Research Paper Volume 12, Issue 2 pp 1643—1655

Figure 3. miR-223-3p is a direct target of hsa_circ_0070963. (A) The luciferase activity of hsa_circ_0070963 after transfection with pGL3-Basic-hsa_circ_0070963 combined with each miRNA mimic in HEK-293T cells. (B) The mRNA level of miR-223-3p was analyzed in hsa_circ_0070963-overexpressing LX2 cells. (C) Hsa_circ_0070963 in LX2 cell lysates was pulled down and enriched with an hsa_circ_0070963 specific probe and then detected by qRT-PCR. (D) MiR-223-3p in LX2 cell lysates was pulled down and enriched with an hsa_circ_0070963 specific probe and then detected by qRT-PCR. (E) Pull-down assay to validate the direct interaction between hsa_circ_0070963 and miR-223-3p. Biotin-coupled NC is not complementary to hsa_circ_0070963. (F) The product of (D) was detected using qRT-PCR, followed by agarose gel electrophoresis. (G) Predicted binding sites of miR-223-3p and hsa_circ_0070963 and mutated hsa_circ_0070963. (H) Relative luciferase activity was determined at 48 h after transfection with miR-223-3p NC/mimic/inhibitor or hsa_circ_0070963 wild-type (WT)/Mutant (Mu) in HEK293T cells. Data are presented as means ± SD of three experiments (^*p < 0.05, ^**p < 0.01, and ^***p < 0.001).