Figure 3.

ATXN1 as a target gene of miR-125b-5p in HCC cells. (a) Target Scan predicted two putative binding sites of miR-125b-5p (position 2439-2445 and 4097-4104, shown in blue) in the ATXN1 3′-UTR region. The mutated sequences (shown in red) of putative binding sites 1 and 2 are shown as ATXN1 3′-UTR Mutant 1 or Mutant 2, respectively. The indicated sequences (Wild type and Mutant 1 and 2) of ATXN1 3′-UTR were cloned to psiCHECK2 vector. (b) psiCHECK2 vectors containing the wild type or mutant sequences, or the empty vector were co-transfected with miR-125b-5p. Renilla luciferase activity was normalized to firefly luciferase. Fold-change values were normalized to empty vector and mimic control-treated cells. Data are expressed as the mean ± standard deviation (SD; n = 4). * Statistically significant difference versus control miRNA treatment (unpaired Student’s t-test, p < 0.05). (c) HEK293T cells were co-transfected with microRNA mimics and luciferase reporter plasmids driven by the promoter fragments of ATXN1. Luciferase activities in these cells were measured using the Dual-Luciferase Reporter Assay System. Data are expressed as the mean ± standard deviation (SD; n = 4). No significant difference between control and miR-125b-5p mimics treated cells (unpaired Student’s t-test). (d) The relative expression level of ATXN1 mRNA in PLC/PRF5-miR125b or PLC/PRF5-miNC was determined by RT-PCR. (e) The expression of ATXN1 protein in PLC/PRF5-miNC and PLC/PRF5-miR125b cells was examined by western blot analysis. Densitometry analysis indicates relative protein levels from one representative of three independent experiments. Numbers below the bands represent protein expression normalized to β-actin. (f) The relative mRNA levels of Snail in PLC/PRF5 cells transfected with siRNA-ATXN1(PLC/PRF5-siATXN1) or control siRNA (PLC/PRF5-siNC) were determined by RT-PCR. (g) Schematic diagram of miR-125-5p-ATXN1-axis and subsequent induction of EMT. ** p < 0.01.