水甘油通道蛋白9短发夹RNA的构建与筛选及其对非酒精性脂肪性肝病细胞模型的作用

Construction of short hairpin RNA targeting aquaglyceroporin 9 and screening its effect on molecular mechanisms of nonalcoholic fatty liver disease using a cell model system

目的构建人水甘油通道蛋白9（AQP9）短发夹RNA（shRNA），筛选出沉默效果明显的重组质粒，检测AQP9的shRNA对非酒精性脂肪性肝病（NAFLD）细胞模型的作用。方法设计合成针对人AQP9的小干扰RNA，退火形成双链shRNA后插入pGensil-1质粒中，并将其转染L02肝细胞，逆转录一聚合酶链反应（RT-PCR）及Westem blot筛选出沉默效果明显的重组质粒。经油酸诱导L02细胞脂肪变性建立NAFLD细胞模型，通过油红O染色，测定甘油三酯、游离脂肪酸及甘油含量，检测重组质粒对NAFLD细胞模型的作用。数据比较采用方差分析。结果经RT-PCR及Westem blot筛选，pshRNA—AQP9a转染组mRNA及蛋白质相对表达率为25．10％士1．19％及25．41％±1．96％，与未处理L02细胞组的39．33％±1．69％及35．08％±1．89％相比，均明显降低∽值均〈0．01）；将重组质粒pshRNA-AQP9a转染NAFLD细胞模型能够降低其AQP9的mRNA及蛋白质表达量；油红O染色结果显示，油酸／pshRNA-AQP9a转染组细胞内脂质含量明显减少。油酸／pshRNA-AQP9a转染组细胞内甘油三酯、游离脂肪酸及甘油含量分别为（2．738±0．231）mmol／L、（1．182±0．168）mmol／L和（0．730±0．084）mmol／L，油酸组分别为（3．218土0．220）mmol／L、（1．538±0．193）mmol／L及（1．024±0．148）mmol／L，油酸／pshRNA-AQP9a转染组均明显降低（尸值均〈0．05）。结论降低AQP9表达量能够减轻NAFLD细胞模型的脂肪变l生程度，为进一步研究AQP9对NAFLD的基因治疗奠定基础。

Objective To 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. Methods Small 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 ofAQP9 mRNA and protein （relative to β-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. Results Ofthe 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.±1.9% 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）. Conclusion The 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 of AQP9 in NAFLD pathogenesis and its potential as a gene therapy strategy.