Down-regulation of endogenously expressed PLAG1 with

siRNA (PLAG1_6) in HUH6 cells was compared with cells treated with a nontargeting (ntg) control siRNA. MiRNA arrays revealed only miR-492 as a sufficiently and significantly (P ≤ 0.05) deregulated miRNA candidate that warranted further confirmation by quantitative TaqMan miRNA assays. Down-regulation of PLAG1 by 3.4-fold in HUH6 cells triggered the down-regulation of miR-492 by an average of 2.2-fold in comparison to the ntg-control (Fig. 1A). In HepT1 cells, a PLAG1 knockdown of 3.1-fold resulted in a miR-492 reduction of 2.4-fold. A second siRNA sequence (PLAG1_5) produced a PLAG1 knockdown of 4-fold in HUH6 cells and reduced the miR-492 level by 1.6-fold (data not shown), excluding the possibility of an off-target effect. The association of PLAG1 and miR-492 was confirmed AZD9291 molecular weight by overexpression experiments (Fig. 1B). HUH6 and HepT1

clones stably overexpressing PLAG1 to a 4 to 5-fold range exhibit a comparable 4 to 5-fold up-regulation of miR-492 expression. A BLAST (Basic Local Alignment Search Tool, NCBI) search for miR-492 against the human genomic and transcript database revealed a 100% identical sequence alignment with KRT19 (Chr.17; ENST00000361566) as well as with the pseudogene of KRT19 (Chr.12;29 NT_019546.15). Thus, both genes represent potential sites of origin for miR-492. Figure 2A depicts the gene structure of KRT19 as well as the location of the miR-492 precursor, which is spread out over the first two exons and thus interrupted by intron1. Closer analyses Autophagy signaling inhibitors of genomic KRT19 revealed seven putative PLAG1 binding sites (GRGGC(6-8nts)GGG identified12) in the 3′ downstream regions of KRT19 as well as in intron 1 (Fig. 2A,B). The pseudogene, however, does not exhibit any PLAG1 consensus sequences. These data suggest that PLAG1 might regulate MCE公司 the level of KRT19 expression, which in turn could coregulate the release of miR-492 from its processed transcript. We therefore tested the ability of KRT19

mRNA to give rise to miR-492. Figure 2A depicts the part of KRT19, termed “miR-492 vector” (including miR-492 precursor and ≈100bp additional bases up- and downstream) that was cloned into a lentiviral miRNA expression vector, pMif-cGFP-Zeo, which enables miRNA to be expressed in its correct environment. HepT1 cells transiently transfected with this miRNA expression vector indeed showed an up-regulation of miR-492 up to 150-fold compared to the empty vector 24 hours after transfection (Fig. 2C). These data provide experimental evidence that miR-492 can be processed from the KRT19 coding sequence and we define KRT19 as a novel precursor for hsa-miR-492 (Fig. 2D). This sequence slightly differs (7% difference) from a precursor previously submitted to miRBase (MI0003131, original miR-492 precursor), but yields an identical mature miRNA.