[23] 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 buy Omipalisib cells, and no significant changes in cell viability were observed (Supporting Fig. S6A). Next we IWR 1 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.[30] Consistent with previous findings linking PPAR-α inhibition with this website steatosis, small molecule-based antagonism of PPAR-α signaling in Huh7 cells can induce triglyceride (TG) accumulation (Supporting Fig. S8).[22] 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,[22] 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.[22] 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).