title: Identification of MicroRNAs that control lipid droplet formation and growth in hepatocytes via high-content screening.
abstract: Hepatic lipid droplets (LDs) are associated with metabolic syndrome, type 2 diabetes, hepatitis C, and both alcoholic and nonalcoholic fatty liver disease. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the level of translation. Approximately 1000 different miRNA species are encoded within the human genome, and many are differentially expressed by healthy and diseased liver. However, few studies have investigated the role of miRNAs in regulating LD expression. Accordingly, a high-content assay (HCA) was performed in which human hepatocytes (Huh-7 cells) were transiently transfected with 327 unique human miRNAs; the cells were then fixed, labeled for nuclei and lipid droplets, and imaged with an automated digital microscopy workstation. LD expression was analyzed on a cell-by-cell basis, using automated image analysis. Eleven miRNAs were identified that altered LDs. MiR-181d was the most efficacious inhibitor, decreasing LDs by about 60%. miRNA-181d was also confirmed to reduce cellular triglycerides and cholesterol ester via biochemical assays. Furthermore, a series of proteins was identified via miRNA target analysis, and siRNAs directed against many of these proteins also modified LDs. Thus, HCA-based screening identified novel miRNA and protein regulators of LDs and cholesterol metabolism that may be relevant to hepatic diseases arising from obesity and alcohol abuse.
authors.affiliation: Vala Sciences Inc., San Diego, CA, USA. whittaker@valasciences.com
authors.forename: Ross
authors.initials: R
authors.lastname: Whittaker
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Loy
authors.forename: Eugene
authors.initials: E
authors.lastname: Sisman
authors.forename: Eigo
authors.initials: E
authors.lastname: Suyama
authors.forename: Pedro
authors.initials: P
authors.lastname: Aza-Blanc
authors.forename: Randall S
authors.initials: RS
authors.lastname: Ingermanson
authors.forename: Jeffrey H
authors.initials: JH
authors.lastname: Price
authors.forename: Patrick M
authors.initials: PM
authors.lastname: McDonough
mesh_terms: D045744:Cell Line, Tumor; D002784:Cholesterol / Q000378:metabolism; D015723:Gene Library; D022781:Hepatocytes / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D050356:Lipid Metabolism; D035683:MicroRNAs / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D015203:Reproducibility of Results; D014280:Triglycerides / Q000378:metabolism
chemical_list: D035683:MicroRNAs; D034741:RNA, Small Interfering; D014280:Triglycerides; D002784:Cholesterol