The Role of RBM47 Inactivation in Colorectal Cancer Progression

我们之前已经发现RNA结合基序蛋白47 （RBM47）是一种推测的结直肠癌（CRC）转移抑制剂：RBM47的低表达与间质样细胞状态、肝转移和生存不良显著相关。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相关。sirna介导的RBM47抑制诱导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的下调促进了移植CRC细胞的转移形成。在此，我们建议进一步研究RBM47在转移性结直肠癌小鼠遗传模型中的作用和功能。值得注意的是，我们对该小鼠模型中已发表的RNA-Seq结果的分析表明，RBM47在正常结肠中表达最高，在非侵袭性crc中表达降低，在侵袭性crc和肝转移中表达最低。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远处转移的发生率。此外，我们将研究一个原位移植模型，该模型由患者来源的转移性CRC注射到NOD/SCID/ γ (NSG)小鼠中，该模型概括了CRC进展的整个腺瘤/腺癌/肝转移序列。为了研究RBM47在该模型中的作用，我们将通过内窥镜将RBM47基因敲除或异位表达的人CRC类肿瘤注射到nsg小鼠的结肠壁上，然后监测原发肿瘤的形成和转移。为了确定RBM47在结直肠癌转移中功能的下游介质，我们将利用从上述小鼠模型的原发肿瘤和转移瘤中分离的类肿瘤。这些将进行下一代测序，以全面分析RBM47调控的RNA编辑和选择性剪接事件。此外，我们计划鉴定和表征维持上皮组织中RBM47表达的其他转录和转录后机制。我们的生物信息学分析发现转录因子FOXA1、几个microrna以及CpG DNA甲基化是RBM47表达的潜在调节因子。这些潜在的调控将通过多重免疫组织学和甲基化特异性PCR在细胞系和CRC患者材料中得到验证和进一步研究。最后，我们将分析来自三个不同CRC患者队列的样本中RBM47蛋白表达、其下游效应物和RBM47启动子甲基化与远处转移形成、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.