OBJECTIVETo construct a short hairpin (sh)RNA targeting aquaglyceroporin 9 (AQP9) that effectively silences gene expression in liver cells in order to investigate of the role of AQP9 in nonalcoholic fatty liver disease (NAFLD) pathogenesis using an in vitro cell model system.

METHODSSmall interfering (si)RNAs were designed against the human gene sequences encoding AQP9 (NCBI GenBank Accession No. AB008775) and unrelated control sequences, synthesized, annealed to form double-strands, and inserted into the pGenesil- 1 shRNA-expression plasmid. The silencing effects of the four pshRNA-AQP9 constructs (a-d) and the pshRNA-negative control construct were investigated by transfecting into the L02 human normal liver cell line and detecting expression of AQP9 mRNA and protein (relative to beta-actin) by reverse transcription-PCR and western blotting. The NAFLD cell model was established by treating L02 cells with oleic acid to induce fatty degeneration. After transfecting the NAFLD cell model with various constructs, the effects on NAFLD-related features were investigated by staining with Oil Red O (to detect lipid droplets) and performing enzymatic assays (to quantitate triglyceride (TG), free fatty acid (FFA) and glycerol content). The significance of intergroup differences was assessed by analysis of variance test.

RESULTSOf the four pshRNA-AQP9 constructs, pshRNA-AQP9a produced the most robust silencing effect on AQP9 mRNA (25.1 - 1.2% vs. untransfected: 39.3 +/- 1.7% and pshRNA-negative control: 39.4 +/- 1.5%, P < 0.01) and protein (25.4 - 2.0% vs. untransfected: 35.1 +/- 1.9% and psh-RNA-negative control: 35.6 +/- 2.3%, P < 0.01). Oleic acid-induced L02 cells showed enhanced AQP9 mRNA and protein expression, and increased intracellular content of lipid, TG, FFA, and glycerol, which were significantly reduced by pshRNA-AQP9a transfection (all P <0.05).

CONCLUSIONThe new pshRNA-AQP9a construct can efficiently reduce AQP9 expression in cultured human liver cells and relieve steatosis-related features in an NAFLD cell model, pshRNA-AQP9a represents a novel tool for studying the role ofAQP9 in NAFLD pathogenesis and its potential as a gene therapy strategy.