NSCLC合并骨转移患者突变基因筛选及GMDS对NSCLC细胞增殖、凋亡的影响

发布时间：2018-05-07 09:47

  本文选题：NSCLC + NGS　； 参考：《中国人民解放军军事医学科学院》2017年硕士论文

【摘要】：【研究背景】肺癌又称原发性支气管肺癌,是具有高发病率及高死亡率的肺部恶性肿瘤。国家癌症中心计算,我国2015年新发肺癌患者73.3万人,死亡61万人。肺癌是男性肿瘤发病率和死亡率的第一位,是女性肿瘤发病率的第二位和死亡率的第一位。在世界范围内,肺癌是男性肿瘤发病率和死亡率的第一位,是女性肿瘤发病率的第三位和死亡率的第二位。按病理类型可将肺癌分为腺癌、鳞癌、大细胞癌、小细胞癌,其中腺癌、鳞癌、大细胞癌又统称为非小细胞肺癌(non-small cell lung cancer,NSCLC)。由于肺癌早期没有明显症状,绝大部分病人就诊时已经是局部晚期或已发生转移,失去了手术根治的机会。骨骼是其常见的血行转移部位之一,一半的肺癌骨转移患者并发骨相关事件。一旦发生骨相关事件,患者生存时间可缩短一半。NSCLC约占整个肺癌的75%-80%。NSCLC预后总体较差,5年生存率约15%-16%。肺癌的标准治疗包括手术、放疗、化疗等综合治疗,目前化疗已发展至瓶颈期。随着分子靶向药物及免疫检查点抑制剂的引入,肺癌的治疗策略发生了巨大的变化,迎来了新的曙光。肺癌的发生发展是个复杂的过程,多个驱动基因异常参与其中,目前主要的可用于非小细胞肺癌治疗的靶向基因包括表皮生长因子受体(epidermal growth factor receptor,EGFR)敏感突变、间变性淋巴瘤激酶(anaplasticlymphoma kinase,ALK)融合、c-ros肉瘤致癌基因1(c-ros oncogene 1,ROS1)融合,其他的还包括人表皮生长因子受体2(human epidermal growth factor receptor 2,HER2)突变、BRAF V600E突变、高水平MET扩增或MET14外显子跳跃突变、RET重排等。然而仍然还有许多未知的驱动基因参与其中。寻找出新的驱动基因或治疗靶点是目前肺癌研究的热点。肿瘤基因组图谱数据库(the cancer genome atlas,TCGA)收集了大量人类肿瘤特别是肺癌的测序结果,旨在通过系统分析,绘制出人类肿瘤的基因变异图谱,为癌症的诊断、治疗提供帮助,同时以期寻找新的治疗肿瘤的方法和策略。我们对TCGA数据库进行分析,筛选出了483个肿瘤相关的基因,希望通过二代测序技术(next generation sequencing,NGS)在临床NSCLC患者组织样本中对这些基因进行测序,检测基因的变异情况,以期发现新的与肺癌特别是肺癌骨转移相关的突变基因;同时,对TCGA中肺腺癌核糖核酸测序结果分析显示,GDP甘露糖-4,6-脱水酶(GDP-mannose-4,6-dehydratase,GMDS)是其中差异性最显著的基因之一。GMDS是岩藻糖基化过程中的关键酶之一,与GDP-4-酮-6-脱氧甘露糖-3,5异构酶-4-还原酶(GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase,FX)共同参与GDP岩藻糖的合成。在肺癌、结直肠癌、乳腺癌、卵巢癌、肝癌、胰腺癌等多个肿瘤中存在岩藻糖基化异常,岩藻糖基化异常与肿瘤的发生发展密切相关。GMDS位于6号染色体,编码372氨基酸,其功能缺失导致白细胞粘附缺陷症的发生。结直肠癌及其转移灶中均存在GMDS突变,而转移灶GMDS突变明显高于原发灶,其可能与疾病的进展相关。结直肠肿瘤细胞系HCT116中GMDS外显子缺失导致功能缺陷,从而使其逃脱自然杀伤细胞的免疫监视。同时岩藻糖基化中的其他关键酶也与肿瘤的发生发展密切相关,与原发性结直肠肿瘤细胞系sw480相比,转移性结直肠肿瘤细胞系sw620高表达FX,增强了肿瘤-内皮细胞粘附作用,肺腺癌细胞系CL1-5高表达FUT8,增加了肿瘤增殖、侵袭、转移能力。GMDS在NSCLC中的表达情况及其在肿瘤发生发展中的作用目前还未见报道,因此,我们希望通过免疫组化检测临床NSCLC患者中GMDS表达情况,同时在细胞水平研究GMDS对NSCLC发生发展的作用及其可能的机制,以探讨GMDS可否作为NSCLC治疗的新靶点。【研究目的】从TCGA数据库中筛选出了483个与肿瘤相关的基因,检测其在NSCLC合并骨转移患者中的变异情况,以期寻找与肺癌特别是骨转移密切相关的基因。观察NSCLC患者癌组织中GMDS表达与癌旁是否存在差异,NSCLC细胞系中GMDS表达情况,同时采用慢病毒介导的RNAi沉默GMDS,观察抑制GMDS表达对细胞增殖、凋亡的影响。【研究方法】采用Hiseq2000_PE75二代测序平台,检测8例NSCLC同时合并骨转移患者中483个肿瘤相关基因的变异情况,以期寻找与NSCLC特别是与骨转移相关的基因,寻找新的潜在治疗靶点。应用免疫组织化学(immunohistochemistry,IHC)方法检测10例肺腺癌患者癌组织及癌旁组织GMDS表达情况,利用聚合酶链式反应(polymerase chain reaction,PCR)、实时聚合酶链式反应(real time polymerase chain reaction,RT-PCR)及蛋白印记(western-blot,WB)检测NSCLC细胞系中GMDS表达情况。慢病毒介导的RNA干扰(RNA interference,RNAi)技术沉默GMDS后,使用RT-PCR检测干扰效率。GMDS表达被抑制后,MTT检测细胞活力,流式细胞仪检测细胞周期及细胞凋亡。【结果】第一部分:8例NSCLC合并骨转移患者中,共检测出3620个基因突变,多个与肺癌相关的信号通路基因均存在突变;同时存在16个突变一致性基因,6例(75%)患者存在HNF1α1394_1395ins8突变,5例(62.5%)患者存在APC A818G突变,4例(50%)患者存在CD22 G1703A突变。对HNF1α及CD22行Sanger测序,结果显示与NGS测序结果一致。第二部分:10例肺腺癌患者癌组织与癌旁组织GMDS IHC检测,癌组织GMDS表达为3.597±1.908,癌旁组织GMDS表达为0.453±1.119,癌组织GMDS表达明显高于癌旁组织(P值0.01)。RNA干扰后,A549细胞干扰组GMDS表达量为0.28±0.017,明显低于对照组的1.03±0.097(P值0.01);H1299细胞干扰组GMDS表达量为0.19±0.022,明显低于对照组的1.00±0.004(P值0.01)。细胞活力方面,GMDS-Si RNA组H1299细胞在96,120小时OD值分别为0.464±0.019,0.453±0.038,低于对照组细胞的0.815±0.051,0.969±0.012(P值0.01);GMDS-Si RNA组A549细胞在96,120小时的OD值为0.744±0.005,0.858±0.041,低于对照组细胞的1.105±0.026,1.441±0.042(P值0.01)。细胞周期分布方面,GMDS-Si RNA组H1299细胞在G1期的比例为46.15±1.20%,高于对照组的27.96±0.93%(P值0.01);在S期的比例为50.80±0.82%,低于对照组的59.40±0.75%(P值0.01);在G2期的比例为3.05±0.41%,低于对照组的12.64±1.21%(P值0.01)。GMDS-Si RNA组A549细胞在G1期的比例58.2±1.32%,高于对照组的54.83±0.41%(P值0.05);在G2期的比例10.62±0.80,低于对照组的14.42±0.42%(P值0.01)。凋亡方面,GMDS-Si RNA组H1299细胞中凋亡细胞占到14.90±0.39%,明显高于对照组的3.83±0.23%(P值0.01)。GMDS-Si RNA组A549细胞中凋亡细胞占到23.01±0.45%,明显高于对照组的3.59±0.13%(P值0.01)。【结论】NGS结果显示多个肺癌相关信号通路基因突变参与了NSCLC的发生发展,同时HNF1α(1394_1395ins8)、CD22(G1703A)、APC(A818G)等高频一致性突变可能与NSCLC骨转移相关。GMDS在肺腺癌组织中高表达,沉默NSCLC细胞系GMDS表达后,明显抑制了细胞增殖,促进了细胞凋亡,GMDS表达可能可以作为NSCLC增殖、凋亡的预测指标,GMDS也许可以作为NSCLC治疗的潜在靶标。
[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.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
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本文编号：1856439