Figure 3

The miR-19a-19b-20a sub-cluster suppresses TGFBR2 protein expression post-transcriptionally by targeting the Tgfbr2 3′ UTR sequence in primary lung fibroblasts. (A) miR-19a, 19b and 20a expression measured in hCD271⁺ fibroblasts transduced with miR-19a-19b-20a sub-cluster vector was higher than that observed in hCD271⁺ fibroblasts transduced with control vector according to qPCR. (B) Tgfbr2 mRNA-expression levels in transduced fibroblasts analyzed by qPCR. (C) TGFBR2 protein levels in the transduced fibroblasts analyzed based on MFI. Cells were stained with PE-conjugated anti-hCD271 and APC-conjugated anti-TGFBR2 antibodies. The MFIs of APC conjugates in hCD271⁺ cells were measured by flow cytometry. (D–F) mmu-miR-19a-3p, 19b-3p and 20a-5p directly targets Tgfbr2 3′ UTR. (D) Diagram of the complementarity between each miRNAs, the wild-type Tgfbr2 3′ UTR, and the each mutant Tgfbr2 3′ UTR sequences. (E) Luciferase-reporter analysis of the interaction between each miRNAs and the Tgfbr2 3′ UTR. The each of control/ sub-cluster-expression vector, and each of the sensor/mutant vectors were co-transfected into HEK293T cells using Lipofectamine LTX and cultured for 24 h. (F) The relative ratio of sensor vector to mutant vector in (E). Graphs show the mean ± SEM (n = 4; A,C,E and F) and (n = 3; B). (A–C and F) *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t-test. Cohen’s effect size d in these data was >0.8.