【作者】 杨超；

【导师】 郑勇斌；

【作者基本信息】 武汉大学 ， 外科学， 2022， 博士

【摘要】 结直肠癌(Colorectal cancer,CRC)是全球发病率第三高的恶性肿瘤,也是癌症导致死亡的第二大原因。在过去几十年里我国经济建设取得巨大成就,伴随着的是癌症谱从发展中国家向发达国家转变,CRC负担显著增加。据统计从1990年到2019年的30年间,我国CRC发病率和死亡率上升幅度分别为144.01%和36.15%。CRC疾病负担已经成为我国目前急需要解决的重大公共卫生课题。因此深入探究CRC发生发展的机制,对于改善CRC患者预后具有重要意义。代谢重编程是恶性肿瘤的重要标志,但目前对代谢重编程研究主要集中在糖代谢和氨基酸代谢上,而对脂质代谢关注不足。据报道,脂质代谢参与了 CRC的发生发展及治疗抵抗,因此有必要对CRC中的脂质代谢重编程进行深入研究,找到参与脂质代谢的关键基因,为开发新的治疗策略提供潜在的代谢靶点。本研究将从以下三个部分展开:第一部分结直肠癌关键脂质代谢基因筛选与风险评分构建目的:筛选CRC中关键的脂质代谢相关基因(Lipid metabolism-related genes,LMRGs)并计算LMRGs风险评分;探究LMRGs风险评分的临床应用价值及与免疫微环境的关系。方法:1.分别从TCGA和GEO数据库中下载CRC的研究队列数据,并从MSigDB获得LMRGs;2.筛选TCGA和GEO队列中共有的LMRGs,并分别通过差异分析和单因素Cox回归分析获得CRC中具有预后价值的差异LMRGs;再利用LASSO Cox回归模型筛选出CRC中关键的LMRGs并构建LMRGs风险评分;3.分析LMRGs风险评分与CRC患者生存预后、临床病理特征间的关系;4.利用CIBERSORT算法计算CRC组织中22种免疫细胞的丰度,评估LMRGs风险评分与免疫细胞丰度间的关系;5.构建整合了 LMRGs风险评分和患者临床病理特征的列线图模型,并评估列线图模型的临床应用价值;6.利用GSEA探究不同LMRGs风险评分组间潜在分子机制。结果:1.从公共数据库中下载了 TCGA-CRC和GEO-GSE39582两个CRC研究队列,一共包含了 1023例CRC样本;从MSigDB—共得到了 1044个LMRGs;2.在TCGA-CRC队列中有729个差异表达的基因;单因素Cox回归分析表明有57个LMRGs具有预后价值;最终,一共得到了 47个预后相关的差异表达 LMRGs;3.利用LASSO Cox回归模型,一共筛选到4个CRC中关键的LMRGs,即CPT2、PROCA1、CCKBR 和 FDFT1;在 TCGA-CRC 和 GEO-GSE39582 队列中高LMRGs风险评分患者的总体生存期均显著短于低LMRGs风险评分患者;单因素和多因素Cox回归分析的结果显示LMRGs风险评分是CRC的独立预后因素;LMRGs风险评分与患者的年龄和性别无关,但与肿瘤恶性程度正相关。4.LMRGs风险评分与大多数免疫细胞的丰度负相关。具体来说,在高LMRGs风险评分组中M2样巨噬细胞(P<0.001)明显富集,而在低LMRGs风险评分组中B细胞(P=0.012)、CD4+T细胞(P=0.008)和树突细胞(P=0.004)丰度更高;5.构建了包括年龄、性别、肿瘤分期和LMRGs风险评分的列线图模型,该列线图具有很好的临床应用潜力。在TCGA-CRC队列中,预测患者1、3和5年生存期 ROC 的 AUC 值分别为 0.7652、0.8058 和 0.7972。在 GEO GSE39582队列中,该列线图预测患者1、3和5年生存期ROC的AUC值分别为0.7325、0.7280和 0.7173;6.GSEA结果显示与肿瘤恶性生物学行为相关的信号通路,如Hedgehog、KRAS、Wnt/β catenin、EMT、Angiogenesis 和 TGF-β等信号通路在高 LMRGs 风险评分组中显著富集。结论:1.本研究一共筛选出4个CRC中关键的LMRGs,分别为CPT2、PROCA1、CCKBR和FDFT1,是CRC中潜在的治疗靶点;2.LMRGs风险评分是CRC患者独立的预后因素,可对CRC患者进行准确的危险度分层;3.LMRGs风险评分与CRC患者的肿瘤免疫微环境相关。第二部分AMFR依赖的CPT2通过下调CYP1A1抑制结直肠癌进展目的:检测CPT2在临床标本和细胞系中的表达情况并分析其与临床病理特征和预后的关系;在体内、外实验中研究CPT2的功能,并探究CPT2发挥功能的分子机制。方法:1.利用QPCR、WB和IHC检测在CRC和配对正常组织中CPT2表达水平;分析CPT2表达水平与患者临床病理特征和预后的关系;2.构建沉默和过表达CPT2的CRC稳转细胞株,通过表型实验探究CPT2的分子功能;3.使用二代转录组测序方法寻找CPT2下游作用靶点;构建瞬转过表达和沉默CYP1A1的CRC细胞模型;通过挽救实验探究CYP1A1在CPT2分子功能中的作用;4.通过蛋白半衰期实验和Co-IP实验对CPT2的上游调控分子进行探究;构建沉默和过表达AMFR的瞬转CRC细胞模型探究AMFR与CPT2间的关系;5.利用拯救实验探究AMFR、CPT2和CYP1A1三者间的关系。结果:1.和正常组织相比,CPT2在CRC组织中显著下调;CPT2表达水平与患者的性别和年龄无关,但随着肿瘤恶性程度增加,CPT2表达降低;高表达CPT2的患者预后更好;2.过表达CPT2抑制了 CRC细胞的增殖、侵袭和迁移能力;而在沉默CPT2后,CRC细胞的增殖、侵袭和迁移能力都得到了显著提升;3.在沉默CPT2后一共有901个表达差异的基因,其中CYP1A1表达差异最显著。QPCR和WB实验证实CPT2负性调控CYP1A1表达;功能实验发现CYP1A1促进了 CRC细胞的恶性生物学行为;挽救实验发现CPT2在CRC中的生物学功能部分是由CYP1A1介导的;4.蛋白半衰期实验发现CPT2受泛素-蛋白酶体途径调控;UbiBrowser网站预测AMFR是调控CPT2的泛素E3连接酶;Co-IP实验表明AMFR能够与内源性CPT2结合;过表达AMFR显著增加了 CPT2的泛素水平从而促进了内源性CPT2蛋白的降解,而沉默AMFR则延缓了 CPT2的降解;回复实验发现CPT2部分介导了 AMFR在CRC中的促癌功能;5.过表达CPT2部分回复了 AMFR诱导的CYP1A1增加。结论:1.CPT2在CRC中低表达,其表达水平与肿瘤恶性程度负相关;高表达CPT2是CRC预后良好的标志;2.CPT2抑制了 CRC的恶性生物学行为;3.AMFR/CPT2/CYP1A1轴在CRC中发挥重要作用,是CRC潜在的治疗靶点。第三部分CPT2参与糖、脂代谢重编程并通过P53/SLC7A11轴促进结直肠癌发生铁死亡目的:探究CPT2通过参与CRC代谢重编程发挥抑癌作用的分子机制。方法:1.利用WB分析CPT2沉默和过表达对糖、脂代谢关键蛋白表达的影响;利用气相色谱-质谱代谢组学方法分析过表达CPT2对CRC中-长链脂肪酸含量的影响;2.利用JC-1探针检测CRC细胞中的线粒体膜电位,并通过透射电镜观察线粒体形态;3.利用MtSOX探针,O2·-探针和丙二醛含量来评估CRC细胞中氧化状态;利用Fe2+探针评估CRC细胞中铁池水平;4.通过表型实验观察铁死亡抑制剂处理对CPT2分子功能的影响;5.利用二代转录组测序方法寻找CPT2下游差异的铁死亡调控基因和可能的调控通路,并利用QPCR和WB进行验证;6.构建沉默P53的瞬转CRC细胞模型,探究P53在CPT2促进CRC发生铁死亡中的作用;结果:1.过表达CPT2促进了线粒体氧化磷酸化、脂肪酸从头合成和脂质转运相关蛋白的表达;但抑制了葡萄糖转运和细胞无氧糖酵解相关的蛋白表达。代谢组数据显示过表达CPT2后CRC细胞中中-长链脂肪酸总含量更高,特别是不饱和脂肪酸;2.JC-1探针检测发现在过表达CPT2后线粒体膜电位出现丢失;透射电镜观察到在CPT2沉默的CRC细胞中线粒体形态大小正常,结构完整;而在CPT2过表达后,CRC细胞中线粒体凝固皱缩,部分线粒体外膜断裂;3.过表达CPT2显著增加了 CRC细胞中线粒体来源的ROS、O2·-和脂质过氧化水平;同时过表达CPT2促进了 Fe2+在细胞内聚集;4.铁死亡抑制剂部分逆转了 CPT2对CRC细胞增殖、侵袭和迁移能力的抑制;5.转录组数据显示SLC7A11可能是CPT2促进CRC发生铁死亡的作用靶点;SLC7A11抑制剂显著促进了过表达CPT2诱导的CRC细胞铁死亡;6.富集分析提示P53通路可能介导了 CPT2发生铁死亡;挽救实验发现沉默P53挽救了 CPT2诱导的CRC细胞铁死亡,机制上CPT2通过P53介导的方式下调SLC7A11表达从而促进CRC发生铁死亡;结论1.CPT2促进了线粒体氧化磷酸化和脂质合成及脂质摄入,但抑制了细胞无氧糖酵解;2.CPT2对CRC细胞的抑制作用部分是由铁死亡介导的;机制上,CPT2通过P53/SLC7A11轴促进CRC细胞发生铁死亡;更多还原

【Abstract】 Colorectal cancer(CRC)is the third most common malignancy worldwide and the second leading cause of death from cancer.In the past few decades,our country has made great achievements in economic construction.With the transformation of the cancer spectrum from developing countries to developed countries,the burden of CRC has increased significantly.According to statistics,in the 30 years from 1990 to 2019,the incidence and mortality of CRC in our country increased by 144.01%and 36.15%,respectively.The disease burden of CRC has become a major public health issue that urgently needs to be solved in our country.Therefore,clarifying the mechanism of tumorigenesis and development of CRC is of great significance for preventing and improving the prognosis of CRC.Metabolic reprogramming is an important marker of malignant tumors.At present,the research on metabolic reprogramming mainly focuses on glucose and amino acid metabolism,while insufficient attention is paid to lipid metabolism.It has been reported that lipid metabolism is involved in the tumorigenesis,development and treatment resistance of CRC.Therefore,it is necessary to conduct in-depth research on lipid metabolism reprogramming in CRC to identify key genes involved in lipid metabolism,which will provide potential metabolic targets for the development of new therapeutic strategies.This study consists of the following three parts:PART Ⅰ Screening and risk score construction of key lipid metabolism genes in colorectal cancerObjective:To screen the key lipid metabolism-related genes(LMRGs)in colorectal cancer(CRC)and calculate the LMRGs risk score.To explore the clinical application value of the LMRGs risk score and its relationship with the immune microenvironment.Methods:1.Download the cohort data of CRC from the TCGA and GEO databases,respectively,and obtain LMRGs from MSigDB;2.LMRGs co-existing in TCGA and GEO cohort were screened,and differential LMRGs with prognostic value in CRC were obtained by difference analysis and univariate Cox regression analysis,respectively.LASSO Cox regression model was used to screen out the key LMRGs in CRC and construct LMRGs risk score.3.Analyze the relationship between LMRGs risk score,survival prognosis and clinicopathological features of CRC patients;4.CIBERSORT algorithm was used to calculate the abundance of 22 immune cells in CRC tissues,and to evaluate the relationship between LMRGs risk score and the abundance of immune cells;5.Build a nomogram integrating LMRGs risk score and clinicopathological characteristics,and evaluate the clinical application value of this nomogram;6.GSEA was used to explore the potential molecular mechanisms between different LMRGs risk score groups.Result:1.Two CRC cohorts TCGA-CRC and GEO-GSE39582 were downloaded,including 1023 CRC samples.1044 LMRGs were obtained from MSigDB;2.There were 729 LMRGs with differential expression in the TCGA-CRC cohort.Univariate Cox regression analysis showed that 57 LMRC had prognostic value.A total of 47 differentially expressed LMRGs related to prognosis were finally obtained.3.Four key LMRGs in CRC were screened by LASSO Cox regression model,namely CPT2,PROCA1,CCKBR and FDFT1.The overall survival of patients with high LMRGs risk scores in the TCGA-CRC and GEO-GSE39582 cohorts was significantly shorter than that with low LMRGs risk score.Univariate and multivariate Cox regression analysis showed that LMRGs risk score was an independent prognostic factor for CRC.LMRGs risk score was not associated with patient age and gender,but was positively correlated with tumor malignancy.4.The LMRGs risk score was negatively correlated with the abundance of most immune cells,specifically,M2-like macrophages(P<0.001)were significantly enriched in the high LMRGs risk score group,while in the low LMRGs risk score group B cells(P=0.012),CD4+T cells(P=0.008)and dendritic cells(P=0.004)were more abundant;5.Construct a nomogram model including Age,Gender,Tumor stage,and LMRGs risk score,which has good potential for clinical application in CRC.6.GSEA analysis found that signaling pathways closely related to tumor malignant biological behavior,such as Hedgehog pathway,KRAS pathway,Wnt/β-catenin pathway,EMT,Angiogenesis and TGF-β,were significantly enriched in the high LMRGs risk score group.Conclusion:1.In this study,four key LMRGs in CRC were selected,namely CPT2,PROCA1,CCKBR and FDFT1,which were potential therapeutic targets in CRC.2.The LMRGs risk score was an independent prognostic factor for CRC patients,which could accurately provide risk stratification for CRC patients.3.The LMRGs risk score was correlated with the tumor immune microenvironment in CRC.PART Ⅱ AMFR-dependent CPT2 inhibits CRC progression by downregulating CYP1A1Objective:To detect the expression level of CPT2 in clinical specimens and cell lines and analyze the relationship with clinicopathological characteristics and prognosis.To study the molecular function of CPT2 in vitro and in vivo,and explore the molecular mechanism of CPT2 function.Method:1.CPT2 expression levels in CRC and adjacent normal tissues were detected by QPCR,WB and IHC.The relationship between CPT2 and clinicopathological features and prognosis of patients was analyzed.2.CRC stable cell lines with silent and overexpressed CPT2 using lentivirus-mediated RNA interference technology were constructed to explore the molecular function of CPT2 through phenotypic experiments.3.Downstream targets of CPT2 were identified by second-generation transcriptome sequencing.CRC cell models of overexpressed and silencing of CYP1A1 were constructed.The role of CYP1A1 in CPT2 oncology function was explored by rescue experiments.4.The upstream regulatory molecules of CPT2 were investigated by protein half-life measurement and Co-IP assay.CRC cell models with transient silencing and overexpression of AMFR were constructed to explore the relationship between AMFR and CPT2.5.Use rescue experiment to explore the regulatory relationship among AMFR,CPT2 and CYP1A1.Result:1.Compared with normal tissues,CPT2 is significantly downregulated in CRC tissues.CPT2 expression level is not related with sex and age,but CPT2 expression decreases with the increase of tumor malignancy.Patients with high CPT2 expression have better prognosis.2.Overexpression of CPT2 inhibited the proliferation,invasion and migration of CRC cells;after silencing CPT2,the proliferation,invasion and migration of CRC cells were significantly improved.3.After silencing CPT2,there were 901 differentially expressed genes,among which CYP1A1 had the most significant difference.QPCR and WB confirmed that CPT2 negatively regulates the expression of CYP1A1.Functional experiments found that CYP1A1 can promote the proliferation,invasion and migration of CRC cells.Rescue experiments found that the biological function of CPT2 in CRC was partly mediated by CYP1A1.4.Protein half-life experiment found that CPT2 was regulated by ubiquitin-proteasome pathway.UbiBrowser website predicted that AMFR was an ubiquitin E3 ligase that regulates CPT2.Co-IP showed that AMFR can bind to endogenous CPT2.Overexpression of AMFR significantly increased the ubiquitin level of CPT2 and promoted the degradation of endogenous CPT2 protein,while silencing AMFR delayed the degradation of CPT2.Rescue experiment found that CPT2 partially mediated the cancer-promoting function of AMFR in CRC.5.Overexpression of CPT2 successfully rescued the AMFR-induced CYP1A1 increase.Conclusion:1.CPT2 was lowly expressed in CRC,and its expression level was negatively correlated with the degree of malignancy of the tumor.High expression of CPT2 was a marker of good prognosis of CRC.2.CPT2 inhibits the malignant biological behavior of CRC;3.AMFR/CPT2/CYP1A1 axis played an important role in CRC and was a potential therapeutic target of CRC.PART Ⅲ CPT2 is involved in the reprogramming of glucose and lipid metabolism and promotes ferroptosis in CRC through the P53/SLC7A11 axisObjective:To explore the molecular mechanism by which CPT2 suppresses CRC by participating in metabolic reprogramming.Method:1.The effect of CPT2 on the expression of key proteins in glucose and lipid metabolism was analyzed by WB.The effect of overexpression of CPT2 on the content of long-chain fatty acids in CRC was analyzed by targeted metabolomics method.2.The mitochondrial membrane potential in CRC cells was detected by JC-1 probe,and the mitochondrial morphology was observed by transmission electron microscope.3.The MtSOX probe,superoxide anion probe and malondialdehyde content were evaluated the oxidation level in CRC cells.4.To observe the effect of ferroptosis inhibitor treatment on the molecular function of CPT2 through phenotypic experiments.5.Found the differential ferroptosis regulatory genes and possible regulatory pathways downstream of CPT2 by second-generation transcriptome sequencing,and verified by QPCR and WB.6.To construct a transient CRC cell model with silenced P53 to explore the role of P53 in CPT2 promoting CRC ferroptosis;Result:1.Overexpression of CPT2 promoted the expression of mitochondrial oxidative phosphorylation,de novo fatty acid synthesis and lipid transport-related proteins,but inhibited glucose transport andanaerobic glycolysis.Metabolome data showed that the total content of medium-long-chain fatty acids,especially unsaturated fatty acids,was higher in CRC cells after overexpression of CPT2.2.JC-1 probe detection found that mitochondrial membrane potential was lost after overexpression of CPT2.Transmission electron microscopy observed that mitochondria in CPT2-silenced CRC cells were normal in size and structure;mitochondria were coagulated in CRC cells shrinking,part of the mitochondrial outer membrane is broken after CPT2 overexpression.3.Overexpression of CPT2 significantly increased the mitochondrial-derived ROS,O2-and lipid peroxidation levels in CRC cells;at the same time,overexpression of CPT2 promoted intracellular accumulation of Fe2+.4.The ferroptosis inhibitor significantly reversed the inhibition of CPT2 on the proliferation,invasion and migration of CRC cells.5.Transcriptome data showed that SLC7A11 is an important target of CPT2 to inhibit CRC through ferroptosis.SLC7A11 inhibitor significantly promoted CRC cell ferroptosis induced by overexpression of CPT2.6.Enrichment analysis suggested that the P53 pathway may mediate ferroptosis in CPT2.Rescue experiment found that silencing P53 rescued CPT2-induced ferroptosis in CRC cells.Mechanistically,CPT2 down-regulated the expression of SLC7A11 in a P53-mediated manner to promote ferroptosis in CRC.Conclusion:1.CPT2 promoted mitochondrial oxidative phosphorylation and lipid synthesis and lipid uptake,but inhibits anaerobic glycolysis.2.The inhibitory effect of CPT2 on CRC cells is partly mediated by ferroptosis;Mechanistically,CPT2 promotes ferroptosis through the P53/SLC7A11 axis.更多还原