Fig. 3. HPGD is directly targeted by miR-146b-3p.

(a) Representative results of cervical cancer miRNA microarray analysis. (b) Luciferase assay of HEK293T cells co-transfected with negative control miRNA (Neg. Ctrl.) or mimics of several miRNAs together with the pGL3-HPGD 3' UTR or pGL3-HPGD CDS luciferase reporter. (c) 293T cells were co-transfected with the 3 × Flag-HPGD construct and miR-922 mimic for 48 h. Cells were collected and immunoblotted with the indicated antibodies. (d) HEK293T cells were co-transfected with the 3 × Flag-HPGD construct and mimic of miR-146b-3p for 48 h. Cells were collected and immunoblotted with the indicated antibodies. (e) HEK293T cells were co-transfected with the 3 × Flag-HPGD construct along with pEGFP and a mimic of miR-106b-3p for 48 h. Cells were collected and immunoblotted with the indicated antibodies. (f) Luciferase assay of HEK293T cells co-transfected with pGL3-HPGD CDS reporter together with increasing amounts (5, 10, and 20 nM) of negative control miRNA (Neg. Ctrl.) or a mimic of miR-146b-3p (miR-146b-3p) for 24 h. (g) HeLa cells were transfected with increasing amounts (10, 20 and 50 nM) of miR-146b-3p mimic or negative control for 48 h, collected, and western blot performed with the indicated antibodies. (h) Putative binding site of miR-146b-3p in the CDS region of HPGD and mutagenesis of the target site in miR-146b-3p. (i) Western blot of HPGD expression in HeLa cells transfected with a negative control mimic (Neg. Ctrl; 20 nM), miR-146b-3p mimic (miR-146b-3p; 20 nM) or miR-146b-3p mutant mimic (miR-146b-3p mut; 20 nM) for 48 h. (j) Luciferase activity in HEK293T cells co-transfected with miR-146b-3p mimic (miR-146b-3p; 10 nM), miR-146b-3p mutant mimic (miR-146b-3p mut ; 10 nM) or a negative control (Neg. Ctrl; 10 nM) and the HPGD CDS reporter construct for 48 h. The quantified results are presented as means ± SD. Three independent experiments, each with four technical replicates, were performed. ** P < 0.01 for Student's t-test. n.s, not significant