DOI QR코드

DOI QR Code

Studying the Effect of Downregulating Autophagy-Related Gene LC3 on TLR3 Apoptotic Pathway Mediated by dsRNA in Hepatocellular Carcinoma Cells

  • Wang, Guilan (Department of Pathological Anatomy, Nantong University) ;
  • Zhang, Maona (Department of Pathological Anatomy, Nantong University) ;
  • Li, Yunlong (Department of Pathological Anatomy, Nantong University) ;
  • Zhou, Jiaming (Department of Pathological Anatomy, Nantong University) ;
  • Chen, Li (Department of Pathological Anatomy, Nantong University)
  • Received : 2015.12.30
  • Accepted : 2016.05.12
  • Published : 2017.01.15

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 ${\beta}$ (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.

Acknowledgement

Supported by : Science Foundation of Nantong City, Science Foundation of Jiangsu Higher Education Institutions, Nantong University

References

  1. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 2000;19:5720-8. https://doi.org/10.1093/emboj/19.21.5720
  2. Levine B, Mizushima N, Virgin HW. Autophagy in immunity and inflammation. Nature. 2011;469:323-35. https://doi.org/10.1038/nature09782
  3. Chen GY, Yang HJ, Lu CH, Chao YC, Hwang SM, Chen CL, et al. Simultaneous induction of autophagy and toll-like receptor signaling pathways by graphene oxide. Biomaterials. 2012;33:6559-69. https://doi.org/10.1016/j.biomaterials.2012.05.064
  4. Chen L, Xu YY, Zhou JM, Wu YY, E Q, Zhu YY. TLR3 dsRNA agonist inhibits growth and invasion of HepG2.2.15 HCC cells. Oncol Rep. 2012;28:200-6.
  5. Han KJ, Su X, Xu LG, Bin LH, Zhang J, Shu HB. Mechanisms of the TRIF-induced interferon-stimulated response element and NF-kappaB activation and apoptosis pathways. J Biol Chem. 2004;279:15652-61. https://doi.org/10.1074/jbc.M311629200
  6. McAllister CS, Lakhdari O, Pineton de Chambrun G, Gareau MG, Broquet A, Lee GH, et al. TLR3, TRIF, and caspase 8 determine double-stranded RNA-induced epithelial cell death and survival in vivo. J Immunol. 2013;190:418-27. https://doi.org/10.4049/jimmunol.1202756
  7. Amaravadi RK, Lippincott-Schwartz J, Yin XM, Weiss WA, Takebe N, Timmer W, et al. Principles and current strategies for targeting autophagy for cancer treatment. Clin Cancer Res. 2011;17:654-66. https://doi.org/10.1158/1078-0432.CCR-10-2634
  8. Mizushima N, Yoshimori T, Levine B. Methods in mammalian autophagy research. Cell. 2010;140:313-26. https://doi.org/10.1016/j.cell.2010.01.028
  9. Kroemer G, Levine B. Autophagic cell death: the story of a misnomer. Nat Rev Mol Cell Biol. 2008;9:1004-10. https://doi.org/10.1038/nrm2529
  10. Inomata M, Niida S, Shibata K, Into T. Regulation of Toll-like receptor signaling by NDP52-mediated selective autophagy is normally inactivated by A20. Cell Mol Life Sci. 2012;69:963-79. https://doi.org/10.1007/s00018-011-0819-y
  11. Xu Y, Jagannath C, Liu XD, Sharafkhaneh A, Kolodziejska KE, Eissa NT. Toll-like receptor 4 is a sensor for autophagy associated with innate immunity. Immunity. 2007;27:135-44. https://doi.org/10.1016/j.immuni.2007.05.022
  12. Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol. 2010;11:373-84. https://doi.org/10.1038/ni.1863
  13. Mizushima N. Autophagy: process and function. Genes Dev. 2007;21:2861-73. https://doi.org/10.1101/gad.1599207
  14. Biswas N, Liu S, Ronni T, Aussenberg SE, Liu W, Fujita T, et al. The ubiquitin-like protein PLIC-1 or ubiquilin 1 inhibits TLR3-Trif signaling. PLoS One. 2011;6:e21153. https://doi.org/10.1371/journal.pone.0021153
  15. Salaun B, Coste I, Rissoan MC, Lebecque SJ, Renno T. TLR3 can directly trigger apoptosis in human cancer cells. J Immunol. 2006;176:4894-901. https://doi.org/10.4049/jimmunol.176.8.4894
  16. Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell. 2010;140:805-20. https://doi.org/10.1016/j.cell.2010.01.022
  17. Yamamoto M, Takeda K. Current views of toll-like receptor signaling pathways. Gastroenterol Res Pract. 2010;2010:240365.
  18. Yang Y, Liao B, Wang S, Yan B, Jin Y, Shu HB, et al. E3 ligase WWP2 negatively regulates TLR3-mediated innate immune response by targeting TRIF for ubiquitination and degradation. Proc Natl Acad Sci U S A. 2013;110:5115-20. https://doi.org/10.1073/pnas.1220271110
  19. Yang ZJ, Chee CE, Huang S, Sinicrope FA. The role of autophagy in cancer: therapeutic implications. Mol Cancer Ther. 2011;10:1533-41. https://doi.org/10.1158/1535-7163.MCT-11-0047
  20. Delgado MA, Elmaoued RA, Davis AS, Kyei G, Deretic V. Toll-like receptors control autophagy. EMBO J. 2008;27:1110-21. https://doi.org/10.1038/emboj.2008.31
  21. Guo Z, Chen L, Zhu Y, Zhang Y, He S, Qin J, et al. Doublestranded RNA-induced TLR3 activation inhibits angiogenesis and triggers apoptosis of human hepatocellular carcinoma cells. Oncol Rep. 2012;27:396-402.
  22. Xu YY, Chen L, Zhou JM, Wu YY, Zhu YY. Inhibitory effect of dsRNA TLR3 agonist in a rat hepatocellular carcinoma model. Mol Med Rep. 2013;8:1037-42. https://doi.org/10.3892/mmr.2013.1646
  23. Shi CS, Kehrl JH. MyD88 and Trif target Beclin 1 to trigger autophagy in macrophages. J Biol Chem. 2008;283:33175-82. https://doi.org/10.1074/jbc.M804478200
  24. Nomi N, Kodama S, Suzuki M. Toll-like receptor 3 signaling induces apoptosis in human head and neck cancer via survivin associated pathway. Oncol Rep. 2010;24:225-31.