受非编码区易感位点调控的新型CASP8剪接异构体促进食管癌癌变机制研究

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers in China and represents the 4th leading causes of cancer death. Environmental and genetic factors are all play crucial roles in carcinogenesis of ESCC. Recently, genetic components underlying ESCC have been explored by genome-wide association studies (GWAS) in Japanese and Chinese populations. Multiple common variants at 12 independent loci that have moderate effects with population heterogeneity are associated with ESCC risk. Our pervious study also found that 5 single nucleotide polymorphisms (SNPs) at 2p33.1 was significantly associated with the risk of ESCC in 1528 cases and 2106 controls of northern Chinese descent. The main effect locus located in the deep intron of CASP8 gene. The combined P value was 1.84×10-40 with a combined odds ratio (95% CI) of 1.34 (1.28-1.40). Further expression quantitative trait loci (eQTL) analysis and bioinformatics analysis based on RNA structure suggested that the locus affected the splicing of primary mRNA of CASP8 gene. The subsequent minigene reporter assay showed that its risk allele led to a new alternative splicing isoform of caspase-8 (designated as caspase-8X). In order to clarify the roles of this non-coding susceptibility locus in ESCC, we aim to focus on: (1) Explore the relationship between the locus and alternative splicing model of CASP8 via splicing minigene reporter assay; (2) Identify the splicing factors that directly regulate the splicing of caspase-8X using RNA pulldown followed by mass spectrometry identification. The functional validation will unravel the mechanism underlying the alternative splicing of caspase-8X; (3) Further investigate the in vitro and in vivo functions of caspase-8X in tumorigenesis of ESCC. The results will increase our understanding of the complex genetic architecture of ESCC, reveal the biological functions of the susceptibility loci in deep intronic region regulate tumorigenesis, and provide new targets for the prevention and therapy of ESCC.

我们前期采用全基因组关联分析（GWAS）发现位于2q33.1区域的5个SNP位点与中国汉族人群的食管鳞状细胞癌（ESCC）显著相关。表达数量性状位点（eQTL）分析和RNA结构功能预测提示，位于深部内含子区域的主效位点与一个新的CASP8剪接异构体（Caspase-8X）相关。为解析该位点与食管癌变的关系，探讨食管癌发病机理，本项目拟开展 ⑴剪接异构体体外功能验证分析（Splicing Minigene Reporter Assay），明确该易感位点与CASP8基因选择性剪接的效应；⑵ 用RNA pulldown和质谱分析鉴定该易感位点的RNA结合蛋白，解析SNP影响CASP8基因选择性剪接的机理；⑶ 通过功能实验，确认新型剪接异构体Caspase-8X促进食管细胞癌变的作用。本研究发现并解析位于深部内含子区域的SNP易感位点促进食管细胞癌变的机理，为食管癌防治提供新靶标。

食管鳞癌是我国常见恶性肿瘤，尽管发现了许多食管癌的易感基因，但致病的分子基础尚不清楚。本项目探讨了由RNA剪接和结合蛋白异常相关的食管上皮细胞癌变的机理。发现⑴Caspase-8L和Caspase-8X剪接异构体具有引发食管癌细胞凋亡逃逸和促进食管癌细胞增殖的功能；⑵单链DNA/RNA 结合蛋白PURα通过参与形成细胞应激颗粒，抑制IGFBP3的mRNA翻译启始而促进食管癌进展；⑶转录因子BACH1通过促进EMT和血管生成而促进食管癌发生发展。同时，BACH1高表达可诱导食管癌细胞铁死亡，这种由BACH1诱导的细胞铁死亡可以驱动肿瘤细胞选择淋巴转移。早期淋巴结转移时外周血就会出现针对BACH1的血清自身抗体，且BACH1诱导的铁死亡促进淋巴结转移但却抑制血行转移。机制研究提示，高表达BACH1的肿瘤细胞其单不饱和脂肪酸生成受阻，易受趋化作用进入淋巴道以逃逸铁死亡；⑷其他消化肿瘤相关机理研究，如采用高通量蛋白质组学分析和“发现-确认-验证”多阶段标志物筛选流程，定量分析和多重验证了健康对照和不同转移风险的CRC患者尿液和组织样本，发现了结直肠癌诊断和转移的尿液蛋白标志物，揭示了其中RAD23B和CORO1C促进CRC侵袭转移的分子机制；分析HCC患者配对血液样本中的CTCs和ctDNA检测肝癌术后微小残留病灶（MRD）。结果表明，术后CTCs和ctDNA是HCC复发的独立重要危险因素；研究了蛋白乙酰化诱导的PCK同工酶转换在肝癌系统治疗耐药和代谢重塑中的作用机制，发现索拉非尼耐药细胞中发生了广泛的蛋白磷酸化调控网络和蛋白质乙酰化修饰的异常改变，耐药细胞表现为更活跃的能量代谢表型。这种代谢适应是通过磷酸烯醇式丙酮酸羧激酶异构体2（PCK2）和异构体1（PCK1）的PCK2-K491和PCK1-K473位点的乙酰化，造成两种蛋白质稳定性改变所介导的；胰腺癌肿瘤微环境中分泌蛋白THBS2促进肿瘤进展。胰腺癌细胞与CAFs间的TGF-β1-THBS2调控环路通过激活肿瘤细胞的integrin αvβ3/CD36-MAPK通路促进胰腺癌细胞的增殖、存活和细胞粘附。