胆固醇转运蛋白NPC1L1对结直肠癌细胞增殖的调控作用及分子机制研究

发布时间：2018-07-09 18:35

本文选题：尼曼-匹克C1型类似蛋白 + 核因子kappa　；参考：《第三军医大学》2016年博士论文

【摘要】：一、研究背景代谢异常是恶性肿瘤的重要特征。代谢改变不仅为肿瘤细胞输送维持恶性生物学特性所需的营养物质和能量,也向其源源不断传递不受限制的生长信号。对于肿瘤代谢研究的关注始于二十世纪初Warburg效应的发现:正常细胞主要通过氧化磷酸化和三羧酸循环分解葡萄糖获得能量,而肿瘤细胞即使在供氧充足的情况下也更倾向于利用糖酵解分解葡萄糖,伴随产生大量的乳酸。经过近一个世纪对肿瘤代谢的探索,以Warburg效应为代表的代谢重编程研究早已经突破了糖酵解和三羧酸循环的局限,许多代谢通路包括脂肪酸代谢,谷氨酰胺代谢,丝氨酸代谢和胆固醇代谢等,都被发现在肿瘤细胞中有代谢改变的现象。其中胆固醇代谢异常在肿瘤发生发展中的作用逐渐受到重视,但对其分子机制的研究还比较缺乏。胆固醇在人体中发挥非常重要的生理功能,它不直接提供能量,却是合成胆汁、维生素D和甾体激素的必须原料,更重要的是它是构成细胞膜的主要成分。细胞膜表面由丰富胆固醇和鞘磷脂组成的微结构域脂筏作为功能平台聚集各种信号分子,直接参与并调控了许多重要的信号转导通路。正常生理状态下的胆固醇代谢包括外源和血浆胆固醇的吸收摄取、内源胆固醇合成以及胆固醇的流出三个方面,共同维持细胞及人体内的胆固醇动态平衡。尼曼-匹克C1型类似蛋白(Niemann-Pick C1-like 1,NPC1L1)是外源胆固醇转运的关键蛋白,是临床上广泛应用的降脂药物依折麦布的作用靶点。2008年,一项大型临床研究报告显示,服用含有依折麦布药物的患者肿瘤发生率明显高于安慰剂组(105vs.70,P=0.01)。尽管人们对于这一结论尚存在争议,但很快动物实验中也发现类似的现象:在致癌剂诱导的乳腺癌动物模型中依折麦布能够促进肿瘤的恶性增殖,然而机制尚不清楚。基于多种肿瘤中发现的胆固醇代谢异常现象,NPC1L1在胆固醇代谢中的重要作用以及NPC1L1靶点药物依折麦布的致癌争议,我们推测胆固醇转运蛋白NPC1L1可能在肿瘤的发生发展中扮演重要角色。结直肠癌是消化道常见的恶性肿瘤,而脂质代谢包括胆固醇代谢的异常与其发生发展密切相关,我们对175例临床结直肠癌患者标本的检测发现,NPC1L1在结直肠癌组织中的表达明显低于对应癌旁组织,其表达与临床预后呈显著负相关。因此我们进一步推测,NPC1L1的低表达对于结直肠癌的发生和发展可能具有重要调控作用。二、研究目的:1.研究NPC1L1在人结肠癌组织中的表达及临床意义。2.研究NPC1L1对结直肠癌细胞恶性增殖能力的作用和分子机制。3.探讨自噬抑制剂联用TW-37对逆转NPC1L1缺失引起的结直肠癌细胞增殖表型的作用和分子机制。三、研究材料与方法:1.结直肠癌组织芯片的免疫组织化学染色用于检测NPC1L1在结直肠癌及癌旁组织的表达及临床相关性。2.NPC1L1基因敲除的APCmin/+肿瘤小鼠模型用于研究NPC1L1表达缺失对肠道肿瘤发生发展的影响。3.CRISPR/Cas9技术构建NPC1L1基因敲除的人结直肠癌细胞系用于体外增殖能力检测及机制研究。4.基于慢病毒载体建立NPC1L1敲减及过表达的人结直肠癌细胞系用于体外增殖能力检测及机制研究。四、研究结果:1.人结直肠组织中表达NPC1L1,与正常组织相比,人结直肠癌癌组织低表达。NPC1L1在癌组织中的低表达与临床预后不良密切相关。2.NPC1L1敲除的APCmin/+肿瘤小鼠模型肠道肿瘤数量和肿瘤大小明显增加,肿瘤负荷显著加重。3.CRISPR/Cas9技术构建NPC1L1基因敲除的人结直肠癌细胞系,基于慢病毒载体构建NPC1L1敲减和过表达的人结直肠癌细胞系,使用克隆形成、CCK-8实验和裸鼠移植瘤实验证实NPC1L1表达具有调控肿瘤细胞恶性增殖的能力。4.NPC1L1敲除降低结直肠癌细胞细胞内胆固醇水平,同时抑制LXRα信号通路和激活SREBP2信号通路。使用特异性药物激活LXRα或抑制SREBP2对NPC1L1敲除引起的核因子kappa B(Nuclear Factor Kappa B,NF-κB)信号通路和增殖表型没有明显影响。使用环糊精和环糊精/胆固醇调控细胞内胆固醇水平对NF-κB信号通路也没有明显影响。此外,回复表达野生型NPC1L1和L216A突变的NPC1L1对NF-κB信号通路和增殖表型具有相同的逆转作用。5.NPC1L1缺失诱导的细胞自噬拮抗TW-37的促凋亡作用。自噬抑制剂联用TW-37能够显著逆转NPC1L1缺失引起的结直肠癌细胞增殖表型,自噬抑制增加结直肠癌细胞对TW-37诱导的细胞凋亡的敏感性。五、结论:1.NPC1L1在人结直肠癌组织中低表达,其表达缺失与临床预后不良相关。2.NPC1L1调控人结直肠癌细胞的恶性增殖。3.NPC1L1通过调控NF-κB信号通路作用于肿瘤细胞增殖表型。4.NPC1L1通过非胆固醇依赖方式调控NF-κB信号通路和肿瘤细胞增殖。5.自噬抑制剂联用TW-37显著抑制NPC1L1缺失引起的结直肠癌细胞增殖表型。
[Abstract]:First, the study of metabolic abnormalities is an important feature of malignant tumors. Metabolic changes not only deliver nutrients and energy for cancer cells to maintain malignant biological properties, but also transmit unrestricted growth signals to them. The attention to tumor metabolism research began with the discovery of the Warburg effect in early twentieth Century: normal thin. Cells are mainly obtained by oxidative phosphorylation and three carboxylic acid cycle decomposition of glucose, and tumor cells are also more inclined to use glycolysis to decompose glucose even if the supply of oxygen is sufficient. It is associated with the production of large amounts of lactic acid. After nearly a century of exploration of tumor metabolism, the metabolic reprogramming study represented by the Warburg effect has long been studied Through the limitations of glycolysis and three carboxylic acid cycles, many metabolic pathways, including fatty acid metabolism, glutamine metabolism, serine metabolism and cholesterol metabolism, have been found to have metabolic changes in tumor cells. The research is still scarce. Cholesterol plays a very important physiological function in the human body. It does not provide energy directly. It is the essential ingredient of the synthetic bile, vitamin D and steroid hormones. More importantly, it is the main component of the cell membrane. The surface of the cell membrane is made up of the lipid rafts, a microstructural domain consisting of cholesterol and sphingomyelin, which is composed of cholesterol and sphingomyelin. The functional platform aggregates a variety of signal molecules and directly participates in and regulates many important signal transduction pathways. The cholesterol metabolism in normal physiological state includes three aspects of exogenous and plasma cholesterol absorption, endogenous cholesterol synthesis and cholesterol efflux, and the dynamic balance of cholesterol in human cells and in human body. Niemann - the PEAK C1 - like protein (Niemann-Pick C1-like 1, NPC1L1) is the key protein of exogenous cholesterol transport. It is a clinically widely used lipid lowering drug based on the target of C1. A large clinical study showed that the incidence of cancer in patients with eip-mb drugs was significantly higher than that of the placebo group (105vs.70, P=0.). 01. Despite the controversy over this conclusion, a similar phenomenon has been found in animal experiments: in the animal model of carcinogen induced breast cancer, the mechanism is not clear. However, the mechanism is not clear. Based on the abnormal cholesterol metabolism found in many kinds of tumors, NPC1L1 is in the metabolism of cholesterol. The important role of the drug and the carcinogenic controversy of the NPC1L1 target drug, according to the carcinogenic dispute of MB, we speculate that the cholesterol transporter NPC1L1 may play an important role in the development of the tumor. Colorectal cancer is a common malignant tumor in the digestive tract, and the lipid metabolism, including cholesterol metabolism, is closely related to its development and development. We have 175 cases of cancer. The detection of clinical colorectal cancer specimens showed that the expression of NPC1L1 in colorectal cancer tissues was significantly lower than that of the adjacent cancerous tissues, and the expression was negatively correlated with the clinical prognosis. Therefore, we further speculate that the low expression of NPC1L1 may have important regulatory role in the occurrence and development of colorectal cancer. Two, the purpose of this study is to study 1. NP Expression and clinical significance of C1L1 in human colon cancer tissue.2. study the role of NPC1L1 on the malignant proliferation of colorectal cancer cells and molecular mechanism.3. explore the role and molecular mechanism of the autophagy inhibitor combined with TW-37 to reverse the proliferation phenotype of colorectal cancer cells induced by NPC1L1 deletion. Three, research materials and methods: 1. colorectal carcinoma tissue core Immunohistochemical staining for detecting the expression of NPC1L1 in colorectal cancer and para cancer tissue and the clinical relevance of.2.NPC1L1 gene knockout in APCmin/+ tumor mice model to study the effect of NPC1L1 expression deletion on the development of intestinal cancer,.3.CRISPR/Cas9 technology to construct NPC1L1 gene knockout human colorectal cancer cell lines Detection and mechanism of proliferation ability in vitro.4. based on lentivirus vector to establish NPC1L1 knockout and overexpressed human colorectal cancer cell lines used in vitro proliferation ability detection and mechanism study. Four. Results: 1. human colorectal tissues express NPC1L1, compared with normal tissues, the low expression of.NPC1L1 in human colorectal cancer tissues is in the cancer tissue The number and size of the intestinal tumor in the.2.NPC1L1 knockout APCmin/+ mouse model with low expression and poor clinical prognosis is significantly increased. The tumor load significantly aggravates the.3.CRISPR/Cas9 technique for the construction of human colorectal cancer cell lines with NPC1L1 knockout, and the construction of NPC1L1 knockout and overexpressed human colorectal cancer based on the lentivirus vector Cell lines, using clone formation, CCK-8 experiment and nude mice transplanted tumor verified that NPC1L1 expression has the ability to regulate the malignant proliferation of tumor cells,.4.NPC1L1 knockout reduces the cholesterol level in the cells of colorectal cancer cells, inhibits the LXR alpha signaling pathway and activates the SREBP2 signaling pathway. Using specific drugs to activate LXR a or inhibit SREBP2 to N The nuclear factor kappa B (Nuclear Factor Kappa B, NF- kappa B) signaling pathway and the proliferation phenotype were not significantly affected by PC1L1 knockout. Cholesterol levels in cyclodextrin and cyclodextrin / cholesterol were not significantly affected by NF- kappa B signaling pathway. The pathways and proliferating phenotypes have the same reversal effect on the apoptosis inducing effect of autophagy induced by.5.NPC1L1 deletion of TW-37. Autophagy inhibitor combined with TW-37 can significantly reverse the proliferation phenotype of colorectal cancer cells induced by NPC1L1 deletion, and autophagy inhibits the sensitivity of colorectal cancer cells to TW-37 induced apoptosis. Five, conclusion: 1.N Low expression of PC1L1 in human colorectal cancer tissue, its lack of expression is associated with poor clinical prognosis..2.NPC1L1 regulates malignant proliferation of human colorectal cancer cells by regulating NF- kappa B signaling pathway in tumor cell proliferation phenotype,.4.NPC1L1 regulation of NF- kappa B signaling pathway and tumor cell proliferation by non cholesterol dependent manner. Phage inhibitor combined with TW-37 significantly inhibited the proliferation phenotype of colorectal cancer cells induced by NPC1L1 deletion.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34

【相似文献】