3A, right panel) indicated that the TSS is located 500 basepairs

3A, right panel) indicated that the TSS is located 500 basepairs (bp) upstream of the first base of pre-miR-216a (Fig. 3B). Next, a luciferase reporter plasmid, pGL3-216PA, driven by the genomic region ≈3.4 kb upstream of the TSS of pri-miR-216a was constructed to help study the transcriptional regulation of pri-miR-216a by the AR pathway PF-562271 concentration (Fig. 3C). We found that the reporter activity in HepG2 cells was stimulated by AR in a ligand-dependent manner (Fig. 3D, lane 4 versus lane 3), which was further enhanced by HBx (Fig. 3D, lane 8 versus lane 4), but HBx alone did not affect the

reporter activity (Fig. 3D, lanes 5 and 6). The effects of AR and HBx on the reporter activity showed the same trend as their effect on the endogenous pri-miR-216a (Fig. 2C,D), suggesting this 5′ genomic region is responsive to the AR-mediated up-regulation of pri-miR-216a. Serial deletion constructs

derived from pGL3-216PA were generated to map the subgenomic region responsible for the AR-mediated transcriptional regulation (Fig. 4A). The luciferase activity of each reporter construct was examined for their responsiveness to HBx-stimulated AR. The enhancement effect on the reporter activity by R1881 treatment remained in all the reporter constructs except pGL3-216PE (Fig. 4B). Because this pGL3-216PE construct deletes 300 bp (−603 to −304 bp) more than the pGL3-216PD construct, click here which remains responsive to AR regulation (Fig. 4A), the genomic region from −603 to −304 bp was the element responsible for the AR-mediated transcriptional regulation of pri-miR-216a. Two putative ARE sites are predicted

within this region: from −349 to −335 bp and −499 to −485 bp upstream of the TSS of pri-miR-216a, respectively (Fig. 4C). To evaluate the critical role of these two putative sites for AR-mediated transcriptional regulation, we mutated the critical nucleotides of an individual site in pGL3-216PD and examined the resulting effect (Fig. 4C; pGL3-216PD-mut1 上海皓元 and pGL3-216PD-mut2, GT > CA). The increased reporter activity for wildtype pGL3-216PD by HBx-stimulated AR was diminished in pGL3-216PD-mut1 but not in the pGL3-216PD-mut2 construct (Fig. 4D), suggesting that the candidate ARE site at −349 to −335 bp upstream of TSS is responsible for the AR-mediated regulation. ChIP analysis was used to further address whether the ligand-stimulated AR can directly bind to this ARE site. The cells transfected with either the AR or the green fluorescent protein (GFP) control expression constructs were processed for ChIP by antibodies against AR or immunoglobulin G (as a negative control), and then a specific primer set flanking this ARE site was used for PCR amplification. The specific PCR product was only detected in the cells transfected with the AR expression construct in a ligand-dependent manner (Fig. 4E, lane 5).

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