利用 CRISPR/Cas 基因筛选技术研究 Wnt/β-catenin 信号通路的调控因子

Study Of Regulatory Factors Of Wnt/β-catenin Signaling Using CRISPR/Cas Screen Technology

谭莹

11749221

硕士

生物工程

谭莹

2019-05-22

2019-06-20

哈尔滨工业大学

深圳

Wnt/β-catenin 信号通路在进化中高度保守。在胚胎发育过程中，其在细胞 分化、增殖、迁移等过程中发挥着关键作用。在成体组织中，其对成体干细胞 自我更新能力的维持、组织的损伤修复发挥着重要的作用。Wnt 信号的失调不 但对肿瘤的发生至关重要，而且对晚期肿瘤的转移形成起到了关键作用。因此， Wnt 信号通路是非常具有吸引力的抗肿瘤药物靶点。本课题的主要研究目标是 系统性寻找参与 Wnt 信号通路的重要调控分子。 本课题结合 Wnt/β-catenin 信号通路报告细胞系和 CRISPR/Cas 全基因组筛 选技术，通过两轮筛选及生物信息学分析，寻找参与 Wnt 信号通路下游调控的 信号分子。为了进一步验证筛选结果，选取多个筛选基因，并将其逐个在报告 细胞中敲除。在基因敲除后的报告细胞内，通过 RT-QPCR 技术检测 Wnt 下游 基因 Axin2 mRNA 的表达，通过 WB 技术检测β-catenin 蛋白的表达，进而验证 筛选得到的基因是否对 Wnt 信号通路的调控起到关键作用。 第一轮筛选富集了 460 个基因，其中已知的 Wnt 信号分子占 3.5%，第二 轮富集了 29 个基因，已知的 Wnt 信号分子占 20.7%。表明我们第二轮筛选更 有效的富集了与 Wnt 信号通路相关的基因。该两轮文库的筛选，鉴定出 APC、 CSNK1A1、SKP1、CUL1、GSKIP、DKKL1、IFI44L、KCNC2 和 NOT7 共 9 个负调控 Wnt 信号通路活性的基因。其中，APC、CSNK1A1、SKP1、CUL1、 GSKIP 和 DKKL1 基因可以负调控 Wnt 信号通路已有文献报道。IFI44L、KCNC2 和 NOT7 对 Wnt 信号通路的负调控作用则没有任何相关报道。 研究结果表明，基于 CRISPR/Cas 的全基因组遗传筛选和报告基因技术相 结合，可以广泛用来研究参与不同信号通路调控的基因，以回答任何可应用表 型分离的生物学问题。此次研究发现了 3 个从未被报道的与 Wnt 信号通路相关 的基因（IFI44L，KCNC2 和 NOT7）。到目前为止，其具体的作用机制仍在研 究。

Wnt/β-catenin signaling pathway is highly conserved in evolution. During embryonic development, it plays a key role in cell differentiation, proliferation, migration and other processes. In adult tissues, it plays an important role in the maintenance of adult stem cell self-renewal ability and tissue damage repair. The dysregulation of Wnt signaling is not only critical for tumorigenesis, but also plays a key role in the metastasis of advanced tumors. Therefore, Wnt signaling pathway is a very attractive anti-tumor drug target. The main research objective of this project is to systematically search for important regulatory factors involved in Wnt signaling pathway,and to study the functional mechanism of these novel signaling molecules, and to study its significance in tumor cells. This study combines the Wnt/β-catenin signaling pathway reporting cell line and CRISPR/Cas genome-wide screening technology, through two rounds of screening and bioinformatics analysis, to identify signal molecules involved in the downstream regulation of Wnt signaling pathway. To verify the screening results, multiple screened genes were selected and knocked out one by one in the reporter cells. In the reporter cells after gene knockout, we measured the expression of the Wnt downstream gene Axin2 by RT-QPCR and the expression of β-catenin at the protein level by WB, thereby verifying whether the screened signal molecules are indeed regulating downstream of the Wnt signaling pathway. The first round of screening enriched 460 genes, of which the known Wnt signaling molecules accounted for 3.5%, the second round enriched 29 genes, and the known Wnt signaling molecules accounted for 20.7%. This indicates that our second round of screening is more efficient in enriching genes associated with the Wnt signaling pathway. Through two rounds of library screening, a total of 9 genes(APC, CSNK1A1, SKP1, CUL1, GSKIP, DKKL1, IFI44L, KCNC2 and NOT7) negatively regulating Wnt signaling pathway were identified. Among them, APC, CSNK1A1, SKP1, CUL1, GSKIP and DKKL1 genes are known negatively regulating Wnt signaling pathway. There is no report on the other three genes(IFI44L, KCNC2 and NOT7) on the negative regulation of Wnt signaling pathway .

Wnt CRISPR 筛选

Wnt CRISPR 筛选

中文

联合培养

南方科技大学

谭莹. 利用 CRISPR/Cas 基因筛选技术研究 Wnt/β-catenin 信号通路的调控因子[D]. 深圳. 哈尔滨工业大学,2019.