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. 2022 Apr 5;20:154. doi: 10.1186/s12967-022-03305-x

Fig. 5.

Fig. 5

Circ_0036412 up-regulates GLI2 expression by competitively binding to miR-579-3p to regulate the proliferation and cell cycle of HCC cells. A StarBase predicted the miRNAs interacting with circ_0036412 and GLI2. The Venn diagram showed the common miRNAs both binding to circ_0036412 and GLI2. B RNA pulldown assay followed by qRT-PCR in HuH-7 and Hep 3B cells detected the enrichments of miR-1245b-5p and miR-579-3p in Bio-circ_0036412 group compared with control group (Student’s t test). C RNA pulldown assay followed by qRT-PCR in HuH-7 and Hep 3B cells evaluated the combination between miR-579-3p and circ_0036412 (One-way ANOVA, Dunnett). D, E Luciferase reporter assays were conducted to testify the interaction between circ_0036412 and GLI2 3′UTR in 293 T cells (Two-way ANOVA, Tukey). F RIP assay followed by qRT-PCR in HuH-7 and Hep 3B cells detected the existence of miR-579-3p and circ_0036412 and GLI2 in RISC (Student’s t test). G The expression of miR-579-3p was detected by qRT-PCR in HuH-7 and Hep 3B cells after circ_0036412 knockdown (One-way ANOVA, Dunnett). H RNA pulldown assay in HuH-7 and Hep 3B cells detected the enrichment of GLI2 in Bio-NC, Bio-miR-579-3p WT, Bio-miR-579-3p MUT and Bio-miR-579-3p WT + si-circ_0036412-1 groups (One-way ANOVA, Tukey). I Luciferase reporter assay assessed the luciferase activity of pmirGLO, pmirGLO-GLI2 3′UTR, pmirGLO-GLI2 3′UTR + pcDNA3.1 and pmirGLO-GLI2 3′UTR + pcDNA3.1-circ_0036412 after the co-transfection with mimics NC or mir-579-3p mimics into 293 T cells (Two-way ANOVA, Tukey). J–K CCK-8 and EdU assays evaluated cell proliferation in HuH-7 cells after the transfection of si-NC, si-circ_0036412-1, si-circ_0036412-1 + inhibitor NC or si-circ_0036412-1 + miR-579-3p inhibitor (One-way ANOVA, Tukey). L Flow cytometry analysis testified cell cycle in HuH-7 cells after the transfection of si-NC, si-circ_0036412-1, si-circ_0036412-1 + inhibitor NC or si-circ_0036412-1 + miR-579-3p inhibitor (One-way ANOVA, Tukey). M, N The mRNA levels of CDC2 and CCNB1 were detected by qRT-PCR in HuH-7 cells after the transfection of si-NC, si-circ_0036412-1, si-circ_0036412-1 + inhibitor NC or si-circ_0036412-1 + miR-579-3p inhibitor (One-way ANOVA, Tukey). O–P The mRNA and protein levels of GLI2 were detected by qRT-PCR (One-way ANOVA, Tukey) and western blot in HuH-7 cells after the transfection of si-NC, si-circ_0036412-1, si-circ_0036412-1 + inhibitor NC or si-circ_0036412-1 + miR-579-3p inhibitor. **P < 0.01