04) increased cellular GPAM levels (Fig. 5B). To determine if miR-27b modulates PPARG transcriptional activity, we performed PPARG

binding assays with nuclear extracts from transfected Huh7 cells (Supporting Methods). Inhibition of endogenous miR-27b resulted in a significant (P = 0.01) increase in PPARG binding to immobilized response elements (Supporting Fig. S2). It should be noted that, whereas overexpression of miR-27b significantly reduced (39% loss, P = 0.002) secreted ANGPTL3 levels (Fig. 5A) after 48 hours, Acalabrutinib cellular GPAM protein levels and PPARG transcriptional activity were not affected (Fig. 5B; Supporting Fig. S2). These observations are likely explained at least in part by the stability and temporal dynamics of each protein. Next we searched for canonical 3′ UTR seed-based miR-27b target sites within each of the six genes (Materials and Methods). As expected, SREBF1, which did not change (mRNA level) in response to overexpression of either miR-27b mimic or its antagomiR, did not harbor any canonical miR-27b seed sites (Fig. 4F). Three out of the five genes that were repressed by miR-27b (PPARG, NDST1, and GPAM) contained one or more seed sites within their 3′ UTRs (Fig. 4A-E). GPAM harbors two highly conserved and one moderately conserved miR-27b target site within its 3′

UTR (Fig. 4). To determine if miR-27b directly targets GPAM through one of these predicted sites, we performed reporter gene (luciferase) assays. A portion of the GPAM 3′ UTR, containing one putative miR-27b site, was http://www.selleckchem.com/products/R788(Fostamatinib-disodium).html cloned downstream of firefly luciferase (Materials and Methods). Dual transfection with miR-27b in HEK293 cells significantly (P = 0.001) reduced firefly luciferase activity (Supporting Fig. S3). After site-directed mutagenesis to eliminate

the putative miR-27b site (Materials and Methods), miR-27b failed to knock-down firefly luciferase activity, indicating that the site is directly involved in miR-27b mediated regulation of GPAM (Supporting Fig. S3). ANGPTL3 was the only down-regulated gene that did not harbor any miR-27b seed sites in its 3′ UTR. To further investigate the observed strong miR-27b-mediated regulation of ANGPTL3 (Fig. 4C, 5A), we expanded the search to two recently discovered classes of target selleck chemicals sites: (1) 3′ UTR centered sites and (2) open reading frame (ORF) sites (Materials and Methods). As its name implies, 3′ UTR centered sites base pair to the center of the miRNA sequence,37 as opposed to the 5′-end seed region. Functional ORF sites are typically preceded by a stretch of rare codons,38 which can cause ribosomal pausing,39 thereby allowing miRNA silencing complexes to form stable interactions with the target site without ribosomal interference. We developed and implemented computational strategies to predict 3′ UTR centered sites based on the strength of base pairing to the center of a miRNA, and ORF seed sites based on a metric that evaluates codon rarity in the preceding sequence (Materials and Methods).