Abstract
Abstract Introduction: The use of emerging sequencing and genomics technologies has recently identified many mutated and/or differentially expressed genes in hepatocellular carcinoma (HCC). However, it is still difficult to identify true driver genes for HCC because of the large number of passenger mutations and other gene-regulatory mechanism such as epigenetic regulation, SNP and non-coding RNA. To overcome this difficulty, we previously performed a transposon mutagenesis screen in a mouse HBV model of HCC and identified 2881 candidate driver genes (Bard-Chapeau EA et al., Nat Genet 2014). In the present study, we focused on one of the candidate cancer genes, RNF125, and validated its novel tumor suppressive role in the mouse and human liver. Methods: To examine a role of RNF125 gene in liver tumor development, mouse non-tumorigenic immortalized liver progenitor cells (LPCs) and two human liver cancer cell lines (SNU-398 and HepG2) were subcutaneously injected into athymic nude mice after transduction of lentiviral shRNA targeting murine Rnf125 or human RNF125 gene. To study the effect of RNF125 gene silencing in cellular proliferation, WST-1 assay was performed after transfection of RNF125 siRNA into SNU-398 and HepG2 cell lines. To study the therapeutic potential of RNF125 gene in HCC, we constructed two stable human liver cancer cell lines (Hep3B and PLC/PRF/5) expressing human RNF125 and investigated the effect of its ectopic expression on cell proliferation and in vivo tumor formation. To evaluate the clinical relevance of RNF125 gene dysregulation, publically available microarray gene expression data of HCC was analyzed (GEO:GSE14520). Results: Transposon Insertion pattern of mutagenesis screen suggested frequent inactivation of Rnf125 gene in transposon-mediated mouse liver tumors, predicting that RNF125 gene may function as a tumor suppressor. Non-tumorigenic mouse LPCs started to form tumors in vivo upon knockdown of Rnf125 gene. Inhibition of RNF125 gene also significantly accelerated in vivo tumor formation of both SNU-398 and HepG2 cell lines. Knockdown of RNF125 gene significantly increased cell proliferation in these cell lines in vitro. Overexpression of RNF125 gene in Hep3B and PLC/ORF/5 cell lines significantly decreased cell proliferation in vitro and suppressed in vivo tumor formation. In microarray data of human HCC, RNF125 gene expression was significantly downregulated in tumor tissues compared to adjacent non-tumor tissues. Conclusion: RNF125 gene is a novel tumor suppressor gene for human HCC and plays an important role in cellular proliferation. RNF125 gene is frequently inactivated in human HCC and therefore its functional recovery could be a potential therapeutic target of HCC. Citation Format: Takahiro Kodama, Zhubo Wei, Michiko Kodama, Nancy Jenkins, Neal Copeland. Sleeping Beauty mutagenesis screening identifies a novel tumor suppressor gene involved in liver tumor development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2045. doi:10.1158/1538-7445.AM2015-2045
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