The tumor suppressor, miR-198, affects central pathways in tumorigenesis and in the immune response of hepatocellular carcinoma cells

Aims: miRNA are small, non-coding RNA molecules that inhibit gene expression by targeting various transcripts. In previous studies we have shown that miR-198 is significantly down-regulated in HCV positive hepatocellular carcinomas (HCCs), affecting markedly the cell growth of hepatoma cells. Methods: In order to characterize the function of miR-198 we performed three different approaches. First, we screened databases, which predict putative target transcripts of miRNAs by algorithm calculations. In the second approach, we overexpressed miR-198 in hepatoma cells. Cell lysates were used for immunoprecipitation of the Argonaute 2 (Ago2) protein, which is responsible for the recognition and binding of the miRNA to the target mRNA. Afterwards, we analysed the transcripts bound to the immunoprecipitated Ago2 miRNA complex by cloning and subsequent sequencing of 200 clones. In the third approach miR-198 treated hepatoma cells, were used for gene expression profiling by microarrays. Finally, we used different Spotfire based software in order to interpret the results by bioinformatics. Results: Screening of different databases identified a panel of putative transcripts of miR-198, predominantly involved in regulation of cell growth and differentiation. Sequencing of clones established from miR-198 treated hepatoma cells and immunoprecipitation of the miRNA transcript complex identified MAP kinases and transcription factors that are participated in tumorigenesis. Additionally, a lot of signal transduction processes such as the TGF-β and the Wnt pathway were shown by expression profiling to be influenced by miR-198. In particular, bioinformatic analyses revealed that the expression of the JAK/Stat1 pathway members is significantly altered by miR-198 overexpression. Conclusion: Comprehensive analyses of miR-198 function revealed that miR-198 is not only involved in wide spectrum tumorigenic pathways, but also in interferon-induced signalling of the immune defence.