Figure 4: PBX3 is a common target of let-7c, miR-200b, miR-222 and miR-424.

(a) Venn diagram shows candidate mRNAs that were predicted to be directly targeted by all the four miRNAs by in silico analysis of upregulated genes in the α2δ1⁺ HCC TICs with predicted target genes of each of the four miRNAs using the miRWalk algorithm. (b,c) qRT–PCR and western blot assays were carried out to detect the expression of endogenous PBX3 at both mRNA (b) and protein (c) levels, respectively, in Hep-12 cells infected with lentiviruses harboring indicated miRNAs. (d,e) The expression of endogenous PBX3 at both mRNA (d) and protein (e) levels in FACS-sorted α2δ1⁻ Huh7 cells after indicated miRNAs were knocked down by TuD RNAs. (f) The effects of PBX3 on tumour formation of Hep-12 cells expressing indicated miRNAs. The numbers above each bar indicate the tumour-formation rates that are expressed as tumours formed/sites transplanted. The bars are means±s.d. of tumour weights. *Student’s t-test. (g) Sequence alignment of the seed sequence of indicated miRNAs with portions of the PBX3 3′-UTR (UTR WT) as predicted using the TargetScan software. The sequences designed as ‘Mut’ are mutated sequences in the matched seed sites that were used for creating firefly luciferase reporter constructs. (h) Dual-luciferase reporter assay in Hep-12 cells transiently co-transfected with the indicated constructs. Data are the average±s.d. of three independent experiments. *Student’s t-test. (i–l) The inverse correlation of indicated miRNAs against PBX3 mRNA expression was determined across purified α2δ1⁺ and α2δ1⁻ subsets from indicated sources. Data represented fold difference of α2δ1⁺ cells over their negative counterparts in the same categories. Error bars indicate s.d. of three independent experiments. (m–p) The relationship between the expression of indicated miRNAs and PBX3 mRNA in clinical HCC tissues.