Protein and mRNA level analysis of genes are functionally associated with miR-24. A: miR-24 was transfected into MIN6 cells for 48 h, and then total protein was extracted for analysis of Hnf1a, Neurod1, Pdx1, Parp1, Cdk4, Cyclind3, p27, p15, Pten, Cycind1, kir6.1, and β-tubulin by Western blot. Protein levels remaining in cells relative to the negative control were quantitatively analyzed
by Quantity One 4.2.1 (Bio-Rad). B: A decrease of Cyclind3 was detected first at 24 h posttransfection of miR-24 (50 nmol/L) and that for Cdk4 was at 48 h. Meanwhile, the peak expression of p15 was at 24 h. C: Transfection with miR-24 at 10 and 50 nmol/L downregulated Cyclind3 and Cdk4 protein levels and upregulated the p15 level. D: Seven cell cycle–associated genes (Ccnd1, Ccnd2, Ccnd3, Cdk4, p15, p21, and p27) and genes of two components of ATP-sensitive potassium (KATP) channels (Kcnj8 and Kcnj11) were determined by qRT-PCR. Ccnd3, Cdk4, and p27 were decreased, whereas p15 and p21 were increased (*P < 0.05 or **P < 0.01 vs. actin). E: mRNA levels of Ccnd3 and Cdk4 were also downregulated in islets isolated from ICR mice (**P < 0.01 vs. actin).