Department of Pathology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan City, 704, Taiwan.
Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
BMC Cancer. 2023 Oct 24;23(1):1025. doi: 10.1186/s12885-023-10655-2.
The Wnt/β-catenin signaling pathway plays an important role in embryogenesis and tumorigenesis. In human cancer, abnormal activity of Wnt/β-catenin signaling pathway induces overexpressed of downstream genes, and initiate oncogene. There are several target genes known to be key players in tumorigenesis, such as c-myc, cyclin D1, MMPs or survivin. Therefore, identifying the target genes of Wnt/β-catenin signaling pathway is important to understanding Wnt/β-catenin-mediated carcinogenesis. In this study, we developed a combined bioinformatics and experimental approach to find potential target genes.
Luciferase reporter assay was used to analyze the promoter activity of RMI2. WST1 cell proliferation assays and transwell assays were performed to determine the proliferation and migration capacities of RMI2 overexpressing or knockdown stable hepatic cells. Finally, xenograft experiments were performed to measure the tumor formation capacity in vivo.
The results showed that RMI2 mRNA was upregulated after LiCl treatment and Wnt3a-conditioned medium in a culture of SK-hep-1 cell lines. A chromatin immunoprecipitation (ChIP) assay showed that the β-catenin/T cell-specific factor (TCF) complex binds to the putative TCF binding site of the RMI2 promoter. We then found a TCF binding site at - 333/- 326 of the RMI2 promoter, which is crucial for β-catenin responsiveness in liver cell lines. RMI2 was overexpressed in hepatoma tissue and cell lines, and it promoted the migration and invasion of HCC cells. Moreover, RMI2 upregulated the expression of epithelial-mesenchymal transition (EMT) markers and the Wnt3a/β-catenin-related genes, but silencing RMI2 had the opposite effects. Notably, the expression of RMI2 was positively correlated with the clinical data of HCC patients who had significantly shorter overall survival (OS) and disease-free survival (DFS) (Both: P < 0.05). In addition, a total of 373 HCC patients' data from the Caner Genome Atlas project (TCGA) were used to validate our findings.
Taking all these findings together, we determined that RMI2 was a new target gene of the Wnt/β-catenin signaling pathway. We also found that RMI2 promotes EMT markers, HCC cell invasion, and metastasis, which indicated that RMI2 is a potential target for preventing or at least mitigating the progression of HCC.
Wnt/β-catenin 信号通路在胚胎发生和肿瘤发生中起着重要作用。在人类癌症中，Wnt/β-catenin 信号通路的异常活性导致下游基因过度表达，并引发癌基因。有几个已知的靶基因是肿瘤发生的关键参与者，如 c-myc、cyclin D1、MMPs 或 survivin。因此，鉴定 Wnt/β-catenin 信号通路的靶基因对于理解 Wnt/β-catenin 介导的致癌作用非常重要。在这项研究中，我们采用了一种组合的生物信息学和实验方法来寻找潜在的靶基因。
使用荧光素酶报告基因检测分析 RMI2 的启动子活性。WST1 细胞增殖试验和 Transwell 试验用于测定 RMI2 过表达或敲低稳定的肝细胞的增殖和迁移能力。最后，进行异种移植实验以测量体内肿瘤形成能力。
结果表明，LiCl 处理和 Wnt3a 条件培养基培养的 SK-hep-1 细胞系中 RMI2 mRNA 上调。染色质免疫沉淀（ChIP）试验表明，β-catenin/T 细胞特异性因子（TCF）复合物结合到 RMI2 启动子的推定 TCF 结合位点。我们随后在 RMI2 启动子的-333/-326 处发现了一个 TCF 结合位点，这对于肝细胞系中的 β-catenin 反应性至关重要。RMI2 在肝癌组织和细胞系中高表达，并促进 HCC 细胞的迁移和侵袭。此外，RMI2 上调上皮-间充质转化（EMT）标志物和 Wnt3a/β-catenin 相关基因的表达，而沉默 RMI2 则产生相反的效果。值得注意的是，RMI2 的表达与 HCC 患者的临床数据呈正相关，这些患者的总生存期（OS）和无病生存期（DFS）明显缩短（均 P<0.05）。此外，我们还使用癌症基因组图谱项目（TCGA）中 373 名 HCC 患者的数据来验证我们的发现。
综上所述，我们确定 RMI2 是 Wnt/β-catenin 信号通路的一个新靶基因。我们还发现 RMI2 促进 EMT 标志物、HCC 细胞侵袭和转移，这表明 RMI2 是预防或至少减轻 HCC 进展的潜在靶点。
