Figure 2: Identification of candidate miRNAs that control α2δ1⁺ HCC TICs.

(a) Schematic overview of the strategy for identification of α2δ1⁺ HCC TIC miRNAs based on the Hep-11 and Hep-12 cell lines. (b) Heat map of miRNA expression showing the miRNAs deregulated in the TIC-enriched Hep-12 cells versus the non-tumorigenic Hep-11 cells (P<0.01). Clustering was performed with average linkage and uncentred correlation using the Partek Genomics Suite (Partek SG Pte Ltd, Singapore). (c) The effect of indicated miRNAs on the colony formation efficiency of Hep-12 cells in soft agar. Pools of Hep-12 cells stably transfected with each indicated pri-mRNA were plated into 0.3% agar at 1,000 cells per well (n=6). Error bars represent s.d. **Student’s t-test. (d) qRT–PCR results demonstrate that the indicated miRNAs are induced significantly at 48 h after 2 μg ml⁻¹ DOX was added into the culture medium of Hep-12 cells, which were stably infected with tet-on pri-miRNA expression lentiviruses. Error bars represent s.d. (e). Tumour formation by Hep-12 cells stably infected with inducible pri-miRNA expression lentiviruses were assayed after transplanting 10⁴ cells in Matrigel subcutaneously. To induce the miRNA expression, mice in the DOX(+) group were fed with water containing 2 mg ml⁻¹ DOX. The numbers above each bar indicate the tumour-formation rates that are expressed as tumours formed/sites transplanted. (f) The spheroid formation efficiency of Hep-12 cells after the expression of indicated miRNAs was induced with 2 μg ml⁻¹ DOX (n=6). Data are means±s.d. of three independent experiments. *Student’s t-test.