The experiments were performed at least 3 times independently. migration of melanoma cells Then, we further evaluated the role of circZNF609 in the migration/invasion of the melanoma cells. Transwell assay showed that the depletion of circZNF609 remarkably attenuated the migration and invasion of A375 and SK-MEL-28 cells (Figure 2A and ?and2B).2B). In addition, wound healing assays revealed that the circZNF609 knockdown significantly enhanced wound proportion in the A375 and SK-MEL-28 cells (Figure 2C and ?and2D),2D), suggesting that circZNF609 enhances invasion and migration of melanoma cells. Open in a separate window Figure 2 CircZNF609 enhances invasion and migration of melanoma Flurazepam dihydrochloride cells. (ACD) The A375 and SK-MEL-28 cells were treated with the circZNF609 shRNA or control shRNA. (A and B) The cell migration and invasion were tested by transwell assays in the cells. (C and D) The migration and invasion were analyzed by wound healing assays in the cells. The wound healing proportion was shown. Data are presented as mean SD. Statistic significant differences were indicated: * 0.05, ** 0.01. CircZNF609 inhibits DNA damage in melanoma cells Next, we determined the impact of circZNF609 on the DNA damage in the A375 and SK-MEL-28 cells. The comet assays showed that the tail length was increased by circZNF609 depletion in the A375 and SK-MEL-28 cells (Figure 3A and ?and3B).3B). Meanwhile, H2AX expression was up-regulated by circZNF609 shRNA in the A375 and SK-MEL-28 cells (Figure 3C and ?and3D),3D), indicating that circZNF609 inhibits DNA damage in melanoma cells. Open in a separate window Figure 3 CircZNF609 inhibits DNA damage in melanoma cells. (ACD) The A375 and SK-MEL-28 cells were treated with the circZNF609 shRNA or control shRNA. (A and B) The DNA damage was analyzed by comet assays in the cells. (C and D) The protein expression of H2AX, H2AX and -actin was determined by Western blot analysis in the cells. The results of Western blot analysis were quantified by ImageJ software. Data are presented as mean SD. Statistic significant differences were indicated: * 0.05, ** 0.01. CircZNF609 represses DNA damage by sponging miR-138-5p in melanoma cells Next, we tried to explore the mechanism of circZNF609-mediated melanoma. We found the interaction between circZNF609 and miR-138-5p in a bioinformatic analysis by using ENCORI (starbase.sysu.edu.cn) (Figure 4A). Then, we treated the A375 and SK-MEL-28 cells with control mimic Flurazepam dihydrochloride or miR-138-5p mimic, and the efficiency was confirmed in the cells (Figure 4B). The miR-138-5p mimic significantly inhibited the luciferase activities of circZNF609but failed to affect the circZNF609 with the miR-138-5p-binding site mutant in the cells (Figure 4C and ?and4D).4D). Meanwhile, the depletion of circZNF609 was able to reduce the expression of miR-138-5p in the A375 and SK-MEL-28 cells (Figure 4E and ?and4F).4F). Moreover, the comet assays revealed that the tail length was enhanced by Flurazepam dihydrochloride circZNF609 depletion in the A375 and SK-MEL-28 cells, while the miR-138-5p inhibitor blocked the phenotype (Figure 4G and ?and4H).4H). Besides, H2AX expression was elevated by circZNF609 shRNA Rabbit Polyclonal to FBLN2 in the A375 and SK-MEL-28 cells, in which the miR-138-5p inhibitor reversed this effect (Figure 4I and ?and4J4J). Open in a separate window Figure 4 CircZNF609 inhibits DNA damage by sponging miR-138-5p in melanoma cells. (A) The interaction of circZNF609 and miR-138-5p was analyzed by bioinformatic analysis based on ENCORI (starbase.sysu.edu.cn). (BCD) The A375 and SK-MEL-28 cells treated with control mimic or miR-138-5p mimics. The expression levels of miR-138-5p were tested by qPCR in the cells. (C and D) Luciferase activities of circZNF609 (circZNF609 WT) and circZNF609 with the miR-138-5p-binding site mutant (circZNF609 MUT) were examined by luciferase reporter gene assays in the cells. (E and F) The A375 and SK-MEL-28 cells were treated with the circZNF609 shRNA or control shRNA. The expression of miR-138-5p was analyzed by qPCR assays in the cells. (GCJ) The A375 and SK-MEL-28 cells were treated with the control shRNA or circZNF609 shRNA, or co-treated with circZNF609 shRNA and miR-138-5p inhibitor. (G and H) The DNA damage was analyzed by comet assays in the cells. (I and J) The protein expression of H2AX, H2AX and -actin was determined by Western blot analysis in.