Research Paper Volume 11, Issue 24 pp 12043—12056

Figure 3. circAHNAK1 can act as a sponge for miR-421. (A) The expression levels of nuclear control (18S), cytoplasmic control (β-actin) and circAHNAK1 were detected; (B) Prediction of the binding site of miR-421 in the circAHNAK1 sequence; (C) The expression of miR-421 in TNBC cell line; (D) Luciferase assay of cells co-transfected with the miR-421 mimics and the circ-AHNAK1 wild type or mutant luciferase reporter; (E) GFP-MS2-RIP assay detects enrichment of miR-421after MS2bs-circAHNAK1-WT, MS2bs-circAHNAK1-Mut or control transfection, respectively; (F) Effect of miR-421 mimics transfection on proliferation of circAHNAK1 overexpressing cells by CCK-8; (G) Effect of miR-421 mimics transfection on circAHNAK1 overexpressing cells by clone formation assay; (H) Quantitative clone formation by ImageJ software; (I) Transwell invasion assay assessed the impact on cell invasion;(J) ImageJ software quantifies the number of invading cells;(K)Wound healing assay for detecting changes in cell migration ability; (L) ImageJ software quantifies the extent of wound closure; (M) Xenograft model to evaluate tumor proliferation in vivo; (N) Comparison of tumor weight; (O)Representative images of luciferase signaling of lung metastasis in vivo; (P) Representative images of HE staining of lung metastatic nodule sections; (Q) Quantification of the number of lung metastatic nodules.