Figure 4. CircRNA Cdr1as regulated AFP, the target gene of miR-1270. (A) Putative binding site of miRNA in the AFP sequence. The putative miRNAs recognition sites were cloned downstream of the luciferase gene and named pGL3-AFP-WT. (B) The luciferase activity in HepG2 and SMMC-7721 cells after co-transfection of plasmid (pGL3-AFP-WT or pGL3-AFP-MUT) and miRNA-1270 mimics was detected by dual-luciferase reporter gene assay. (C) Relative expression of AFP in HCC cell lines compared to control human normal liver cell line HL-7702. (D) Protein levels of AFP in human normal liver cell line HL-7702, HepG2 and SMMC-7721 cell lines. (E) qRT-PCR detection of the relative expression of AFP in paired HCC tumor and paired para-carcinoma tissues (n=42). (F) Correlation between miR-1270 and AFP in HCC samples. (G) Correlation between circRNA Cdr1as and AFP in HCC samples. (H) After HepG2 cells were treated with circRNA Cdr1as shRNA (with inhibitor NC or with miR-1270 inhibitors), Western blotting analysis were adopted to measure the AFP protein level, with GAPDH as a control. (I) SMMC-7721 cells were transfected with circRNA Cdr1as overexpression lentiviral vector (with mimics-NC or with miR-1270 mimics), and Western blotting was adopted to detect the AFP protein expression level compared with the control. MiR-1270 inh/mimics means transfection with miR-1270 inhibitor/mimics, sh-circRNA Cdr1as/circRNA Cdr1as means transfection with circRNA Cdr1as shRNA/circRNA Cdr1as overexpressing vector. Results were presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001. All of the experiments were performed in triplicate.