RUNX2和TEAD1在调控肿瘤EMT过程中的功能与机制研究

随着人口老龄化的加剧，全球癌症发病率与死亡率不断增高，至2017年，癌症已成为导致人类死亡的第二大因素，仅次于心脑血管疾病。同时，近年来国内癌症的负担不断加大，中国癌症的发病率及死亡率均是全球第一，其中中国发病人数占全球发病人数的21.1%，死亡人数占全球死亡人数的23.9%。因此，亟需开发出新的癌症治疗方法与手段以解决目前所面临的问题。癌症治疗新方法的提出依赖于对癌症发病机制的深度探索，而目前关于癌症治疗的难点在于癌症治疗过程中病灶的转移以及化疗药物耐药性的产生。上皮间质转化（EMT）对肿瘤的转移和耐药的产生都具有促进作用，其中EMT的发生是肿瘤开始转移的标志性事件。本研究建立了以肿瘤细胞系为工具研究细胞异质性的方法，同时联合多组学测序技术探明了RUNX2促进细胞迁移的具体机制；发现了在临床上RUNX2的表达量与肿瘤是否发生转移呈正相关，与多种癌症病人的生存期呈负相关的现象；发现了在Wnt-high细胞与Wnt-low细胞中TEAD1基因存在着不同的剪接形式，这种剪接形式可以被ESRP1与MBNL1调节，并且发现TEAD1不同剪接异构体在Wnt-high细胞中具有不同的功能。

通过单细胞测序发现在SW480细胞中具有两群EMT状态且Wnt信号通路活性都不同的细胞群。利用以绿色荧光蛋白（GFP）为信号的Wnt信号报告系统标记SW480细胞，并通过流式细胞仪将性状不同的两群细胞分离出来，分别命名为Wnt-high细胞和Wnt-low细胞。通过qPCR技术检测Wnt下游基因AXIN2与LEF1的mRNA表达水平，确认Wnt-high细胞具有高的Wnt信号通路活性，Wnt-low细胞具有低的Wnt信号通路活性；通过Transwell实验检测发现Wnt-high细胞具有强的细胞迁移能力，Wnt-low细胞具有弱的细胞迁移能力。对Wnt-high细胞与Wnt-low细胞进行ATAC-seq与mRNA-seq测序，通过分析发现两个活性不同且表达特性不同的转录因子RUNX2与TEAD1：RUNX2基因在Wnt-high细胞和Wnt-low细胞中转录水平不同，而TEAD1基因在Wnt-high细胞和Wnt-low细胞中剪接偏好性不同。

在Wnt-high细胞中，利用shRNA干扰RUNX2基因的表达，可以降低细胞的体外迁移能力和体内转移能力；在Wnt-low细胞中，过表达RUNX2，可以促进细胞的体外迁移能力和体内转移能力。此外，在SW480细胞中Wnt信号通路可以调控RUNX2基因的表达：在Wnt-low细胞中，利用CHIR99021激活细胞内Wnt信号通路活性，可以促进RUNX2基因的表达；在Wnt-high细胞中，利用shRNA干扰β-catenin基因的表达，可以抑制RUNX2基因的表达。为了进一步研究RUNX2促进SW480细胞迁移能力的作用机制，多种组学高通量测序技术被应用，包括在Wnt-high细胞中检测了基于RUNX2蛋白的ChIP-seq，以及在Wnt-high细胞干扰RUNX2基因的表达后检测mRNA-seq。综合以上测序结果分析，受RUNX2直接调控的基因需满足以下条件：在ChIP-seq数据中具有RUNX2蛋白结合位点；在ATAC-seq数据中Wnt-high细胞中峰的强度要高于Wnt-low细胞；在Wnt-high细胞中mRNA水平表达量高于Wnt-low细胞；在Wnt-high细胞中，当下调RUNX2基因的表达时，该基因的表达量降低。通过分析共发现401个基因直接受到RUNX2的调控，其中有210个基因与EMT相关，包括PSG1，EPAS1，MPZL2等。通过临床数据分析发现在结肠癌的临床分期病例中，临床Ⅳ期的RUNX2基因的表达量显著高于临床Ⅱ期；在肿瘤发生转移的病人中RUNX2表达量高于未发生转移的病人。同时，在RUNX2表达量更高的结肠癌患者中，生存率更低，并且在神经胶质瘤、肾癌、膀胱癌以及胃癌中也是类似的情况，RUNX2的表达量与患者生存率呈负相关。

通过mRNA-seq的分析发现TEAD1基因的外显子5-6-7处的剪接形式在Wnt-high与Wnt-low细胞之间有巨大差异，这种差异也存在于干细胞与分化的细胞之间。在TEAD1基因中，外显子5与外显子6是一对具有不同功能的互斥外显子，外显子7是一个仅编码4个氨基酸的微小外显子，其在不同细胞中具有不同的组合形式。通过对TEAD家族基因蛋白序列分析发现，不同TEAD家族成员在TEAD1基因外显子5-6-7处的序列都可以由TEAD1基因外显子5、6、7不同组合而成。两个已知对EMT有调控作用的剪接调控基因ESRP1与MBNL1对TEAD1的可变剪接有调控作用。在Wnt-low细胞中，TEAD1所有的剪接异构体均可促进细胞的迁移；在Wnt-high细胞中，只有TEAD1-468剪接异构体可以促进细胞的迁移。通过mRNA-seq分析推测，在Wnt-high细胞中，TEAD1-468剪接异构体可能通过促进ZEB1、KRT81、LCN2、SPARC、CX3CL1以及TLE1等EMT相关基因的表达来促进细胞的迁移；TEAD1-4578剪接异构体可能通过促进IL24基因的表达，以及抑制CLU、CTGF、FSCN1、MPP7基因的表达来抑制细胞的迁移。

综上，本研究在SW480细胞中探索了以肿瘤细胞系作为研究对象来研究肿瘤异质性的可行性，同时探究了多组学相互结合的应用场景，找到了RUNX2促进EMT的作用机制。通过对TEAD1的深入研究发现TEAD1不同剪接异构体在Wnt-high细胞中具有不同的功能。本研究促进了对肿瘤异质性的探索，同时为肿瘤转移的诊断、治疗提供了新的理论基础和潜在的治疗靶点。

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