Journal: British journal of cancer

Article Title: Hepatitis B virus X protein downregulates expression of the miR-16 family in malignant hepatocytes in vitro.

doi: 10.1038/bjc.2011.190

Figure Lengend Snippet: 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 mg PCDNA3.1-hbx or 4 mg 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 30UTR 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) 30UTR 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-30UTR 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. (*Po0.05, **Po0.01, ***Po0.001; Student’s t-test, NPar tests; n ¼ 3).

Article Snippet: The antibodies used in western blotting were HBx (sc-71239; Santa Cruz Biotechnology, Santa Cruz, CA, USA), b-actin (sc-130301; Santa Cruz), c-Myc (9402; Cell Signaling Technology (CST), Boston, MA, USA), CCND1 (2926; CST), and GAPDH (BA2913; Wuhan Boster Biological Technology, Wuhan, China). miRNA microarrays The method for the miRNA microarray analysis was described in our previous report, except for the addition of an updated miRNA probe set (the NCode Human miRNA Microarray Probe Set V3þ , Invitrogen) composed of 703 known and 393 predicted human miRNAs in the present work (Yu et al, 2007).

Techniques: Expressing, Infection, In Vitro, Quantitative RT-PCR, Stable Transfection, Control, Plasmid Preparation, Transfection, Activity Assay, Luciferase, Mutagenesis, Western Blot