【作者】 刘佳；

【导师】 李春；

【作者基本信息】 天津大学 ， 生物化工， 2020， 硕士

【摘要】 代谢重编程是癌症的十大特征之一,肿瘤细胞通过加快代谢速率或改变代谢路径以满足其持续增殖和生长的要求。胆固醇作为细胞膜的重要组分,其高速合成是肿瘤细胞快速增殖的必要条件。研究表明,靶向胆固醇合成关键酶HMGCR的他汀类药物可降低胆固醇合成速率,抑制多种肿瘤的发展,然而通过直接将胆固醇转化至其他代谢物进而遏制肿瘤生长的研究仍较少。在多个物种体内,胆固醇与胆固醇合成中间产物7脱氢胆固醇(7-DHC)可以互相转化。已有研究表明7-DHC对黑色素瘤具有明显的抑制作用,7-DHC可以显著地抑制黑色素瘤细胞体外增殖并诱导凋亡,因此胆固醇体内转化可能是黑色素瘤潜在的治疗策略之一。合成生物学中的基因线路设计构建在疾病治疗中具有巨大潜力,其运用了工程化的设计思路,构建标准化的元器件和模块,合成全新的人工生命系统调控生物体的行为,具有精确、灵活、适用范围广等特点。对于代谢异常引起的相关疾病,工程化基因线路的体内构建有望实现精准、可控地重构代谢路径,实现体内自稳态,为代谢异常的疾病(如糖尿病,心血管疾病以及肿瘤疾病)提供新的治疗方案。本研究以黑色素瘤为模型,基于CRISPR-Cas9系统以及胆固醇7-去饱和酶构建了一种精准、可控的基因线路,靶向胆固醇代谢重编程,通过体内催化胆固醇转化为7-DHC实现对黑色素瘤的抑制。研究内容主要分为三部分:输出信号的筛选、输入信号的筛选和基因线路的构建及验证。鉴于尚未在高等哺乳生物中发现胆固醇7-去饱和酶,本研究根据进化保守原则,最终5种真核生物中筛选出家蚕来源的胆固醇7-去饱和酶NVD-BM作为输出信号,NVD-BM可有效地催化黑素瘤细胞中的胆固醇转化为7-DHC,导致7-DHC在肿瘤细胞中积累,从而显著地抑制小鼠模型中黑色素瘤的生长。转录因子是一类可结合到启动子上调控下游基因表达的内源性信号分子,细胞中转录因子的表达受到严格调控,异常表达可导致多种肿瘤发生。因此特定肿瘤中高表达的转录因子是基因线路的理想输入信号。本研究通过分析黑色素瘤和正常皮肤样本的转录组数据,筛选到十个在黑色素瘤中表达上调的转录因子,经过双萤光素酶实验优化两两组合的双输入信号,最终确定将调控报告基因表达效率最高的转录因子RELA和STAT1作为输入信号。接着我们结合CRISPR-Cas9系统和TET-ON开关,在黑色素瘤细胞中构建了一种新型的合成基因线路,双输入信号“与”门通过感应表达上调的转录因子RELA和STAT1,从而激活NVD-BM在黑色素瘤细胞中的表达,催化胆固醇转化为7-DHC,进而抑制黑色素瘤细胞的增殖和迁移速率。本研究首次在肿瘤中构建了以转录因子为输入信号,异源胆固醇7-去饱和酶NVD-BM为输出信号的基因线路。NVD-BM的表达可以显著抑制黑色素瘤的生长,同时机制上我们解析了NVD-BM通过抑制Akt1-ser473位点上丝氨酸的磷酸化进而抑制Akt1/NF-κB信号通路的活化。本研究的结果证实了双转录因子作为输入信号的基因线路可精确控制胆固醇去饱和酶的表达,可实现肿瘤体内代谢路径重构,进而达到杀伤肿瘤的效果。该基因线路的构建思路同时有望应用于其他代谢异常的肿瘤类型,为肿瘤靶向治疗提供全新的策略。更多还原

【Abstract】 Metabolic reprogramming is one of the ten characteristics of tumor.In order to meet the requirements of continuous proliferation and growth,cancer cells must accelerate the metabolic rate,or change the metabolic pathway.As an important component of cell membrane,high synthesis rate of cholesterol is a necessary condition for rapid proliferation of tumor cells.Studies have shown that statins targeting the key enzyme of cholesterol synthesis,HMGCR,can reduce the rate of cholesterol synthesis and inhibit the development of a variety of tumors.However,studies on inhibiting tumor growth by directly converting cholesterol to other metabolites are still rare.Cholesterol and cholesterol synthesis intermediate metabolite 7-dehydrocholesterol(7-DHC)can be transformed each other in multiple species.Previous studies have shown that 7-DHC has a significant inhibitory effect on melanoma,and 7-DHC can significantly inhibit the proliferation of melanoma cells in vitro and induce apoptosis,so the transformation of cholesterol in vivo may be one of potential melanoma treatment strategies.The genetic circuits of synthetic biology,which use the engineering design idea to build standardized components and modules,and synthesizes the new artificial life system to regulate biological behavior,are characterized by precision and flexibility and widely used range can play a huge potential in the treatment of tumor and other diseases.Therefore,the construction of engineered genetic circuits is expected to realize the accurate and controllable reconstruction of the metabolic pathway in vivo,enables homeostasis in the body and provides new treatments for metabolic disorders such as diabetes,cardiovascular disease and tumors.In this study,based on the CRISPR-Cas9 system and cholesterol 7-deasturase,a precise and controlled genetic circuit was constructed in melanoma,which can target the reprogramming of cholesterol metabolism and mediate the transformation of cholesterol into 7-DHC,so as to achieve the inhibition of melanoma.The contents of this study are mainly divided into three parts: the screening of output signal,the screening of input signals,and the construction and verification of gene circuits.In consideration of unsuccessful identification of cholesterol 7-desaturase in higher mammals,this study finally identified the source of silkworm cholesterol 7-desaturase NVD-BM as output signal from five species of eukaryotes by evolutionary conservatism,which can catalyze the conversion of cholesterol to 7-DHC in melanoma cells,so that 7-DHC can accumulate in cells and significantly inhibit the growth of melanoma.Transcription factors are endogenous signaling molecules that bind to promoters to regulate the expression of downstream genes.The expression of transcription factors is strictly regulated in cells,and abnormal expression can lead to multiple tumorigenesis.Therefore,highly expressed transcription factors are ideal input signals for genetic circuits in specific tumors.In this study,ten highly expressed transcription factors were selected in melanoma via analyzing transcriptome data of melanoma and normal skin tissues.According to the dual luciferase assay,pair-wise input signals were optimized that two transcription factors,RELA and STAT1,with the highest efficiency in regulating reporter gene expression,were finally selected as input signals.Then a new type of genetic circuit was constructed in melanoma cells based on CRISPR-Cas9 system and TET-ON switch.Through inducing the up-regulated transcription factors RELA and STAT1 in melanoma,dual-input “AND” gate activated the expression of NVD-BM that catalyzed cholesterol into 7-DHC,which inhibited the proliferation and migration rate of melanoma cells.In this study,for the first time,a genetic circuit was constructed in the tumor with transcription factors as the input signals and heterogenous cholesterol 7-deasturase NVD-BM as the output signal.NVD-BM can significantly inhibit the growth of melanoma,at the same time,we verified that NVD-BM suppressed the activation of Akt1/NF-κB pathway by inhibiting phosphorylation of serine at Akt1-ser473.The results confirmed that genetic circuit can precisely control the expression of cholesterol7-desaturase to realize the reconstruction of metabolic pathway,and thus achieve the effect of killing tumors.Meanwhile,the construction idea of genetic circuit is expected to be applied to other tumor types with abnormal metabolism,providing a new idea for targeted therapy of tumors.更多还原