Figure 3.

miRNA-101 directly inhibits COX-2 expression at the posttranscriptional level. A, COX-2 protein levels in pairs of BPH1^Cvec/BPH1^CmiR101 and BPH1^CAFTD+siRNA/BPH1^CAFTD cell lines were evaluated by Western blotting and semiquantified on the basis of COX-2/β-actin relative intensity. The percentage indicates the relative intensity of COX-2. B, COX-2 mRNA levels of BPH1^Cvec and BPH1^CmiR101 cells were determined by quantitative RT-PCR and the results represented the mean ± SD of 2 independent experiments with triplicates. C, a RNA sequence map to show the 3′-UTR of COX-2 mRNA carries a complementary site (NM_000963 3′-UTR: 1,735–1,741) for the seed region of miRNA-101. D, luciferase report assay was applied to determine the miR-101 binding target. The luciferase activity was detected after transfection of 3′-UTR-Luci vector or EGFP-miR-101 vector alone, or combination of the two vectors into 293T cells. The P value was compared with vector control BPH1^Cvec cells (*, P < 0.05) and the results represented the mean ± SD of 2 independent experiments with triplicates. E, the COX-2 protein levels were analyzed by Western blotting and the amount of protein was normalized by comparing the intensity of the β-actin band.