Fig 4.

HBV can sponge the miR-15a/16 cluster. (A) The predicted miR-15a/16-binding sequences are located at nt 1362 to 1383 of the HBV genome. Perfect matches are indicated by vertical lines. Mutations in HBV were made in the predicted seed region of miR-15a/16-binding sites (UTR-HBV-mut) (upper panel; the mutated nucleotides are shown in bold italics). A putative miR-15a/16-complementary region was found in the HBV mRNAs and is indicated with gray boxes (lower panel). (B) miR-15a/16-binding sequences in different genotypes of HBV were pulled from the database, and the homology among them was determined. The seed region of miR-15a/16 is shown in bold italics. (C) HepG2 cells were cotransfected with a miR-15a/16 mimic, or a randomized oligonucleotide as a control, and the dual-luciferase reporter plasmid UTR-HBV-wt or UTR-HBV-mut. The cell lysates were harvested for firefly luciferase and Renilla luciferase activity assays. The relative luciferase activity was quantified. (D) HepG2 cells were transfected with pHBV1.3 or pHBV1.3-mut for 48 h. The total RNA was prepared for real-time PCR to measure miR-15a/16 levels. (E to H) HepG2 cells were transfected with pHBV1.3 or pHBV1.3-mut. Forty-eight hours after transfection, the total RNA was extracted and subjected to real-time PCR for HBV transcripts (E) and miR-15a/16 levels (H). (F) The supernatants were collected at the indicated time points for ELISA to detect HBV surface antigen. (G) The DNAs extracted from cells were subjected to Southern blotting for HBV DNA. RC and DL, relaxed circular and dendrimer DNAs, respectively. (I) HepG2 cells were transfected with the indicated amounts of pHBV1.3. The total HBV transcripts (left) and miR-15a/16 expression (right) were quantified by real-time PCR. *, P < 0.05; **, P < 0.01.