miR-9 regulated the expression of KLF5, and the miR-9 mimic attenuated the expression of KLF5 in VSMCs. A and B. Validation of miR-9 targets in VSMCs. VSMCs were transfected with miR-9 inhibitor and control oligo by nucleofection. 48 h after transfection, the expression of KLF5 was analyzed with qRT-PCR. HG: high glucose, LG: low glucose. The results showed that the expression of KLF5 was positively regulated by the miR-9 inhibitor in both C-12511 cells and VSMCs from db/db mice. C and D. The representative figures of immunoblot analysis show the expression of KLF5 in C-12511 and VSMCs isolated from db/db mice under different conditions. HG: high glucose, LG: low glucose, HG + I: high glucose + inhibitor, HG + con: high glucose + control oligos. E and F. The influence of miR-9 mimics (E) or inhibitors (F) on luciferase activity after the vectors with mutated and wild type 3’ UTR fragments of KLF5 were transfected into the HEK293 cells. Con + WT: control oligos + wild type 3’ UTR fragment, Con + Mut: control oligos + mutational 3’ UTR fragment. The results shown are representatives of 3 independent experiments. Data are presented as mean ± SD; *P < 0.05 vs. control oligo and **P < 0.01 by Student’s t-test. N = 5.