title: Studying the Effect of Downregulating Autophagy-Related Gene LC3 on TLR3 Apoptotic Pathway Mediated by dsRNA in Hepatocellular Carcinoma Cells.
abstract: OBJECTIVE
  The purpose of this study is to examine the role of the double-stranded RNA (dsRNA) activated Toll-interleukin-1 receptor domain-containing adaptor inducing interferon β (TRIF) signal pathway in triggering apoptosis in hepatocellular carcinoma (HCC) cells.
  
  
  METHODS
  First, siRNA targeted autophagy-related gene LC3 (pU6H1-LC3 siRNA and siLC3) and a dsRNA used as a Toll-like receptor 3 (TLR3) ligand was constructed and synthesized, respectively. Then, a human HCC cell line was transfected with dsRNA, siLC3, and cotransfected with siLC3 and dsRNA (siLC3+dsRNA), respectively. Finally, quantification real-time polymerase chain reaction, western blotting, and immunofluorescence staining were used in the HCC line (SMMC7721), and MTT assay, flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling, and transmission electron microscopy were used in an HCC xenograft model of nude mice. Human umbilical vein endothelial cell tube forming assay, color Doppler ultrasonographic flow image examination, and CD34-positive microvessel density were usedin vitroandin vivo.
  
  
  RESULTS
  Compared with untreated cells, the protein and mRNA expression of TLR3 and TRIF was up-regulated, in order, siLC3+dsRNA, dsRNA, and siLC3. Expression of LC3 was obviously down-regulated and the autophagosomes were significantly decreased in siLC3+dsRNA and siLC3, whereas in dsRNA (p < 0.05). LC3 and TRIF colocation was observed in HepG2 cells. Decreased cell viability, increased apoptosis, decrease in xenograft tumor volume, and angiogenesis potential were also observed in order (p < 0.05).
  
  
  CONCLUSIONS
  Suppression of intracellular autophagy resulted in decreased degradation of TRIF protein, which can promote triggering of apoptosis by the TLR3-TRIF pathway. dsRNA and siLC3 could play anticancer roles in coordination.
authors.affiliation: Department of Pathological Anatomy, Nantong University, Nantong, China.
authors.forename: Guilan
authors.initials: G
authors.lastname: Wang
authors.affiliation: Department of Pathological Anatomy, Nantong University, Nantong, China.
authors.forename: Maona
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Department of Pathological Anatomy, Nantong University, Nantong, China.
authors.forename: Yunlong
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Pathological Anatomy, Nantong University, Nantong, China.
authors.forename: Jiaming
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Department of Pathological Anatomy, Nantong University, Nantong, China.
authors.forename: Li
authors.initials: L
authors.lastname: Chen
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D001343:Autophagy / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D018618:Echocardiography, Doppler, Color; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D051196:Toll-Like Receptor 3 / Q000378:metabolism*; D023041:Xenograft Model Antitumor Assays
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; C470409:TICAM1 protein, human; D051196:Toll-Like Receptor 3
keywords: Apoptosis; Autophagy; Hepatocellular carcinoma; LC3; TRIF