CARS has been used extensively for label-free imaging and quantification of hepatic lipid content in biological systems, thereby avoiding perturbations and artifacts that can be introduced by added dyes and staining protocols.[22, 23] Transfection of miR-27a and miR-27b mimics in Huh7 cells induced an increase in both the size and abundance of LDs (Fig. 2A-C). The average LD diameter increased from 540 ± 10 nm to 600 ± 10 nm (n > 1,900 LDs;
P < 0.01) during miR-27b overexpression. Similar results were observed in Huh7.5 cells (Supporting Fig. S5). To exclude the possibility that miR-27 mimics resulted in cytotoxicity, we performed 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assays on transfected Huh7 selleck screening library cells, and no significant changes in cell viability were observed (Supporting Fig. S6A). Next we selleck compound sought to identify the relevant
endogenous targets of miR-27 that might induce lipid accumulation. We examined messenger RNA (mRNA) levels using qRT-PCR to confirm that they are miR-27 targets. Huh7 cells were transfected with miR-27b or control mimics, and qRT-PCR revealed an inverse correlation between miR-27b activity and the mRNA levels of PPAR-α and angiopoietin-like protein 3 (ANGPTL3) (Supporting Fig. S7A), consistent with previous reports.[14, 29] Both of these genes have conserved miR-27 binding sites (Supporting Fig. S7B), and have known links to triglyceride homeostasis.[14, 29] PPAR-α is a key nuclear receptor that transcriptionally activates genes associated with fatty acid oxidation. Consistent with previous findings linking PPAR-α inhibition with Chlormezanone steatosis, small molecule-based antagonism of PPAR-α signaling in Huh7 cells can induce triglyceride (TG) accumulation (Supporting Fig. S8). If miR-27′s induction of hepatic lipid storage relied on inhibition of PPAR-α signaling and the resulting triglyceride accumulation, activating the PPAR-α pathway should reverse the effect.
Treatment with a small molecule PPAR-α agonist, bezafibrate, was sufficient to reverse miR-27-induced lipid accumulation to levels observed in control cells, confirming this hypothesis (Fig. 3). Overall, these observations suggest that miR-27 overexpression induces triglyceride accumulation through repression of PPAR-α expression. Our previous work showed that PPAR-α antagonism is effective at inhibiting HCV replication. To examine if miR-27 has a similar effect, we overexpressed miR-27b in Huh7.5 cells stably expressing the HCV full length replicon (Fig. 4A). Interestingly, ectopic miR-27b expression resulted in a 3-fold down-regulation of HCV RNA (Fig. 4B). A similar down-regulation was observed in HCV NS3 and NS5A proteins by western blot (Fig. 4C). No cytotoxicity was observed during miR-27b overexpression (Supporting Fig. S6B).