2.15 cells at a concentration of 20 nM. HBV RI were isolated at day 4 after transfection and analyzed by southern blot. As compared to control miR-C, HBV replication was significantly enhanced and quantitative real-time PCR showed 3.5-fold up-regulation of HBV RI by miR-1 transfection (Fig. 1A, lane 3). MiR-146 and miR-214 increased HBV replication slightly, whereas miR-210 decreased HBV replication by about 40%. The liver specific miRNA-122, as well as miR-132,
did not Barasertib supplier have a significant effect on HBV replication (Fig. 1A). In Con 1 cells with HCV subgenomic replicon, miR-122 transfection resulted in an increase of HCV RNA copy number,3 whereas other miRNAs had no significant influence on HCV replication (Supporting Information Fig. 1). To further evaluate these miRNAs effects on HBV replication, miRNAs and a replication competent clone of HBV pSM2 were cotransfected into Huh7 and HepG2 cells. Consistently, the amount of HBV RI was increased about 2.5-fold after miR-1 transfection (Fig. 1B). Other miRNAs tested so for had no significant effect on HBV replication in the cotransfection
system (data not shown). Based on these results, we subsequently focused on miR-1. Next we examined the effect of miR-1 on the Trichostatin A solubility dmso different stages of the HBV life cycle. HepG2.2.15 cells were transfected with miR-1 at concentrations ranging from 0.1 to 40 nM. An increased HBV replication was observed at a low concentration of 0.1 nM of miR-1 and up to 5.0-fold at 40 nM (Fig. 2A; Supporting Information Fig. 2A). The up-regulation of HBV replication by miR-1 transfection became recognizable after 2 days, increased with time, and maintained at Interleukin-3 receptor least up to 14 days (Supporting Information Fig. 2B). HBV RNA levels were elevated significantly as determined by real-time RT-PCR (Fig. 2B, upper panel) and northern blot (Fig. 2B, bottom panel). The amount of HBV
progenies released into culture supernatants was also increased in a dose-dependent manner (Fig. 2C). The amount of HBsAg in culture supernatants of transfected HepG2.2.15 cells increased in a similar manner after miR-1 transfection. A slight increase of HBeAg could be observed after transfection with 40 nM of miR-1, whereas HBcAg expression in cytoplasm was clearly increased, as shown by western blot (Fig. 2D). Transfection with miR-C had no influence on the levels of HBV RI, RNAs, proteins, and the production of HBV progenies (Fig. 2A-D). These results confirmed that miR-1 effectively enhanced HBV replication, as well as transcription, gene expression, and progeny viral secretion. To investigate molecular mechanisms of miR-1 effect on HBV replication, we first addressed the sequence specificity of the effect of miR-1.