title: RNAa-mediated overexpression of WT1 induces apoptosis in HepG2 cells.
abstract: OBJECTIVE
  Recent studies have reported that double-stranded RNA (dsRNA) can activate gene expression by targeting promoter sequence in a process termed RNA activation. The present study was conducted to evaluate the potential of WT1 induction by small activating RNA targeting the WT1 promoter (dsWT1) in the treatment of hepatocellular carcinoma.
  
  
  METHODS
  The human hepatocellular carcinoma cell line HepG2 was transfected with dsRNA by liposomes. The expression of mRNA and protein in cells were investigated using real-time reverse real-time quantitative PCR and Western blot, respectively. Cell viability and clonogenicity were determined by MTT assay and clonogenicity assay, respectively. Cell apoptosis was evaluated by flow-cytometric analysis.
  
  
  RESULTS
  Expressions of WT1 mRNA and protein in dsWT1 treated HepG2 cells were significantly elevated. Inhibition of cell viability by dsWT1 was dose-dependent and time-dependent. Reduction of the number and size of colonies formed were found in dsWT1 treated cells. dsWT1 induced significant apoptosis in HepG2 cells. The decreased anti-apoptotic protein Bcl-2 and elevated pro-apoptotic protein Bak expression were detected in dsWT1 treated cells. The level of pro-caspase-3 remarkably decreased and cleaved caspase-3 and PARP fragment were also detected in dsWT1 treated cells.
  
  
  CONCLUSIONS
  These data show that RNAa-mediated overexpression of WT1 may have therapeutic potential in the treatment of hepatocellular carcinoma.
authors.affiliation: Department of General Surgery, Children Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Qin
authors.forename: Yi-Wei
authors.initials: YW
authors.lastname: Lin
authors.forename: Xiang-Yi
authors.initials: XY
authors.lastname: Zheng
authors.forename: Hong
authors.initials: H
authors.lastname: Chen
authors.forename: Qi-Qi
authors.initials: QQ
authors.lastname: Mao
authors.forename: Kai
authors.initials: K
authors.lastname: Yang
authors.forename: Shou-Jiang
authors.initials: SJ
authors.lastname: Huang
authors.forename: Zheng-Yan
authors.initials: ZY
authors.lastname: Zhao
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D053148:Caspase 3 / Q000378:metabolism; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D005434:Flow Cytometry; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D014410:Tumor Stem Cell Assay; D025721:WT1 Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
chemical_list: C494826:BAX protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D025721:WT1 Proteins; D051028:bcl-2-Associated X Protein; D053148:Caspase 3