The prediction and confirmation of the <i>miR-101</i> target.

<p>(A) <i>MiR-101</i> binding sites in the 3′-UTR region of <i>Stmn1</i> in cross-species. (B) Schematic diagram for constructing the <i>miR-101</i> binding site into pGL3 control vector. (C) Confirmation of the target gene of <i>miR-101</i>. T47D cells were co-transfected with pre-miR control, <i>miR-101</i> mimics, anti-miR control or <i>miR-101</i> inhibitor and Stmn1-pGL3 for dual-luciferase assay. PRL-TK containing Renilla luciferase was co-transfected with 3′-UTR of <i>Stmn1</i> for data normalization. (D) Mutation analysis of the <i>miR-101</i> binding site. When the binding site of <i>miR-101</i> in the 3′-UTR of <i>Stmn1</i> was completely deleted (Stmn1-pGL3-Mutant), the luciferase activity was significantly decreased in T47D cells co-transfected with <i>miR-101</i> mimics and Stmn1-pGL3 compared with Stmn1-pGL3-Mutant or pGL3. (E) STMN1 protein level in <i>miR-101</i> mimics or inhibitor-treated T47D cells was detected by western blot. The bands were analyzed using the Quantity One analyzing system (Bio-Rad Laboratory inc.). The expression of β-ACTIN served as an internal control. The black histogram represents the optical densities of the signals quantified by densitometric analysis and represented as STMN1 intensity/β-ACTIN intensity to normalize for gel loading and transfer. (F) The expression of <i>Stmn1</i> mRNA in <i>miR-101</i> mimics and inhibitor-treated T47D cells were detected by qRT-PCR. β-actin serves as an internal reference among different samples and helps normalize for experimental error. The <i>y</i>-axis displays the relative log2 ratio of <i>Stmn1</i> normalized by β-actin. *<i>P</i><0.05. **<i>P</i><0.01.