Figure 4

circ_8521 promotes the expression of LC3A by binding to miR-324. ASchematic diagram of the complementary sequences between circ_8521 and miR-324 and between miR-324 and LC3A mRNA. B PK-15 cells were transfected with biotin-coupled miR-324 mimic. Fold enrichment of circ_8521 was quantified by qRT-PCR after miRNA pull-down (left). qRT-PCR analysis of miR-324 levels was performed to determine transfection efficiency (right). C Schematic diagram showing the binding of miR-324 to wild-type LC3A (LC3A-WT) and mutant LC3A (LC3A-MUT) luciferase reporter vector (upper part). Detection of luciferase activity following the co-transfection of the luciferase reporter vector and miR-324 or control mimic into PK-15 cells for 48 h (lower part). Firefly luciferase activity was normalized to that of Renilla luciferase. PK-15 cells were transfected with circ_8521 siRNAs and NC siRNA served as control (D, E). PK-15 cells were transfected with pEZX-circ_8521 (F, G). PK-15 cells were transfected with the miR-324 mimic and NC mimic served as control (H, I). PK-15 cells were transfected with miR-324 inhibitor and NC inhibitor served as control (J, K). At 24 h post-transfection, cells were infected with SVA for 3, 10.5, or 18 h. D miR-324 (left) and LC3A mRNA (right) levels were detected by qRT-PCR. E LC3A protein was examined by Western blotting. F miR-324 (left) and LC3A mRNA (right) levels were detected by qRT-PCR. G LC3A protein was examined by Western blotting. H miR-324 (left) and LC3A mRNA (right) levels were detected by qRT-PCR. I LC3A protein was examined by Western blotting. J miR-324 (left) and LC3A mRNA (right) levels were detected by qRT-PCR. K LC3A protein was examined by Western blotting. PK-15 cells were co-transfected with miR-324 inhibitor and circ_8521 siRNAs. At 24 h post-transfection, cells were infected with SVA for 12 h. L LC3A mRNA levels were detected by qRT-PCR. M LC3A protein was examined by Western blotting. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.