Cancer Res Treat.  2021 Apr;53(2):506-527. 10.4143/crt.2020.397.

Hepatitis B Virus Core Protein Mediates the Upregulation of C5α Receptor 1 via NF-κB Pathway to Facilitate the Growth and Migration of Hepatoma Cells

• Kong F
• Tao Y
• Yuan D
• Zhang N
• Li Q
• Yu T
• Yang X
• Kong D
• Ding X
• Liu X
• You H
• Zheng K
• Tang R

• KMID: 2514932
• DOI: http://doi.org/10.4143/crt.2020.397

Abstract

Purpose
C5α receptor 1 (C5ΑR1) is associated with the development of various human cancers. However, whether it is involved in the development of hepatitis B virus (HBV)–related hepatocellular carcinoma (HCC) is poorly understood. We explored the expression, biological role, and associated mechanisms of C5AR1 in HBV-related hepatoma cells.
Materials and Methods
The expression of C5ΑR1 mediated by HBV and HBV core protein (HBc) was detected in hepatoma cells. The function of nuclear factor кB (NF-κB) pathway in HBc-induced C5AR1 expression was assessed. The roles of C5ΑR1 in the activation of intracellular signal pathways, the upregulation of inflammatory cytokines, and the growth and migration of hepatoma cells mediated by HBc, were investigated. The effect of C5α in the development of HCC mediated by C5AR1 was also measured.
Results
C5ΑR1 expression was increased in HBV-positive hepatoma cells. Dependent on HBc, HBV enhanced the expression of C5ΑR1 at the mRNA and protein levels. Besides, HBc could promote C5ΑR1 expression via the NF-κB pathway. Based on the C5ΑR1, HBc facilitated the activation of JNK and ERK pathways and the expression and secretion of interleukin-6 in hepatoma cells. Furthermore, C5ΑR1 was responsible for enhancing the growth and migration of hepatoma cells mediated by HBc. Except these, C5α could promote the malignant development of HBc-positive HCC via C5AR1.
Conclusion
We provide new insight into the mechanisms of hepatocarcinogenesis mediated by HBc. C5ΑR1 has a significant role in the functional abnormality of hepatoma cells mediated by HBc, and might be utilized as a potential therapeutic target for HBV-related HCC.

Keyword

Hepatitis B virus; HBc; C5AR1; Hepatocellular carcinoma; Growth; Migration

Figure

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  Fig. 1 The role of HBc on C5AR1 expression in HBV-related hepatoma cells. (A) The role of HBV on the expression of complement receptors in hepatoma cells at mRNA levels detected by real-time PCR. (B) The expression of C5AR1 protein mediated by HBV was measured by western blot. (C) The expression of C5AR1 protein in HBV-negative adjacent tissues (n=40), HBV-positive adjacent tissues (n=50), HBV-negative tumor tissues (n=50), and HBV-related tumor tissues (n=65) assessed by IHC analysis (×400). (D) The role of different viral genes on C5AR1 mRNA expression in hepatoma cells that measured by real-time PCR. (E) The role of different viral genes on C5AR1 protein expression in hepatoma cells. (F) The expression of C5 AR1 mRNA in HBc stably-transfected HCC cells, which transfected with Flag-HBc plasmid and selected by G418. (G) The expression of C5AR1 protein in HBc-positive hepatoma cells. (H) The correlation of C5AR1 protein and HBc protein in HBV-related tumor tissues assessed by IHC analysis, the tumor tissues with low HBc expression (n=24), the tumor tissues with high HBc expression (n=41) (×400). C5AR1, C5α receptor 1; HBc, the cells transfected with HBc plasmids; HBV, the cells transfected with hepatitis B virus (HBV) plasmids; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; Mock, the cells transfected with control plasmids; PCR, polymerase chain reaction. *p < 0.05.

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  Fig. 2 The effect of the NF-κB pathway in activating the promoter of the C5AR1 mediated by HBc in hepatoma cells. (A) The identification of the regulatory region of C5AR1 promoter mediated by HBc via luciferase reporter gene assay. (B) The information of potential NF-κB and Sp1 binding site in C5AR1 promoter in PGL3-P (−233/+100) plasmid. (C) The activation of p65 in HBc-positive hepatoma cells and control cells. (D) The expression of Sp1 in HBc-positive hepatoma cells and control cells. (E) The effect of inhibition of p65 via BAY11-7082 on C5AR1 protein expression in hepatoma cells. (F) The effect of inhibition of Sp1 via Mithramycin A on C5AR1 protein expression in hepatoma cells. (G) The location of p65 in HBc-positive hepatoma cells and control cells. (H) The effect of inhibition of NF-κB via BAY11-7082 on the activity of C5AR1 promoter in PGL3-P (−233/+100) plasmid in HBc-positive hepatoma cells. (I) The information on the mutations in the NF-κB binding site in PGL3-P (−233/+100) plasmid. (J) The influence of the mutation in the NF-κB binding site on activation of C5AR1 promoter in PGL3-P (−233/+100) plasmid. (K) The inhibition of p65 using shRNA plasmids on the expression of p65 protein in HBc-positive hepatoma cells. (L) The effect of inhibition of p65 via shRNA on the activity of C5AR1 promoter in PGL3-P (−233/+100) plasmid in HBc-positive hepatoma cells. (M) The effect of inhibition of p65 by shRNA on C5AR1 protein expression in hepatoma cells. C5AR1, C5α receptor 1; HBc, cells transfected with HBc plasmid; HBc-shC5AR1, HBc-positive hepatoma cells transfected with shRNA plasmid targeting C5AR1; HBc-shNC, HBc-positive hepatoma cells transfected with shRNA control plasmid; Mock, the cells transfected with control plasmid; NF-κB, nuclear factor κB. *p < 0.05.

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  Fig. 3 The role of C5AR1 in intracellular signal pathways and inflammatory cytokines in HBc-positive hepatoma cells. (A) The inhibition of C5AR1 mediated by shRNA in the expression of C5AR1 protein in HBc-positive hepatoma cells. (B) The effect of inhibition of C5AR1 expression mediated by shRNA on the activation of JNK, ERK, p38, and P13K (AKT) pathway in hepatoma cells stably-transfected with HBc. (C) The effect of inhibition of C5AR1 expression mediated by shRNA on the expression of TNF-α, IL-6, and IL-1β in HBc-positive hepatoma cells. (D) The effect of inhibition of C5AR1 expression mediated by shRNA on the secretion of IL-6 in HBc-positive hepatoma cells. C5AR1, C5α receptor 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HBc, cells transfected with HBc plasmid; HBc-shC5AR1, HBc-positive hepatoma cells transfected with shRNA plasmid targeting C5AR1; HBc-shNC, HBc-positive hepatoma cells transfected with shRNA control plasmid; IL, interleukin; Mock, cells transfected with control plasmid; PI3K, phosphoinositide 3-kinase; TNF-α, tumor necrosis factor α. *p < 0.05.

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  Fig. 4 The role of C5AR1 in the growth and migration of HCC cells mediated by HBc. (A) The effect of inhibition of C5AR1 expression by shRNA on the growth of HBc-positive hepatoma cells detected by CCK-8 assay. (B) The effect of inhibition of C5AR1 expression via shRNA on the growth of HBc-positive hepatoma cells assessed by plate clone formation assay. (C) The role of C5AR1 in the growth of hepatoma cells mediated by HBc in nude mice. (D) The inhibition of C5AR1 expression via shRNA on the migration of HBc-positive hepatoma cells detected by transwell assay. (E) The effect of inhibition of C5AR1 expression using shRNA on the migration of HBc-positive hepatoma cells was examined by wound healing assay. C5AR1, C5α receptor 1; CCK-8, Cell Counting Kit-8; HBc, cells transfected with HBc plasmid; HBc-shC5AR1, the HBc-positive hepatoma cells transfected with shRNA plasmid targeting C5AR1; HBc-shNC, HBc-positive hepatoma cells transfected with shRNA control plasmid; HCC, hepatocellular carcinoma; Mock, cells transfected with control plasmid. In Fig. 4A, *p < 0.05, the Mock group compared with HBc group; #p < 0.05, the HBc-shNC group compared with HBc-shC5AR1 group.

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  Fig. 5 C5α gene expression medicated by HBV or HBc, and the role of C5AR1 intracellular signal pathways and inflammatory cytokines in HBc-positive hepatoma cells stimulated by C5α. (A) The serum levels of C5α in HC, CHB patients, HCC patients with HBV infection (HBV-HCC), and HBV-negative HCC patients (non-HBV-HCC) were detected by ELISA. (B) The role of HBV on the expression of C5α at mRNA levels detected by real-time PCR. (C) The role of HBc on the expression of C5α at mRNA levels. (D) The role of C5AR1 in the C5α-mediated activation of intracellular signaling pathways in HBc-positive hepatoma cells (20 ng/mL). (E) The effect of C5AR1 on C5α-induced expression of TNF-α, IL-6, and IL-1β in HBc-positive hepatoma cells that measured by western blot. (F) The effect of C5AR1 on C5α-induced secretion of IL-6 in HBc-positive hepatoma cells detected by ELISA. C5AR1, C5α receptor 1; CHB, chronic hepatitis B; ELISA, enzyme-linked immunosorbent assay; HBc, cells transfected with HBc plasmid; HBc-shC5AR1, HBc-positive hepatoma cells transfected with shRNA plasmid targeting C5AR1; HBc-shNC, HBc-positive hepatoma cells transfected with shRNA control plasmid; HBV, hepatitis B virus; HC, health controls; HCC, hepatocellular carcinoma; IL, interleukin; Mock, the cells transfected with control plasmid; PCR, polymerase chain reaction; TNF, tumor necrosis factor. *p < 0.05.

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  Fig. 6 The role of C5AR1 in the growth and migration of HBc-positive hepatoma cells stimulated by C5α. (A) CCK-8 assay was used to detect the effect of inhibition of C5AR1 using shRNA on the growth of HBc-positive hepatoma cells stimulated by C5α. (B) Plate clone formation assay was utilized to explore the effect of inhibition of C5AR1 via shRNA on the growth of HBc-positive hepatoma cells stimulated by C5α. (C) Transwell assay was used to assess the inhibition of C5AR1 via shRNA on the migration of HBc-positive hepatoma cells stimulated by C5α. (D) Wound healing assay was utilized to examine the effect of inhibition of C5AR1 by shRNA on the migration of HBc-positive hepatoma cells stimulated by C5α. C5AR1, C5α receptor 1; CCK-8, Cell Counting Kit-8; HBc, cells transfected with HBc plasmid; HBc-shC5AR1, HBc-positive hepatoma cells transfected with shRNA plasmid targeting C5AR1; HBc-shNC, HBc-positive hepatoma cells transfected with shRNA control plasmid; shRNA, short hairpin RNA.

Reference

References

1. Xu W, Yu J, Wong VW. Mechanism and prediction of HCC development in HBV infection. Best Pract Res Clin Gastroenterol. 2017; 31:291–8.

2. Wu CC, Chen YS, Cao L, Chen XW, Lu MJ. Hepatitis B virus infection: defective surface antigen expression and pathogenesis. World J Gastroenterol. 2018; 24:3488–99.

3. Diab A, Foca A, Zoulim F, Durantel D, Andrisani O. The diverse functions of the hepatitis B core/capsid protein (HBc) in the viral life cycle: implications for the development of HBc-targeting antivirals. Antiviral Res. 2018; 149:211–20.

4. Gai X, Zhao P, Pan Y, Shan H, Yue X, Du J, et al. Hepatitis B virus core protein enhances human telomerase reverse transcriptase expression and hepatocellular carcinoma cell proliferation in a c-Ets2-dependent manner. Int J Biochem Cell Biol. 2013; 45:1174–85.

5. Liu W, Guo TF, Jing ZT, Yang Z, Liu L, Yang YP, et al. Hepatitis B virus core protein promotes hepatocarcinogenesis by enhancing Src expression and activating the Src/PI3K/Akt pathway. FASEB J. 2018; 32:3033–46.

6. Du J, Bai F, Zhao P, Li X, Li X, Gao L, et al. Hepatitis B core protein promotes liver cancer metastasis through miR-382-5p/DLC-1 axis. Biochim Biophys Acta Mol Cell Res. 2018; 1865:1–11.

7. Sadik CD, Miyabe Y, Sezin T, Luster AD. The critical role of C5a as an initiator of neutrophil-mediated autoimmune inflammation of the joint and skin. Semin Immunol. 2018; 37:21–9.

8. Woodruff TM, Nandakumar KS, Tedesco F. Inhibiting the C5-C5a receptor axis. Mol Immunol. 2011; 48:1631–42.

9. Ajona D, Zandueta C, Corrales L, Moreno H, Pajares MJ, Ortiz-Espinosa S, et al. Blockade of the complement C5a/C5aR1 axis impairs lung cancer bone metastasis by CXCL16-mediated effects. Am J Respir Crit Care Med. 2018; 197:1164–76.

10. Kaida T, Nitta H, Kitano Y, Yamamura K, Arima K, Izumi D, et al. C5a receptor (CD88) promotes motility and invasiveness of gastric cancer by activating RhoA. Oncotarget. 2016; 7:84798–809.

11. Gu J, Ding JY, Lu CL, Lin ZW, Chu YW, Zhao GY, et al. Overexpression of CD88 predicts poor prognosis in non-small-cell lung cancer. Lung Cancer. 2013; 81:259–65.

12. Nitta H, Murakami Y, Wada Y, Eto M, Baba H, Imamura T. Cancer cells release anaphylatoxin C5a from C5 by serine protease to enhance invasiveness. Oncol Rep. 2014; 32:1715–9.

13. Sayegh ET, Bloch O, Parsa AT. Complement anaphylatoxins as immune regulators in cancer. Cancer Med. 2014; 3:747–58.

14. Hu WH, Hu Z, Shen X, Dong LY, Zhou WZ, Yu XX. C5a receptor enhances hepatocellular carcinoma cell invasiveness via activating ERK1/2-mediated epithelial-mesenchymal transition. Exp Mol Pathol. 2016; 100:101–8.

15. Kong F, You H, Zhao J, Liu W, Hu L, Luo W, et al. The enhanced expression of death receptor 5 (DR5) mediated by HBV X protein through NF-kappaB pathway is associated with cell apoptosis induced by (TNF-alpha related apoptosis inducing ligand) TRAIL in hepatoma cells. Virol J. 2015; 12:192.

16. You H, Kong F, Zhou K, Wei X, Hu L, Hu W, et al. HBX protein promotes LASP-1 expression through activation of c-Jun in human hepatoma cells. J Cell Physiol. 2018; 233:7279–91.

17. Kong F, Hu W, Zhou K, Wei X, Kou Y, You H, et al. Hepatitis B virus X protein promotes interleukin-7 receptor expression via NF-kappaB and Notch1 pathway to facilitate proliferation and migration of hepatitis B virus-related hepatoma cells. J Exp Clin Cancer Res. 2016; 35:172.

18. Kong F, Zhou K, Zhu T, Lian Q, Tao Y, Li N, et al. Interleukin-34 mediated by hepatitis B virus X protein via CCAAT/enhancer-binding protein alpha contributes to the proliferation and migration of hepatoma cells. Cell Prolif. 2019; 52:e12703.

19. Kong F, Li N, Tu T, Tao Y, Bi Y, Yuan D, et al. Hepatitis B virus core protein promotes the expression of neuraminidase 1 to facilitate hepatocarcinogenesis. Lab Invest. 2020; 100:1602–17.

20. Wang H, Shi J, Luo Y, Liao Q, Niu Y, Zhang F, et al. LIM and SH3 protein 1 induces TGFbeta-mediated epithelial-mesenchymal transition in human colorectal cancer by regulating S100A4 expression. Clin Cancer Res. 2014; 20:5835–47.

21. Chen Z, Li YX, Fu HJ, Ren YL, Zou L, Shen SZ, et al. Hepatitis B virus core antigen stimulates IL-6 expression via p38, ERK and NF-κB pathways in hepatocytes. Cell Physiol Biochem. 2017; 41:91–100.

22. Mulero MC, Huxford T, Ghosh G. NF-κB, IκB, and IKK: integral components of immune system signaling. Adv Exp Med Biol. 2019; 1172:207–26.

23. Zhang JX, Zha WS, Ye LP, Wang F, Wang H, Shen T, et al. Complement C5a-C5aR interaction enhances MAPK signaling pathway activities to mediate renal injury in trichloroethylene sensitized BALB/c mice. J Appl Toxicol. 2016; 36:271–84.

24. Verghese DA, Chun N, Paz K, Fribourg M, Woodruff TM, Flynn R, et al. C5aR1 regulates T follicular helper differentiation and chronic graft-versus-host disease bronchiolitis obliterans. JCI Insight. 2018; 3:e124646.

25. Chen J, Li GQ, Zhang L, Tang M, Cao X, Xu GL, et al. Complement C5a/C5aR pathway potentiates the pathogenesis of gastric cancer by down-regulating p21 expression. Cancer Lett. 2018; 412:30–6.

26. Song N, Li P, Jiang Y, Sun H, Cui J, Zhao G, et al. C5a receptor1 inhibition alleviates influenza virus-induced acute lung injury. Int Immunopharmacol. 2018; 59:12–20.

27. Kanda T, Wu S, Sasaki R, Nakamura M, Haga Y, Jiang X, et al. HBV core protein enhances cytokine production. Diseases. 2015; 3:213–20.

28. Zhou RS, Wang XW, Sun QF, Ye ZJ, Liu JW, Zhou DH, et al. Anticancer effects of emodin on HepG2 cell: evidence from bioinformatic analysis. Biomed Res Int. 2019; 2019:3065818.

29. Mak LY, Wong DK, Seto WK, Lai CL, Yuen MF. Hepatitis B core protein as a therapeutic target. Expert Opin Ther Targets. 2017; 21:1153–9.

30. Xiang A, Ren F, Lei X, Zhang J, Guo R, Lu Z, et al. The hepatitis B virus (HBV) core protein enhances the transcription activation of CRE via the CRE/CREB/CBP pathway. Antiviral Res. 2015; 120:7–15.

31. Maeda Y, Kawano Y, Wada Y, Yatsuda J, Motoshima T, Murakami Y, et al. C5aR is frequently expressed in metastatic renal cell carcinoma and plays a crucial role in cell invasion via the ERK and PI3 kinase pathways. Oncol Rep. 2015; 33:1844–50.

32. Torresi J, Tran BM, Christiansen D, Earnest-Silveira L, Schwab RHM, Vincan E. HBV-related hepatocarcinogenesis: the role of signalling pathways and innovative ex vivo research models. BMC Cancer. 2019; 19:707.

33. Jekarl DW, Lee S, Kwon JH, Nam SW, Kim M, Kim Y, et al. Complex interaction networks of cytokines after transarterial chemotherapy in patients with hepatocellular carcinoma. PLoS One. 2019; 14:e0224318.

34. Yang YM, Kim SY, Seki E. Inflammation and liver cancer: molecular mechanisms and therapeutic targets. Semin Liver Dis. 2019; 39:26–42.

35. Zaal A, van Ham SM, Ten Brinke A. Differential effects of anaphylatoxin C5a on antigen presenting cells, roles for C5aR1 and C5aR2. Immunol Lett. 2019; 209:45–52.

36. Brandolini L, Grannonico M, Bianchini G, Colanardi A, Sebastiani P, Paladini A, et al. The novel C5aR antagonist DF3016A protects neurons against ischemic neuroinflammatory injury. Neurotox Res. 2019; 36:163–74.