Identification of a two-layer regulatory network of proliferation-related microRNAs in hepatoma cells

To elucidate how microRNA (miRNA)-regulated networks contribute to the uncontrolled growth of hepatoma cells (HCCs), we identified several proliferation-related miRNAs by comparing miRNA expression patterns in clinical HCC samples and growth-arrested HepG2 cells. To explore the molecular functions targeted by these miRNAs, we classified genes differentially expressed in clinical HCC samples into six functional clusters based on their functional similarity. Using target enrichment analysis, we discovered that targets of three proliferation-related miRNAs - miR-101, miR-199a-3p and miR-139-5p - were significantly enriched in the 'transcription regulation' functional cluster. An interactome network consisting of these three miRNAs and genes in the 'transcriptional control' cluster revealed that all three miRNAs were highly connected hubs in the network. All three miRNA-centered subnetworks displayed characteristics of a two-layer regulatory architecture, with transcription factors and epigenetic modulators as the first neighbors and genes involved in cell-cycle progression as second neighbors. The overexpression of miR-101 in HepG2 cells reduced the expression of transcription regulators and genes in cell-cycle progression and suppressed the proliferation and colony formation of HepG2 cells. This study not only provides direct experimental data to support the 'miRNA-centered two-layer regulatory network' model, but our results also suggest that such a combinatorial network model may be widely used by miRNAs to regulate critical biological processes.