Dynamic alteration of protein expression profiles during neoplastic transformation of rat hepatic oval-like cells.

Liver Cancer Institute, Affiliated Zhongshan Hospital of Fudan University, Shanghai, China.
Cancer Science (Impact Factor: 3.48). 03/2010; 101(5):1099-107. DOI: 10.1111/j.1349-7006.2010.01513.x
Source: PubMed

ABSTRACT To explore the molecular basis of neoplastic transformation of hepatic oval cells, a proteomic strategy was utilized to examine the global protein expression alterations during neoplastic transformation of rat hepatic oval-like cells. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-initiated WB-F344 cells were treated with H(2)O(2) for neoplastic transformation. The transformed cells were identified by soft agar assay and MTT assay. The subsequent proteomic separation and identification were performed with 2-DE followed by MALDI-TOF-MS/MS analysis. Of the 148 differentially displayed protein spots analyzed, 121 spots representing 79 distinct proteins were finally identified. The expression levels of interested proteins were validated by western blotting including 40 S ribosomal protein A (RPSA) and cytokeratin 8. Bioinformatics annotations indicated that these identified proteins were enriched with oxidoreduction and stress response; transcription, translation, and protein processing; and energy/metabolism functions. Interestingly, 17 of the identified proteins were also found to be involved in early hepatic differentiation of mouse embryonic stem (ES) cells in our previous study. Twenty-six proteins had been reported as being dysregulated in hepatocellular carcinoma and other cancers. It suggested that these changed proteins may be implicated in neoplastic transformation of WB-F344 cells. The results may provide some clues for understanding the molecular mechanisms of hepatocarcinogenesis as viewed from dysregulation of differentiation.

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    ABSTRACT: Hepatocellular carcinoma (HCC) can be derived from malignant transformed adult hepatic progenitor cells. However, the regulatory factors and molecular mechanisms underlying the process are not well defined. Our previous microRNA (miRNA) microarray analysis revealed a significant decrease of miR-200a level in F344 rat HCC side population (SP) fraction cells versus their normal counterparts. In the present study, we further investigated the effect of miR-200a on hepatic oval cell (HOC) phenotypes. We first confirmed downregulated miR-200a levels in rat hepatoma cells compared with WB-F344 cells. Next, by lentivirus-mediated loss-of-function studies, we showed that stable knockdown of miR-200a confers a mesenchymal phenotype to WB-F344 cells, including an elongated cell morphology, enhanced cell migration ability and expression of epithelial mesenchymal transition (EMT)-representative markers. Concomitantly, several cancer stem cell (CSC)-like traits appeared in these cells, which exhibit enhanced spheroid-forming capacity, express putative hepatic CSC markers and display superior resistance to chemotherapeutic drugs in vitro. Furthermore, bioinformatics analysis, luciferase assays and western blot analysis identified β-catenin (CTNNB1) as a direct and functional target of miR-200a. Knockdown of miR-200a partially activated Wnt/β-catenin signaling, and silencing of β-catenin functionally attenuated anti-miR-200a effects in vitro in WB-F344 cells. At length, in vivo xenograft assay demonstrated the acquisition of tumorigenicity of WB-F344 cells after miR-200a siliencing. Collectively, our findings indicate that miR-200a may function as an important regulatory factor in neoplastic transition of HOCs by targeting the β-catenin pathway.
    PLoS ONE 01/2013; 8(11):e79409. · 3.53 Impact Factor