The Role of RBM47 Inactivation in Colorectal Cancer Progression

我们先前已经确定RNA结合基序蛋白47(RBM47)是结直肠癌(CRC)转移的潜在抑制因子：RBM47低表达与间充质样细胞状态、肝转移和低生存率显著相关。然而，在正常肠上皮和原发肿瘤中发现的RBM47表达升高与上皮样状态有关。SiRNA介导的抑制RBM47诱导的EMT、迁移和侵袭。转录因子Snail和Slug抑制了RBM47的表达，siRNA介导的RBM47在移植瘤细胞中的下调促进了转移的形成。在此，我们建议进一步研究RBM47在转移性结直肠癌遗传小鼠模型中的作用和功能。值得注意的是，我们对在这个小鼠模型中获得的已发表的RNA-Seq结果的分析表明，RBM47在正常结肠中的表达最高，在非侵袭性大肠癌中表达降低，在侵袭性大肠癌和肝转移癌中表达最低。我们计划确定肠上皮细胞特异性缺失Rbm47是否会增加这些小鼠的远处转移发生率。此外，我们将研究一种由患者来源的转移性CRC注射到NOD/SCID/伽马(NSG)小鼠体内的原位移植模型，该模型重述了整个腺瘤/腺癌/肝转移序列的CRC进展。为了研究RBM47在该模型中的作用，我们将在内窥镜下将RBM47基因敲除或异位表达的人结直肠癌类肿瘤注射到NSG-小鼠的结肠壁，然后监测原发肿瘤和转移的形成。为了确定RBM47在结直肠癌转移中作用的下游介质，我们将利用从上述小鼠模型的原发肿瘤和转移模型中分离出来的肿瘤样物质。这些将受到下一代测序的影响，以便全面分析受RBM47调控的RNA编辑和选择性剪接事件。此外，我们计划识别和表征在上皮组织中维持RBM47表达的其他转录和转录后机制。我们的生物信息学分析确定转录因子FOXA1、几个microRNAs以及CpG DNA甲基化是RBM47表达的潜在调节因素。这些潜在的调控将通过多重免疫组织学和甲基化特异性聚合酶链式反应在细胞系和结直肠癌患者材料中得到验证和进一步研究。最后，将在三个不同的结直肠癌患者队列样本中分析RBM47蛋白表达、其下游效应物和RBM47启动子甲基化与远处转移的形成、患者生存和肿瘤进展的关系。本研究的结果将有助于更好地了解结直肠癌的进展，并确定预后标志物和治疗干预点。

We have previously identified the RNA binding motif protein 47 (RBM47) as a putative inhibitor of colorectal cancer (CRC) metastasis: Low RBM47 expression was significantly associated with a mesenchymal-like cellular state, liver metastasis and poor survival. However, elevated RBM47 expression, which was found in normal intestinal epithelia and primary tumors, was associated with an epithelial-like state. siRNA-mediated suppression of RBM47 induced EMT, migration, and invasion. The transcription factors SNAIL and SLUG repressed RBM47 expression and siRNA-mediated down-regulation of RBM47 in xenografted CRC cells promoted metastasis formation. Here we propose to further study the role and functions of RBM47 in a genetic mouse model of metastatic CRC. Notably, our analysis of published RNA-Seq results obtained within this mouse model indicates that RBM47 expression is highest in normal colon, decreased in non-invasive CRCs, and lowest in invasive CRCs and in liver metastases. We plan to determine whether intestinal epithelial cell specific deletion of Rbm47 increases the incidence of distant metastasis in these mice. In addition, we will study an orthotopic transplantation model consisting of patient-derived metastatic CRCs injected into NOD/SCID/gamma (NSG) mice which recapitulates the entire adenoma/adenocarcinoma/liver-metastasis sequence of CRC progression. To study the role of RBM47 in this model, human CRC tumoroids with knockout or ectopic expression of RBM47 will be injected into the colon wall of NSG-mice using endoscopy, followed by monitoring of the formation of primary tumors and metastases. To identify downstream mediators of RBM47 functions in CRC metastasis, we will utilize tumoroids isolated from primary tumors and metastases of aforementioned mouse models. These will be subjected to Next-Generation-Sequencing in order to comprehensively analyze RNA editing and alternative splicing events, which are regulated by RBM47. In addition, we plan to identify and characterize additional transcriptional and post-transcriptional mechanism that maintain RBM47 expression in epithelial tissues. Our bioinformatics analysis identified the transcription factor FOXA1, several microRNAs, as well as CpG DNA methylation as potential regulators of RBM47 expression. These potential regulations will be validated and further studied in cell lines and CRC patient material by using multiplexed immunohistology and methylation-specific PCR. Finally, the association of RBM47 protein expression, its down-stream effectors and methylation of the RBM47 promoter with formation of distant metastases, patient survival and tumor progression will be analyzed in samples from three different CRC patient cohorts. The results obtained here should contribute to a better understanding of CRC progression and identify prognostic markers and therapeutic intervention points.