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. 2015 Jan 6;5:7641. doi: 10.1038/srep07641

Figure 4. miR-574-3p modulates its candidate target CLTC mRNA expression in MCF-7 cells.

Figure 4

(a) Silencing of miR-574-3p induces CLTC mRNA expression. MCF-7 cells were transfected with anti-miR-574-3p or control anti-miR for 48 h, and then expression levels of miR-574-3p were evaluated by qPCR. Data are presented as mean ± SE; **P < 0.01. (b) Validation of miR-574-3p overexpression by transfection of the miR-574-3p precursor. Cells were transfected with pre-miR-574-3p or control pre-miR for 48 h, and then the expression levels of miR-574-3p were evaluated by qPCR. Data are presented as mean ± SE; **P < 0.01. (c) Overexpression of miR-574-3p decreased CLTC mRNA expression. Cells were transfected with pre-miR-574-3p or control pre-miR for 48 h, and then the expression levels of miR-574-3p were evaluated by qPCR. Data are presented as mean ± SE; **P < 0.01. (d) Overexpression of miR-574-3p decreased CLTC protein expression. Cells were transfected with pre-miR-574-3p or control pre-miR for 48 h, and then the expression levels of CLTC protein were evaluated by western blot using anti-CLTC antibody. Loading control was obtained with anti-β-actin antibody. (e) Location of putative miR-574-3p-binding sequences and mutated sites in the 3′-UTR of target genes. (f) Luciferase reporter assay using vectors containing a putative CLTC 3′-UTR binding site for miR-574-3p and a mutated version of the site. The 293T and MCF-7 cells were transiently transfected with psiCHECK2 vectors containing either the wild-type or mutated putative binding sites for miR-574-3p, together with pre-miR-574-3p precursor or control pre-miR for 48 h, and then the luciferase assay was performed. Data are presented as mean ± SE; **P < 0.01.