title: [Effect of RNAi-mediated silencing of SREBP2 gene on inflammatory cytokine-induced cholesterol accumulation in HepG2 cells].
abstract: OBJECTIVE
  To investigate the effect of RNA interference (RNAi)-mediated silencing of the SREBP2 on inflammatory cytokine-induced cholesterol accumulation in HepG2 cells.
  
  
  METHODS
  Short-hairpin (sh)RNA targeting SREBP2 or negative control (NC) shRNA were transfected into HepG2 cells by a liposomal method. G418-selective culturing was used to obtain the SREBP2 shRNA HepG2 and NC shRNA HepG2 cell lines. The two cell lines were cultured in serum-free medium and left untreated (control) or treated with TNF-a (20 ng/ml), low-density lipoprotein (LDL) loading (100 mug/ml), or a combination LDL plus TNF-a treatment. Lipid accumulation was evaluated by oil red O (ORO) staining. Intracellular cholesterol level was measured by enzymatic assay. The mRNA and protein levels of SREBP2 and its downstream target genes, LDL receptor (LDLr), and HMGCoA reductase, were measured by real-time PCR and Western blotting, respectively.
  
  
  RESULTS
  SREBP2 shRNA HepG2 and NC shRNA HepG2 stable cell lines were successfully established. ORO staining and cholesterol quantitative analysis showed that LDL loading significantly increased intracellular cholesterol and that expression of SREBP2 further exacerbated the inflammatory cytokine-induced lipid accumulation, as seen in NC shRNA HepG2 cells. LDL loading of NC shRNA HepG2 decreased the gene and protein expressions of SREBP2, LDLr, and HMGCoA reductase, but the suppressive effect was overridden by inflammatory cytokine. SREBP2 shRNA HepG2 cells showed lower levels of cholesterol accumulation under LDL loading and inflammatory stress conditions. Moreover, the mRNA and protein levels of SREBP2, LDLr, and HMGCoA reductase were much lower than in NC shRNA HepG2 cells under the same conditions.
  
  
  CONCLUSIONS
  Inflammatory cytokine exacerbated cholesterol accumulation in HepG2 via disrupting SREBP2. RNAi-mediated inhibition of SREBP2 expression significantly ameliorated the cholesterol accumulation induced by inflammatory cytokine.
authors.affiliation: Chongqing Medical University, Chongqing, China. zlcyxi@sina.com
authors.forename: Jun-lei
authors.initials: JL
authors.lastname: Liao
authors.forename: Lei
authors.initials: L
authors.lastname: Zhao
authors.forename: Yao
authors.initials: Y
authors.lastname: Chen
authors.forename: Qing
authors.initials: Q
authors.lastname: Li
authors.forename: Yu-yang
authors.initials: YY
authors.lastname: Chen
authors.forename: Xiong-zhong
authors.initials: XZ
authors.lastname: Ruan
authors.forename: Ya-xi
authors.initials: YX
authors.lastname: Chen
mesh_terms: D002784:Cholesterol / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D007249:Inflammation; D034622:RNA Interference; D034741:RNA, Small Interfering; D051782:Sterol Regulatory Element Binding Protein 2 / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D034741:RNA, Small Interfering; C497390:SREBF2 protein, human; D051782:Sterol Regulatory Element Binding Protein 2; D014409:Tumor Necrosis Factor-alpha; D002784:Cholesterol