Fig. 3.

LIN28A is a novel target gene of miR-622 in HCC. (A) MicroRNA recognition elements (MRE) in the LIN28A 3’UTR represent potential miR-622-binding sites and were identified applying the "TargetScan 7.2" database. (B) Quantitative RT-PCR analysis of LIN28A mRNA levels in control-transfected as compared to miR-622-transfected different HCC cell lines (PLC (n = 3); Hep3B (n = 3)). (C) Luciferase LIN28A 3′UTR-reporter (containing a conserved miR-622 MRE) activity in control-miR (CTR) as compared with miR-622-mimic (622)-transfected HCC cells (PLC) (n = 3). (D) Schematic image depicting the pre-let-7-f-1 loop region (as predicted applying RNA-fold [80] with the highlighted consensus motif for recognition and binding of LIN28 to pre-let-7-f-1 which was identified previously [33]. (E) Schematic image depicting the pre-miR-622 stem loop (as predicted applying RNA-fold [80] and miRNAMap [24]) and magnification of the pre-miR-622 loop region containing the identic sequence motif as identified previously for recognition and binding of LIN28 to pre-let-7. (F) Hypothesis image: According to pre-let-7, LIN28A might also bind to the terminal loop of pre-miR-622 with subsequent ZCCHC11-mediated oligouridylation followed by degradation of pre-miR-622. (G) RNA-pulldown (n = 2) depicting binding of LIN28A to the let-7-g hairpin (positive control). No binding was found applying a negative control miR without the potential consensus motif (miR-18b) as well as a miR-622 and a miR-622-mutated (mutated consensus motif) hairpin. Data are presented as the mean ± SEM. Statistical significance was determined by 2-tailed, unpaired t-test (A,B). *P < 0.05, ***P < 0.001.