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. 2015 Jan 1;11(1):48–58. doi: 10.7150/ijbs.9969

Figure 2.

Figure 2

THBS1 as a target gene of let-7f. A, qRT-PCR confirmed transfection efficiency of let-7f and AMO-let-7f in HEK293T cells. After transfected for 48 h, let-7f expression was increased, and AMO-let-7f inhibited let-7f expression to 42% of NC group. * P<0.05 vs NC, n=6 independent batches cells. B, Luciferase assay in HEK293T cells for post-transcriptional inhibition of THBS1 (gene encoding TSP-1 protein). WT THBS1 3'UTR luciferase reporter vector was in structure with full length of THBS1 3'UTR including let-7f binding stie; Mut THBS1 3'UTR luciferase reporter vector was insert with fragments of mutant 7 nucleotide of let-7f binding site in full length THBS1 mRNA 3'UTR (upper). HEK293T cells were transfected with miR-let-7f, AMO-let-7f or a scrambled miRNA (NC) and NC inhibitor as negative control by Lipofectamine™ RNAiMAX. Blank group was treated with Lipofectamine™ RNAiMAX alone. * P<0.05 vs NC, ** P<0.01 vs NC, ## P<0.01 vs let-7f alone; n=6 independent batches of cells for each group. C, Let-7f and AMO-let-7f regulated THBS1 mRNA expression in MCF-7 cells. After transfected with let-7f or AMO-let-7f in MCF-7 cells for 48 hours. THBS1 mRNA expression was significantly decreased to 41% of NC group. Consistently, AMO-let-7f increased THBS1 mRNA expression as well. ** P<0.01 vs NC group, ## P<0.01 vs let-7f alone, n=6 independent batches cells in each group. D, The effect of let-7f or AMO-let-7f on TSP-1 protein expression in MCF-7 cells. Downregulation of TSP-1 protein was observed in let-7f group, and AMO-let-7f increased the expression level of TSP-1 in MCF-7 cells. ** P<0.01 vs NC, ## P<0.01 vs let-7f alone, n=6 independent batches of cells for each group.