Figure 4.

NCK associated protein 1 (NCKAP1) is the target gene of miR‐214 in vascular smooth muscle cells (VSMCs). A through C, NCKAP1 protein level was significantly downregulated by miR‐214 overexpression in VSMCs, as shown in the volcano plot (B), and confirmed by Western blot analyses (C). Reverse transcriptase–quantitative PCR (RT‐qPCR) confirmed miR‐214 overexpression in VSMCs (A). B, Volcano plot showing P values (−log₁₀) vs protein ratio of miR‐214 mimics/control mimics (log₂) of all 1594 proteins fulfilling strict quantitation criteria (red, 14 upregulated proteins with 2‐fold changes; blue, 7 downregulated proteins with 2‐fold changes; purple, significantly changed but such change is <2‐fold on miR‐214 overexpression; ANOVA with P<0.05). D and E, Modulations of miR‐214 expression levels in VSMCs negatively regulates NCKAP1 gene expressions. VSMCs were transfected with miR‐214 mimics (miR‐214) or inhibitor or with respective negative controls. Total RNAs were harvested and subjected to RT‐qPCR analyses with indicated primers. F, All 3 binding sites are important for miR‐214–mediated NCKAP1 gene repression. miR‐214 mimics or negative control were cotransfected into VSMCs with wild‐type NCKAP1 3′ untranslated region (3′UTR) reporter or the indicated single/combined binding site mutants (binding sites 1 [BS1^mut], 2 [BS2^mut], and 3 [BS3^mut] or the combinational mutations [BS1/2/3^mut]). Luciferase activity was measured at 48 hours after transfection. The data presented are representative (C) or show mean+SEM of 3 to 4 independent experiments (C through F). *<0.05, ***<0.001 Compared with miRNA control.