Fig. 6: miR-4756 directly binds to circUBAP2 and the 3′-UTR of IFIT1/IFIT3.

A ENCORI was used to predict miRNAs that circUBAP2 might target, and TargetScan was used to predict miRNAs that might target IFIT1/IFIT3. These two sets of miRNAs intersected at hsa-miR-4756-5p, hsa-miR-1321, hsa-miR-552-3p, hsa-miR-3611, and hsa-miR-24-3p. The expression of miR-4756-5p, miR-1321, miR-552-3p, miR-3611, and miR-24-3p was examined in MHCC-97H and Huh-7 cells with or without CXCL11 treatment. miR-4756 was chosen for further experiments due to its downregulation in both HCC cell lines in response to CXCL11 stimulation. B MHCC-97H and Huh-7 were transfected with si-circUBAP2 and examined for the expression of miR-4756 by real-time PCR. C The expression of miR-4756 was examined in 12 cases of nonmetastatic HCC tissues, metastatic HCC tissues, and para-carcinoma tissues by real-time PCR. D miR-4756 overexpression or inhibition was achieved in MHCC-97H and Huh-7 cells by transfecting miR-4756 mimics or miR-4756 inhibitor; the transfection efficiency was confirmed by real-time PCR. E MHCC-97H and Huh-7 cells were transfected with miR-4756 mimics or miR-4756 inhibitor and examined for the protein levels of IFIT1 and IFIT3 by Immunoblotting. F–H Wild- and mutant-type circUBAP2, IFIT1 3′-UTR, or IFIT3 3′-UTR luciferase reporter plasmids were constructed as described and named as wt-circUBAP2/mut-circUBAP2, wt-IFIT1/mut-IFIT1, and wt-IFIT3/mut-IFIT3. These plasmids were co-transfected in 293T cells with miR-4756 mimics or miR-4756 inhibitor and the luciferase activity was determined. *P < 0.05, **P < 0.01, compared to si-NC + inhibitor NC group. ^##P < 0.01, compared to si-cirUBAP2+miR-4756 inhibitor group.