Figure 2.

hnRNPA1 is a direct target of miR-25-3p and miR-15-5p in ovarian cancer cells. The sites for miR-25-3p and miR-15a-5p targeting of the hnRNPA1 3′-untranslated region (UTR) were predicted. (a) miR-25-3p and miR-15a-5p targeting sequences of the hnRNPA1 3′-UTR is evolutionarily conserved over the three species tested (Homo sapiens: human; Mus musculus: mouse; Rattus novergicus: rat). The targeting sites are underlined. (b) This schematic illustration depicts the localization of the binding sites for miR-25-3p and miR-15a-5p targeting sequences of hnRNPA1, each color indicate an exonic region of the gene and boxes in white represent the 5′ and 3′ UTR. UT, untreated HEK293T cells were co-transfected with wild-type or mutant reporter and the miR-25-3p and/or miR-15a-5p mimic or negative control (NC mimic). After 48 h, luciferase/Renilla activity was measured *P<0.05. (c) The expression of hnRNPA1 was examined by quantitative reverse transcriptase-PCR (d, ****P<0.0001) and western blotting analysis (e) in HeyA8 cells that were co-transfected with miR-25-3p and/or miR-15a-5p mimic (or NC mimic). β-Actin was used as the endogenous control. (f) hnRNPA1 and K-RAS were detected by western blotting after transfection with hnRNPA1 or control siRNA. (g) hnRNPA1 and K-RAS were detected by western blotting after transfection with miR-25-3p and/or miR-15a-5p inhibitors and/or co-transfected with hnRNPA1 or control siRNA. (h) K-RAS was detected by western blotting after transfection with miR-18-3p inhibitor or mimic. (i) Endogenous hnRNPA1 was immunoprecipitated from total HeyA8 extracts and the pulled-down RNA was isolated and reverse-transcribed with the specific primer for miR-18a-3p PCR amplification. (j) Direct interaction between hnRNPA1 and Drosha was shown by pull-down assay in HeyA8 and HeyA8-MDR cells.