Figure 4. Circ-PAN3 sponges miR-153 to regulate cyclin D1 expression in HCC cells. (A) RT-qPCR analysis of circ-PAN3 transcripts in the cytoplasmic or nuclear fractions of SK-HEP-1 (left) and Huh7 (right) cells. (B) Venn diagram showing the predicted miRNA targets of circ-PAN3. (C) The potential binding region and the mutated sequences between circ-PAN3 and miR-153. (D) miR-153 was validated as a target of circ-PAN3 using dual-luciferase reporter assays in SK-HEP-1 (left) and Huh7 (right) cells. (E) Venn diagram showing the predicted mRNA targets of miR-153. (F) The potential binding region and and the mutated sequences between miR-153 and CCND1. (G) Cyclin D1 was validated as a direct target of miR-153 using dual-luciferase reporter assays in SK-HEP-1 (left) and Huh7 (right) cells. (H) RNA pull-down analysis of miR-153 interaction using biotinylated-circ-PAN3 probe and control probe in SK-HEP-1 and Huh7 cells (left). RNA pull-down analysis of miR-153 interaction with the biotinylated-Cyclin D1 probe or control probe in SK-HEP-1 and Huh7 cells (right). (I) The expression levels of miR-153 in SK-HEP-1 and Huh7 cells transfected with circ-PAN3 siRNAs were detected by RT-qPCR. (J) The expression levels of cyclin D1 in SK-HEP-1 and Huh7 cells transfected with circ-PAN3 siRNAs were detected by RT-qPCR (left) and Western blotting (right). (K) The expression levels of miR-153 in SK-HEP-1 and Huh7 cells transfected with miR-153 mimics were detected by RT-qPCR. (L) The expression levels of cyclin D1 in cells transfected with miR-153 mimic were detected by RT-qPCR (left) and Western blotting (right). (M) The expression levels of circ-PAN3 in cells transfected with miR-153 mimic in SK-HEP-1 and Huh7 cells were detected by RT-qPCR. Data are presented as mean ± SEM; n ≥ 3. **p < 0.01; ***p < 0.001; ^##p < 0.01.