from the TCGA database to detect the mutation in patients with NSCLC combined with bone metastasis in order to find the genes closely related to lung cancer, especially bone metastases. To observe the expression of GMDS in the cancer tissues of patients with NSCLC and to the side of the cancer. There is no difference in the expression of GMDS in the NSCLC cell line and the use of RNAi silent GMDS mediated by lentivirus, to observe the effect of inhibition of GMDS expression on cell proliferation and apoptosis. [Methods] the mutation of 483 tumor related genes in 8 cases of NSCLC combined with bone metastases was detected by the Hiseq2000_PE75 two generation sequencing platform. Looking for the genes associated with NSCLC, especially bone metastases, looking for new potential therapeutic targets. Immunohistochemistry (IHC) was used to detect the expression of GMDS in 10 cases of lung adenocarcinoma and para cancerous tissue, using polymerase chain reaction (polymerase chain reaction, PCR), and real-time polymerase chain reaction (real Ti). Me polymerase chain reaction, RT-PCR) and protein imprint (Western-blot, WB) detection of GMDS expression in NSCLC cell lines. After the slow virus mediated RNA interference (RNA interference), the expression was suppressed by the detection of interference efficiency, and the cell viability was detected by the flow cytometry, and the cell cycle and cell withering were detected by flow cytometry. [results] [results] Part 1: in part 1: in 8 cases of NSCLC with bone metastases, 3620 gene mutations were detected. There were multiple mutations in the signal pathway genes associated with lung cancer; there were 16 mutation conformance genes, 6 (75%) patients with HNF1 alpha 1394_1395ins8 mutation, 5 (62.5%) patients with APC A818G mutation and 4 (50%) patients. CD22 G1703A mutation. Sanger sequencing of HNF1 alpha and CD22 showed that the results were consistent with the results of NGS sequencing. The second part: 10 cases of adenocarcinoma of lung cancer tissue and para cancerous tissue GMDS IHC detection, the GMDS expression of the cancer tissue is 3.597 + 1.908, the expression of GMDS in the para cancerous tissue is 0.453 + 1.119, the GMDS expression of the cancer tissue is significantly higher than that of the para cancerous tissue (P value 0.01). The expression of GMDS in A549 cell interference group was 0.28 + 0.017, significantly lower than that of the control group (1.03 + 0.097) (P value 0.01), and the GMDS expression of H1299 cell interference group was 0.19 + 0.022, significantly lower than that of the control group (1 + 0.004 (P value 0.01). In cell viability, the H1299 cells in GMDS-Si RNA group were 0.464 + 0.019,0.453 + 0.038 in 96120 hours, respectively, lower than the control. The cells in the group were 0.815 + 0.051,0.969 + 0.012 (P value 0.01), and the A549 cells in group GMDS-Si RNA were 0.744 + 0.005,0.858 + 0.041 at 96120 hours, lower than those of the control group (1.105 + 0.026,1.441 + 0.042) (P value 0.01). The proportion of GMDS-Si RNA group H1299 cells in G1 stage was 46.15 + 1.20%, higher than 27.96 + 0.93% in the control group. Value 0.01), the proportion in S period was 50.80 + 0.82%, lower than that of the control group (59.40 + 0.75%) (P value 0.01), and the proportion in G2 period was 3.05 + 0.41%, which was lower than that of the control group and 12.64 + 1.21% (P value 0.01).GMDS-Si RNA A549 cells in G1 phase ratio 58.2 + 1.32%, higher than that of the control group (P value); the ratio of G2 period was lower than that of the control group. 42 + 0.42% (P value 0.01). Apoptosis, apoptotic cells in GMDS-Si RNA group H1299 cells accounted for 14.90 + 0.39%, significantly higher than the control group 3.83 + 0.23% (P value 0.01).GMDS-Si RNA group A549 cell apoptotic cells accounted for 23.01 + 0.45%, significantly higher than the control group of 3.59 + 0.13% (P 0.01). [Conclusion] NGS results show a number of lung cancer related signaling pathways Gene mutation is involved in the development of NSCLC, while HNF1 alpha (1394_1395ins8), CD22 (G1703A), APC (A818G) and other high-frequency conformance mutations may be highly expressed in the lung adenocarcinoma tissue associated with NSCLC bone metastasis. After the silencing of GMDS expression of NSCLC cell lines, it obviously inhibits cell proliferation and promotes cell apoptosis. GMDS expression may be considered as a possibility. GMDS, a marker of proliferation and apoptosis, may be a potential target for NSCLC therapy.

【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2
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本文編號：1856439