Studying the Effect of Downregulating Autophagy-Related Gene LC3 on TLR3 Apoptotic Pathway Mediated by dsRNA in Hepatocellular Carcinoma Cells.
- Author:
Guilan WANG
1
;
Maona ZHANG
;
Yunlong LI
;
Jiaming ZHOU
;
Li CHEN
Author Information
- Publication Type:Original Article
- Keywords: Autophagy; Apoptosis; TRIF; LC3; Hepatocellular carcinoma
- MeSH: Animals; Apoptosis; Autophagy; Blotting, Western; Carcinoma, Hepatocellular*; Cell Line; Cell Survival; Endothelial Cells; Flow Cytometry; Fluorescent Antibody Technique; Hep G2 Cells; Heterografts; Humans; In Vitro Techniques; Interferons; Mice; Mice, Nude; Microscopy, Electron, Transmission; Microvessels; Real-Time Polymerase Chain Reaction; RNA, Double-Stranded; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Toll-Like Receptor 3; Tumor Burden; Umbilical Veins
- From:Cancer Research and Treatment 2017;49(1):230-245
- CountryRepublic of Korea
- Language:English
- Abstract: PURPOSE: 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. MATERIALS AND 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 used in vitro and in 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). CONCLUSION: 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.
