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. 2011 May 31;105(1):146–153. doi: 10.1038/bjc.2011.190

Figure 2.

Figure 2

HBx downregulates the expression of the miR-16 family in non-HBV-infected malignant hepatocytes in vitro and upregulates the expression of CCND1 in HepG2 cells. (A) The expression of the miR-16 family was measured using qRT-PCR and normalised by U6 expression in HepG2 cells that stably expressed HBx or control vector. (B–D) The expression of the miR-16 family normalised to U6 was detected by qRT-PCR in (B) HepG2, (C) Huh7, and (D) SK-HEP-1 cells transiently transfected with HBx-expressing plasmid or control vehicle. Each cell line was transfected with 4 μg PCDNA3.1-hbx or 4 μg PCDNA3.1 as a control. Cells were collected for analysis 48 h after each transfection. (E) The activity of the luciferase reporter containing the wild-type 3′UTR of CCND1 was elevated in HepG2-hbx cells. pRL-TK was co-transfected with a firefly luciferase reporter plasmid carrying either the wild-type (WT) or mutant (MUT) 3′UTR of CCND1 into HepG2-hbx or HepG2-vc cells; luciferase activity was analysed 48 h later. pRL-TK expressing Renilla luciferase was used to correct for the differences in transfection and harvest efficiencies between the HepG2-hbx and HepG2-vc cells. The ratio of the luciferase activity of MUT-3′UTR in HepG2-hbx to that in HepG2-vc cells was normalised to 1. (F) A western blot confirmed the induction of CCND1 by HBx in HepG2 cells. The data are presented as mean±s.e. fold. (*P<0.05, **P<0.01, ***P<0.001; Student's t-test, NPar tests; n=3).