Pygo2 is a target gene of miR‐516a‐3p. A, miR‐516a‐3p and its putative binding sequence in the 3′‐UTR of Pygo2 mRNA. Mutant miR‐516a‐3p binding sites were generated in the complementary site for the seed region of miR‐516a‐3p (WT, wild type; Mut, mutant type). B, miR‐516a‐3p effects on luciferase activity in cells that carried the wild type and mutant type 3’‐UTR of Pygo2 mRNA. (C‐D) miR‐516a‐3p expression was changed after transfection of MCF‐7 and MDA‐MB‐231 cells with miR‐516a‐3p mimic or inhibitor. E, Western blot analysis of the expression of Pygo2 protein in MCF‐7 and MDA‐MB‐231 breast cancer cell lines transfected with miR‐516a‐3p NC or miR‐516a‐3p mimic. F, Western blot analysis of the expression of Pygo2 protein in MCF‐7 and MDA‐MB‐231 breast cancer cell lines transfected with miR‐516a‐3p NC or miR‐516a‐3p inhibitor. G, Immunohistochemistry of Pygo2 was shown and compared between tissues with high miR‐516a‐3p level and those with low miR‐516a‐3p level (×400), bar = 50 μm. Data are shown as mean ± SD (**, P < 0.01; ***, P < 0.001)