Figure 3: The effects of let-7c, miR-200b, miR-222 and miR-424 on the self-renewal and tumorigenicity of HCC TICs.

(a) The expression of indicated miRNAs in α2δ1⁺ and α2δ⁻ cells purified from indicated sources were detected using qRT–PCR. The data presented as fold difference of α2δ1⁺ cells over α2δ⁻ counterparts that was defined as 1 (omitted in the histogram). Error bars indicate s.d. (b,c) FACS-sorted α2δ1⁺ fractions were incubated with indicated lentiviruses for 2 h and were plated in 96-well plates at 100 cells per well (n=6) to assay their spheroid formation efficiency (b), or were transplanted into NOD/SCID mice (n=5) at 1,000 cells per site to test their tumorigenicity (c). Scale bar, 2 cm. Data in (b) are means±s.d. of three independent experiments. *Student’s t-test, P<0.01 versus respective controls. (d) The fold difference (2^−ΔΔCt) of indicated miRNAs in Huh7 α2δ1⁻ cells following TuD RNA interference of the indicated miRNAs over the control (infected with empty virus), which was set up as 1. Error bars indicate s.d. of three experiments. (e,f) FACS-sorted α2δ1⁻cells were infected with lentiviruses harbouring TuD RNA expression constructs against indicated miRNAs and were subsequently plated into 96-well plates to assay their primary spheroid formation efficiency (1°) and subsequent expansion ability (2°, e; n=6), or serially transplanted s.c. into NOD/SCID mice at 1,000 cells per site (f; n=5). Scale bar, 2 cm. TFR, tumour-formation rate that is expressed as tumours formed/sites transplanted. Data in (e) are means±s.d. of three independent experiments. *Student’s t-test, P<0.01 versus respective controls. (g) HE staining demonstrating the histology of the tumours formed in f. Scale bar, 150 μm. (h) The frequencies of α2δ1⁺ cells were measured by flow cytometry in Huh7 cells after indicated miRNAs were knocked down by infection with Tud RNA lentiviruses. The data are representative of three experiments. (i,j) Western blot analysis of α2δ1 expression in Huh7 cells after indicated miRNAs were knocked down (i) and in Hep-12 cells after induced expression of indicated miRNAs (j).