Hepatitis B Virus-Encoded X Protein Downregulates EGFR Expression via Inducing MicroRNA-7 in Hepatocellular Carcinoma Cells
Figure 2
The 3′UTR activity of EGFR was reduced by HBx in HCC cells. (a) The mRNA expression of EGFR in HCC cells was examined by RT-qPCR. The EGFR mRNA expression was normalized to actin expression. (b) Hep3Bx cells were treated with either lysosomal inhibitors (bafilomycin A1 and NH4Cl) or proteasomal inhibitors (MG132 and bortezomib) for 6 hrs. EGFR protein expression was analyzed by Western blot. (c) Hep3B cells were transiently transfected with myc-EGFR expression vector along with or without HA-HBx plasmid for 48 hrs. The protein expression of myc-EGFR was examined by Western blot with anti-myc antibody. (d) Hep3B and Hep3Bx cells were transiently transfected with EGFR-3′UTR luciferase plasmid for 48 hrs. Total cells lysates were harvested for luciferase activity analysis. The luciferase activities were normalized to β-gal. Values of luciferase activity were means ± SE of three determinations. Statistical analysis was performed by Student’s -test. * as compared to Hep3B cells. (e) Human embryonic kidney HEK293 cells were transiently transfected with EGFR-3′UTR luciferase plasmid as well as different doses of myc-HBx expression vector for 48 hrs. Total lysates were harvested for luciferase activity analysis. The luciferase activities were normalized to β-gal. Values of luciferase activity were means ± SE of three determinations. Statistical analysis was performed by Student’s -test. *; *** as compared to control group.
