Genomewide miRNA changes were

studied in both sham and PH samples at the indicated time points by a custom microarray platform,19 as described in Supporting Information. A minimum of 2-3 replicates were studied in each group. Array data for each of the different time points have been deposited in the Gene Expression Omnibus under accession number GSE28404. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), Western blots, and immunoflurescence were performed, following the manufacturer’s instructions. Please refer to Supporting Information for additional details. Human RNASEN (Drosha), TARBP2 (TRBP), and PRKRA (PACT)-3′UTRs were amplified and cloned into pSGG prom 3′UTR reporter plasmid (SwitchGear Genomics, Menlo Park, CA) by NheI and XhoI. DICER and DGCR8 3′UTR reporters were purchased from SwitchGear mTOR inhibitor Genomics. Ten individual miRNAs or miRNA Roxadustat clusters were cloned into pcDNA3.1 (Invitrogen, Carlsbad, CA) by HindIII/XbaI or NheI/XhoI. The miR-17-92 expression construct was kindly provided by Dr. He Lin (University of California, Berkeley, CA). The miRNAs included in the constructs and primers used in cloning are shown in Supporting Tables 2 and 3, respectively.

Anti-miR-107, anti-miR-424, and anti-let-7a were purchased from Qiagen (Hilden, Germany). Human hepatoma Huh-7 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) as previously described.20 Cells were plated at 70% density 24 hours before transfection. Primary rat hepatocytes were obtained from male (225-250 MCE g) Sprague-Dawley rats via collagenase perfusion, as previously outlined.21 Please refer to Supporting Information for additional details. Different 3′UTR reporter constructs were cotransfected with miRNA constructs (or anti-miRs) and the SV40-RL internal control plasmid (Promega, Madison, WI) by Lipofectamine 2000 into Huh-7 cells. Cells were harvested 24 hours after transfection, and luciferase

activity was determined by the Dual-Glo Luciferase Assay System (Promega), using a Synergy 2 microplate reader (BioTek, Winooski, VT). A number of different miRNA expression constructs were transfected into Huh-7 cells, and cells were harvested after 24 hours. For cell-cycle and cell-death studies of both Huh-7 cells and primary hepatocytes, please refer to Supporting Information. We analyzed hepatic miRNA expression profiles from both sham and 70% hepatectomized rats from 3 to 72 hours after surgery. Between 300 and 400 miRNAs were expressed at these various time points (Table 1). Comparing sham and PH groups, 208 miRNAs could be detected at all indicated times. Based on their expression levels, we grouped these miRNAs into three sets and classified them as down-regulated (<0.8-fold), unchanged (0.8- to 1.2-fold), and up-regulated (>1.2-fold).