The findings indicate that miR-196 plays a role in the regulation of HMOX1/Bach1 expression and HCV replication in hepatocytes. They also add to the growing evidence that up-regulation of HMOX1 may be beneficial in HCV infection.5, 19, 20 GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HCV, hepatitis C virus; HMOX, heme oxygenase; miRNA, microRNA; MMNC, miRNA mimic negative control; mRNA, messenger RNA; Mut, mutant ; NS, nonstructural; qRT-PCR, quantitative real-time polymerase chain reaction; siRNA, small interfering RNA; UTR, untranslated region; WT, wild-type. R.

Bartenschlager (University of Heidelberg, Heidelberg, Germany) kindly provided 9–13 cells. These cells harbor a replicating HCV NS region with the selleck screening library use of the NS3–NS5B gene regions from the Con1 isolate.21 Huh-7.5 and Con1 subgenomic genotype 1b HCV replicon cell lines were from Apath LLC (St. Louis, MO). Huh-7.5 is a highly permissive, interferon-α–cured Huh-7 human hepatocellular carcinoma cell line RG7204 purchase derivative. The Con1 cell line is a Huh-7.5 cell population containing the full-length HCV genotype 1b replicon. The 9–13, Huh-7.5, and Con1 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% (vol/vol) fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and selection antibiotic 500 μg/mL G418 for 9–13 cells, or 750 μg/mL G418 for Con1 cells. The miRIDIAN

miRNA mimics for has-miR-196, has-miR-16, MCE公司 customized mutant has-miR-196, miRNA mimic negative control (MMNC), and Bach1–small interfering RNA (siRNA) were obtained from Dharmacon (Lafayette, CO). pRL-TK

vector was obtained from Promega. The pRL-TK reporter vector contains a complementary DNA (Rluc) encoding Renilla luciferase as an internal control reporter. pGL3-Bach1 luciferase reporter construct, containing a 1,837-bp fragment of Bach1 3′-UTR, was a kind gift of Dr. Rolf Renne (University of Florida, Gainesville, FL).22 Mutant pGL3-Bach1 was generated by GENEWIZ, Inc. (South Plainfield, NJ). pLSV40-Rluc and pLSV40-GL3/Bach1 reporter vectors were kindly provided by B. R. Cullen (Duke University, Durham, NC). pLSV40-Rluc contains a complementary DNA (Rluc) encoding Renilla luciferase as an internal control reporter and pLSV40-GL3/Bach1 firefly luciferase reporter construct contains the full-length 3′-UTR of Bach1 mRNA.23 Constructs were confirmed by way of restriction enzyme digestion and sequencing. Transfection of miR-196 mimic or Bach-siRNA was performed as described.24 Cotransfection of miRNA mimics and reporters were performed using Lipofectamine 2000 from Invitrogen (Carlsbad, CA) according to the manufacturer’s protocol. Briefly, cells were cotransfected with 0.4 μg/mL of pGL3-Bach1 or mutant pGL3-Bach, with 0.4 μg/mL of pRL-TK, and with 10–50 nM tested miRNAs.