miR-199a-5p Is Upregulated during Fibrogenic Response to Tissue Injury and Mediates TGFbeta-Induced Lung Fibroblast Activation by Targeting Caveolin-1
Figure 3
CAV1 is a direct target of miR-199a-5p.
(A) Position of miR-199a-5p target site in CAV1 3′ UTR and sequence alignment of miR-199a-5p and the CAV1 3′ UTR from various species are shown. The “seed” region with a conserved WC match to the eighth nucleotide of the miRNA is highlighted. Bases that have been mutated in the psiCHECK-2 construct are underlined. (B) Co-transfection of pre-miR-199a-5p or pre-miR-Neg and human CAV1 3′UTR-derived psiCHECK-2 construct (wild type or mutated in the putative miR-199a-5p seed region) in HEK293 cells. Cells were harvested two days after transfection and luciferase activities were analyzed. All renilla luciferase activities were normalized with firefly luciferase activity. * p<0.05. (C) and (D) Normalized expression values of CAV1 in hFL1 lung fibroblasts after transfection with pre-miR-199a-5p, pre-miR-21 or si-CAV1 from microarrays (C, n = 2) or real time PCR (D, n = 3) experiments. Data are expressed as mean ± SEM (n = 2). **p<0.01 (E) Western blot analysis showing the down regulated expression of CAV1 protein after transfection of hFL1 lung fibroblasts with pre-miR-199a-5p, pre-miR-21 or si-CAV1. One representative experiment out of two is shown.