Figure 4.

MiR‐24‐3p targets the 3′‐UTR of MEN1 mRNA. The bovine miR‐24‐3p (bta‐miR‐24‐3p) were predicted to interact with the 3′‐UTR region of MEN1 mRNA at position 559–565 using TargetScan 6.2 and RNAhybrid 2.2 software (a). MiR‐24‐3p, broadly conserved in different species, possibly also interacts with 3′‐UTR regions of MEN1 in human (has‐), mouse (mmu‐), rat (rno‐) and pig (ssc‐), with exact the same seed nucleotide sequence (underlined, b). The 3′‐UTR region (789 bp) of bovine MEN1 gene were construed into luciferase‐expressing plasmid (3′‐UTR). The plasmid was cotransfected with miR‐24‐3p mimics (mimics) and/or miR‐24‐3p inhibitor (inhibitor) into mammary epithelial cells, as well as their corresponding negative controls (mimics NC and/or inhibitor NC). The beta‐galactosidase reporter plasmid (β‐gal) was simultaneously transfected for each every transfection, serving as internal control. Significant suppressed luciferase activity was shown in the cotransfection group of 3′‐UTR plasmid and miR‐24‐3p mimics, indicating bta‐miR‐24‐3p targeted the 3′‐UTR of bovine MEN1. All of the experiments were performed three times for each transfection. Each experiment was performed in triplicates. Different letters (a,b) indicate significant difference (*p < 0.05). 3′‐UTR: 3′ untranslated region; MEN1: multiple endocrine tumor type 1; mRNA: messenger RNA; NC: negative control