Cancer Res Treat.  2017 Jan;49(1):230-245. 10.4143/crt.2015.506.

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

• Wang G
• Zhang M
• Li Y
• Zhou J
• Chen L

• KMID: 2367521
• DOI: http://doi.org/10.4143/crt.2015.506

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.

Keyword

Autophagy; Apoptosis; TRIF; LC3; Hepatocellular carcinoma

MeSH Terms

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
Interferons
RNA, Double-Stranded
RNA, Messenger
RNA, Small Interfering
Toll-Like Receptor 3

Figure

• 

  Fig. 1. Expression of Toll-like receptor 3 (TLR3) and microtubule-associated protein light chain 3 (LC3) mRNA and protein in different cells. (A) The location of TLR3 and LC3 proteins in different cells by immunofluorescence (×200). (B) Expression of TLR3 and LC3 mRNA in different cells by quantitative real-time polymerase chain reaction. (C) Expression of TLR3 and LC3 proteins in different cells by western blotting.

• 

  Fig. 2. (A, B) Screening of siRNAs targeting microtubule-associated protein light chain 3 (LC3) mRNA. Among the three siRNAs, siRNA targeted autophagy–related gene LC3 (siLC3)-2 showed a more intense effect in suppression of LC3 mRNA and protein. The “Liposome” in the graph represented HepG2 cells treated only with liposome, and the expression level of this group was taken as 1. *p < 0.05 compared to liposome.

• 

  Fig. 3. The mRNA and protein expression of Toll-like receptor 3 (TLR3), Toll–interleukin-1 receptor domain-containing adaptor inducing interferon β (TRIF), and microtubule-associated protein light chain 3 (LC3) in all testing HepG2 cells detected by quantification real-time polymerase chain reaction (A) and western blotting (B). The expression levels in the untreated group were taken as 1. The experiment was repeated three times with reproducible results. *p < 0.05 compared to untreated cells. siLC3, siRNA targeted autophagy–related gene LC3.

• 

  Fig. 4. Toll–interleukin-1 receptor domain-containing adaptor inducing interferon β (TRIF) and microtubule-associated protein light chain 3 (LC3) protein expression with co-localization detected by immunofluorescence (×100). FITC and TRITC labeled TRIF (green) and LC3 (red) were localized in the cytoplasm of cells, respectively. The nuclei of cells were counter stained by Hoechst in blue and the merged figure was yellow-green. Compared with untreated cells, TRIF expression was markedly increased in siRNA targeted autophagy–related gene LC3 (siLC3)+dsRNA, dsRNA, and siLC3 treated cells, and LC3 expression was markedly decreased in siLC3+dsRNA and siLC3 treated cells. TRIF and LC3 showed co-localized expression in cytoplasm.

• 

  Fig. 5. Transmission electron microscopy detected the autophagosomes. Electron microscopic analysis of testing cells. Arrows indicate autophagosome with double-membrane structures (×15,000). siLC3, siRNA targeted autophagy–related gene LC3.

• 

  Fig. 6. (3-(4,5)-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) detected cell viability in triplicates at different time-points of each experimental group. siLC3, siRNA targeted autophagy–related gene LC3. *p < 0.05 vs. untreated cells.

• 

  Fig. 7. Apoptosis in HepG2 cells detected by flow cytometry (A), Hoechst33285 (B), and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining (C) (arrows indicating apoptotic nuclei were bright blue in Hoechst33285 staining, and bright green in TUNEL staining, ×200). PI, propidium iodide; siLC3, siRNA targeted autophagy–related gene LC3.

• 

  Fig. 8. Effects of dsRNAs and siRNA targeted autophagy–related gene LC3 (siLC3) on the xenograft tumor of nude mice. The xenograft tumor growth of nude mice was significantly slower in days (A) and the tumor nodules were significantly smaller after the mice were sacrificed (B) in order siLC3+dsRNA, dsRNA, and siLC3 compared with phosphate buffered saline (PBS) controls. *p < 0.05 vs. the PBS control group.

• 

  Fig. 9. Results of immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) detection in xenograft tumor tissues (×400). TLR3, Toll-like receptor 3; TRIF3, Toll–interleukin-1 receptor domain-containing adaptor inducing interferon β; LC3, microtubule-associated protein light chain 3; siLC3, siRNA targeted autophagy–related gene LC3.

• 

  Fig. 10. dsRNA and siRNA targeted autophagy–related gene LC3 (siLC3) inhibited angiogenesis in vitro and in vivo. (A) Effects of dsRNA and siLC3 on human umbilical vein endothelial cell (HUVEC) tube-formation (×100). Both dsRNA and siLC3 inhibited the tube formation of HUVECs on matrigel; however, an obvious organized network of tubes was observed in untreated HUVECs. (B) The tumor blood flow changes in B-model ultrasound diagnosis. Compared to the phosphate buffered saline control group with obvious blood flow in tumor and basilar part, blood flow was moderately inhibited in dsRNA and siLC3 alone, while most significantly reduced in siLC3+dsRNA (the red area for flow toward the probe, and the blue area for flow back to the probe). (C) CD34 expressing positive microvessels in immunohistochemistry staining (×400). The results of CD34 expressing microevessels by immunohistochemistry staining were similar to that in HUVEC tube–formation or B-model ultrasound diagnosis.

Reference

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.

2. Levine B, Mizushima N, Virgin HW. Autophagy in immunity and inflammation. Nature. 2011; 469:323–35.

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.

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.

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.

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.

8. Mizushima N, Yoshimori T, Levine B. Methods in mammalian autophagy research. Cell. 2010; 140:313–26.

9. Kroemer G, Levine B. Autophagic cell death: the story of a misnomer. Nat Rev Mol Cell Biol. 2008; 9:1004–10.

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.

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.

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.

13. Mizushima N. Autophagy: process and function. Genes Dev. 2007; 21:2861–73.

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.

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.

16. Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell. 2010; 140:805–20.

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.

19. Yang ZJ, Chee CE, Huang S, Sinicrope FA. The role of autophagy in cancer: therapeutic implications. Mol Cancer Ther. 2011; 10:1533–41.

20. Delgado MA, Elmaoued RA, Davis AS, Kyei G, Deretic V. Toll-like receptors control autophagy. EMBO J. 2008; 27:1110–21.

21. Guo Z, Chen L, Zhu Y, Zhang Y, He S, Qin J, et al. Double-stranded 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.

23. Shi CS, Kehrl JH. MyD88 and Trif target Beclin 1 to trigger autophagy in macrophages. J Biol Chem. 2008; 283:33175–82.

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.