3A) and miR-206, an miRNA with the identical miR-1 seed-sequence but a different sequence at its 3′ end, were used for comparison with miR-1. Transfection of HepG2.2.15 cells with m-miR-1 and miR-206 did not enhance HBV replication (Fig. 3A). Further, cotransfection of miR-1 and its specific antisense inhibitor anti-miR-1 abolished the increase of HBV RI in HepG2.2.15, whereas the enhancing effect of miR-1 on HBV RI remained unchanged if an
unrelated anti-miR-C was cotransfected (Fig. 3B, lane 3). Consistently, knockdown of argonaute-2 (Ago2), a main component of RNA-induced silencing complex, by specific siRNA appeared to attenuate the effect of miR-1 (Fig. 3C, lane 4). These results suggested that up-regulation of HBV replication was mediated by miR-1-guided RISC formation. A critical feature of a direct interaction between miRNAs and target mRNAs is the presence of the corresponding seed sequences in the target.2 However, Fulvestrant manufacturer the complementary sequence (ACATTCC) of miR-1 seed sequence which was required for its binding to target mRNA was not found in the HBV genomic sequence. Consistently, cotransfection of pMIR-REPORT system Gefitinib cell line with cloned full length or four fragments of HBV genome and miR-1 into HepG2 cells did not result in a decrease of luciferase gene expression
(Supporting Information Fig. 3). Taken together, the data suggest that it is unlikely that miR-1 regulates HBV gene expression and replication by a direct interaction with genomic sequence of HBV. These results suggested that MCE miR-1 may act on specific cellular targets and thereby enhances HBV replication and gene expression in an indirect manner. Previously, a member of class II histone deacetylase (HDAC4) was identified as a cellular target of miR-1.22 Similarly, transfection with miR-1
led to a markedly reduced expression level of HDAC4 protein in HepG2.2.15 cells (Fig. 4A). The reduction of HDAC4 by miR-1 hinted at the potential role HDAC4 on HBV replication, similar to the recent results of HDAC1.23 Indeed, the knockdown of HDAC4 expression by specific siRNAs led to nearly a 2.5-fold increase in HBV replication in HepG2.2.15 cells (Fig. 4B), as well as the use of broad-spectrum HDAC inhibitor TSA (Supporting Information Fig. 4). Furthermore, cotransfection of an HDAC4 expression vector pHDAC4 with miR-1 could attenuate the increased replication of HBV (Fig. 4C). We concluded that HDAC4 is a target of miR-1 and may play a significant role in the action of miR-1 on HBV replication. The modulation of HDAC4 expression by miR-1 may lead to changes of HBV promoter activity. Thus, four pGL3-based luciferase reporter constructs pSP1, pSP2, pCP, and pXP containing the region of HBV SP1, SP2, core, and X promoters were cotransfected with miR-1 into HepG2.2.15 cells. The ectopic expression of miR-1 increased the level of transcription activity of the HBV core promoter about 3.0-fold but had no effect on the other three promoters (Fig. 5A).