A combined bioinformatics and experimental approach identifies RMI2 as a Wnt/β-catenin signaling target gene related to hepatocellular carcinoma

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Abstract
Background: 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. Methods: 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. Results: 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. Conclusions: 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.

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