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. 2016 May 20;6:26166. doi: 10.1038/srep26166

Figure 2. MiR-30 inhibits CaMKIIδ expression by targeting CaMKIIδ 3′UTR in primarily cultured smooth muscle cells.

Figure 2

Rat aorta medial layers were enzymatically dispersed and primarily cultured for 3–7 passages. (a) The miRNA and mRNA levels of miR-30a, miR-30b, miR-30c, miR-30d, miR-30e, SM-MHC, SM-22α and Klf4 were measured by qPCR in aorta and cultured smooth muscle cells. Data were normalized over U6 (miRNA) or GAPDH (mRNA) (n = 3 aorta and 5 cultured SM cells). (b) miR-30c mimic or miR-30e (Invitrogen) (0.2pmol) was eletroporated into primarily cultured SM cells (1 million) and the expression of CaMKIIδ and GAPDH was analyzed by Western blot 3 days post-electroporation. (c) Full length of CaMKIIδ 3′UTR or truncated CaMKIIδ 3′UTR was introduced into pmiR reporter vector (Invitrogen). Full length CaMKIIδ 3′UTR reporter or truncated CaMKIIδ 3′UTR reporter as well as miR-30c and renilla were transfected into HEK293 cells and luciferase activity was measured 3 days after transfection. Values are shown as mean +/- S.E. M., n ≥ 4 and analyzed by two-way ANOVA or t-test. *p < 0.05 **p < 0.01 and *** p < 0.001.