RNF6通过调节RIPK3转录促进NF-κB信号通路在结肠癌进展中的分子机制研究

Gene amplification is a hallmark of cancer and is frequently observed in colorectal cancer (CRC). Whole genome sequencing identified the novel gene amplification of Ring finger protein 6 (RNF6), a RING-domain E3 ubiquitin ligase, also a transcription factor in CRC. In our previous study, we reported RNF6 plays a pivotal oncogenic role by mediating TLE3 ubiquitination and degradation in colorectal tumorigenesis. However, little is known about the transcriptional regulating role of RNF6 in CRC development and progression. The ability of transcription factor to interact with thousands of DNAs makes a group of mRNAs to be selectively dysregulated in cancer. The idea that amplification of RNF6 may bring large-scale changes in global gene expression is conceivable. In the current study, we performed ChIP-sequencing and transcriptome-sequencing analysis to find the transcriptional targets and dysregulated pathways of RNF6. RIPK3 was the crucial downstream target of RNF6 confirmed by ChIP-PCR assay. RIPK3 could activate NF-κB signaling pathway in many tumors. We hypothesis that RNF6 may through transcriptional regulation of RIPK3 to trigger NF-κB activation in colorectal tumorigenesis. In this study we will use ChIP-PCR, co-immunoprecipitation (Co-IP), luciferase reporter and immunofluorescence assays to investigate the transcriptional regulation mechanisms of RNF6 in CRC. It is the first time to investigate the tumorigenecity of conditional expression
 of a homozygous/heterozygous rnf6 
in the mouse intestines. The clinical impact of RNF6 and RIPK3 will be assessed in independent CRC cohorts. The RNF6-RIPK3-NF-κB axis may represent a potential therapeutic target in CRC treatment.

我们前期研究发现RNF6在结肠癌中拷贝数增加导致其异常高表达。RNF6含有指环蛋白结构，具有转录调节和E3泛素化蛋白酶双重功能。我们既往研究揭示了RNF6发挥E3泛素化酶的致癌机制，但是RNF6作为转录因子如何调控基因表达，在结肠癌中如何发挥作用仍不清楚。应用ChIP-seq联合RNA-seq分析，我们首次发现RNF6转录激活RIPK3 mRNA表达，并参与调控NF-κB信号通路，提出RNF6-RIPK3-NF-κB轴可能作为新的信号通路促进结肠癌发生发展。我们已经成功建立结肠特异表达RNF6转基因小鼠模型（Rnf6 fl/fl cdx2cre/+），本项目拟采用染色质免疫共沉淀、蛋白免疫共沉淀、双荧光素酶报告基因等技术，从体内及体外探讨RNF6促进结肠肿瘤形成能力及转录调控机制。本项目研究有助于阐明RNF6介导RIPK3激活NF-κB新途径及机制，为结肠癌个性化靶向治疗提供新的理论依据。

结肠癌是一种具有多种致病机制的异质性疾病，涉及遗传和表观遗传改变。我们前期通过对CRC 组织标本进行全基因组测序识别了RNF6拷贝数（DNA copy number）扩增与其过表达密切相关。RNF6是一种具有致癌作用的环状指蛋白。多项研究表明，RNF6 通过泛素化和靶蛋白降解促进多种肿瘤的发生发展。然而，RNF6 作为转录因子介导的转录调控在CRC进展中的潜在作用尚无报道。.在本研究中，我们建立了结肠特异性RNF6过表达转基因小鼠，并证明了在化学药物（AOM）诱导的结肠癌(Colorectal Cancer, CRC)模型中，与野生型小鼠相比，RNF6过表达促进了结直肠癌的发生发展。为了阐明RNF6的致癌作用是否依赖于其转录活性，我们进行了染色质免疫沉淀测序和RNA测序分析，发现剪接因子SF3B2(Splicing factor 3b subunit 2, SF3B2)可能是RNF6潜在的转录调控靶点。CHIP-PCR、凝胶迁移实验(EMSA)、荧光素酶报告基因实验表明RNF6与SF3B2启动子结合，其过表达后可激活CRC肿瘤细胞、原始CRC类器官和RNF6转基因小鼠中SF3B2的表达。敲除SF3B2可消除RNF6过表达产生的促肿瘤作用，而SF3B2的再表达恢复了RNF6敲除CRC细胞的促生长和迁移/入侵效应，这进一步表明在CRC中，SF3B2是RNF6转录调控的直接作用靶点。应用SF3B2抑制剂Pladienolide B靶向RNF6-SF3B2轴，可在体内、体外抑制RNF6过表达的CRC细胞生长。此外，5-氟尿嘧啶与Pladienolide B联合用药在RNF6高表达CRC中发挥协同作用，致使异种移植模型中的肿瘤消退。这些研究表明，RNF6的促肿瘤作用主要是通过SF3B2的转录上调来实现的, RNF6-SF3B2轴可能是CRC治疗的潜在靶点。.本研究首次发现在CRC中RNF6与SF3B2启动子结合并转录上调SF3B2表达，从而促进CRC的发生发展。用剪接体抑制剂Pladienolide B靶向 RNF6-SF3B2 轴能有效抑制RNF6高表达的CRC 肿瘤细胞生长，特别是与 5-FU 联合使用时。我们的研究发现为CRC提供了一种新的药物靶点和治疗策略。