Research Paper Volume 10, Issue 8 pp 1884—1901

Figure 6. MiR-15a and miR-16-1 mediate the downregulation of ANLN expression in HCC cells. (A) Western blotting analysis of ANLN protein expression in HepG2 and HepG2.215 cells. (B) QPCR analysis of ANLN mRNA expression in HepG2 and HepG2.215 cells. (C) Endogenous ANLN protein turnover in HepG2 and HepG2.215 cells over the course of 10 h following the addition of 200 μg·mL^-1 cycloheximide. GAPDH indicates total protein loading for each sample. The relative fold changes of each sample compared with time 0 are shown below. (D) Potential microRNAs targeting full-length ANLN mRNA are shown using the TargetScan tool (www.targetscan.org). (E) The endogenous expression levels of miR-15a, miR-16-1, miR-195 and miR-424 in HepG2 and HepG2.215 cells were determined using qPCR. U6 was used as an internal control. (F) Western blotting analysis of ANLN expression in HepG2.215 cells treated with miR-15a, miR-16-1, miR-195 and negative control mimics, respectively. The relative fold changes compared with control are shown below. (G) Western blotting analysis of ANLN expression in HepG2 cells treated with miR-15a, miR-16-1, miR-195 and negative control microRNA inhibitors, respectively. The asterisk indicates a nonspecific band. The relative fold changes compared with the control are shown below. (H) Schematic diagram of the reporter constructs containing the predicted miR-15a and miR-16-1 binding sites in the 3’ UTR of ANLN. (I) Treatment of miR-15a and miR-16-1 mimics significantly attenuated the luciferase activity of the ANLN 3’ UTR compared with the control in QGY-7703 cells. *P < 0.05; ***P < 0.001.