Histone H3 activates caspase-1 and promotes proliferation and metastasis in hepatocellular carcinoma

Abstract

Background: As a component of nucleosomes, histone H3 plays an important role in chromosome structure and gene expression. Current studies have mostly focused on the role of histones in epigenetics, but in addition to this, the role of histones themselves in tumor development and microenvironment have been less explored. Methods: Western blot and immunofluorescence were carried out to detect the content and localization of histone H3 in hepatocellular carcinoma. The changes of histone H3 were observed in hypoxia treatment cells, the specific action mechanism of histone H3 was studied by CoIP and other methods. Cell Counting Kit-8 assay, plate cloning assay and transwell assay were used to exam the effect of histone H3 on cell proliferation and metastasis, which were verified by subcutaneous tumors in mice and lung metastasis by tail vein injection in mice. Results: We found that histone H3 was overexpressed in hepatocellular carcinoma tumor tissues compared to adjacent non-tumor tissues, and there was concomitant translocation of histone H3 from the nucleus to the cytoplasm. We found that hypoxia could contribute to this phenomenon of histone H3 translocation from the nucleus to the cytoplasm in hepatocellular carcinoma cells and increased binding levels to TLR9. At the same time, hypoxia induced downstream activation of TLR9 and caspase-1, as well as cleavage and release of the pro-inflammatory cytokines IL-1β and IL-18. We further demonstrated that histone H3 could also promote proliferation and metastasis of hepatocellular carcinoma through TLR9 activation of NLRP3 inflammasome. In addition, overexpression of histone H3 was also confirmed to promote hepatocellular carcinoma proliferation and metastasis in mouse models of hepatocellular carcinoma growth assay and lung metastasis. Conclusions: In hypoxic hepatocellular carcinoma cells, histone H3 can translocate to the cytoplasm and activate caspase-1 via TLR9, thereby producing pro-inflammatory cytokines that promote tumor proliferation and metastasis.

Keywords: Hepatocellular carcinoma; Histone H3; Hypoxia; NLRP3; TLR9.

Figure 1

Overexpression and translocation of histone H3 in HCC (A) Histone H3 protein levels were measured with western blot in paired HCC tumor tissues (T) and adjacent non-tumor tissues (N). Protein expression results were normalized to internal control β-actin. (B) Representative images of IHC showing histone H3 expression in 30 paired HCC tumor tissues and adjacent non-tumor tissues. **P<0.01. (C) Western blot analyzed the expression of histone H3 in the cytoplasm of HCC tissues and adjacent non-tumor tissues. The expression of histone H3 in cytoplasm was measured with in HCC samples and their adjacent nontumor counterparts. (D) Immunofluorescence staining of histone H3 was performed on adjacent non-tumor tissues and HCC tissues. Red, histone H3; Green, F-actin; blue, nuclei.

Figure 2

Hypoxia leads to the translocation of histone H3 from nucleus to cytoplasm in HCC cells and promotes HCC cells metastasis (A-B) Western blot analysis the expression of histone H3 in whole cell protein, cytoplasmic protein and supernatant of Huh7 and G2 cells after normoxic and hypoxic (1% O₂) culture. (C) Huh7 and G2 were stained by immunostaining after normoxia and hypoxia. Red, histone H3; blue, nuclei. Scale bar = 25 µm. (D) The viability of Huh7 and G2 cells under normoxia and hypoxia for 24h was compared by CCK-8 assay. *P<0.05, **P<0.01, N.S. no significance. (E) Transwell migration and invasion studies were performed for 24h under normoxia and hypoxia. The numbers of migratory and invasive cells were quantified in normoxia and hypoxia of Huh7 and G2 cells. Magnification = 200×, scale bar = 50 µm.

Figure 3

Hypoxia and histone H3 activate NLRP3 inflammasome through TLR9 (A) The expression of HIF-1α, TLR9, NLRP3, Caspase-1, IL-1β and IL-18 were determined by western blot in Huh7 and G2 cells exposed to 24h hypoxia. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, N.S. no significance. (B) The expression of caspase-1 in Huh7 and G2 cells treated with 10 μg/ml anti-Histone H3 neutralizing antibody under hypoxia. (C) The IP results showed that the content of histone H3 bound by TLR9 increased after hypoxia. (D) The content of H3 in cytoplasm of Huh7 and G2 cells treated with 30 μg/ml recombinant human histone H3 for 24 hours. (E) The expression of caspase-1 in Huh7 and G2 cells treated with 30 μg/ml recombinant human histone H3 for diverse times. (F) The expression of TLR9, NLRP3, caspase-1, IL-1β and IL-18 were significantly increased in stable histones H3-expressing cells via western blot analysis. (G) Western blot analysis for caspase-1 from stable histones H3-expressing cells after transfected with TLR9 siRNA. Lv-control+si-control group was stable Lv-control HCC cell to be transfected with si-control; Lv-H3+si-TLR9 group was stable histones H3-expressing cells to be transfected with si-TLR9.

Figure 4

Histones H3 promotes HCC cells proliferation and metastasis through TLR9 in vitro (A) CCK-8 assay was performed to determine the proliferation of G2 and MHCC-97H cells with various treatments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. # represents the comparison and analysis between Lv-control+si-control and Lv-control+si-TLR9; represents the comparison and analysis between Lv-control+si-control and Lv-H3+si-control; * represents the comparison and analysis between Lv-H3+si-control and Lv-H3+si-TLR9. (B) Colony-forming assay was performed to determine the proliferation of G2 and MHCC-97H cells with various treatments. (C) G2 and MHCC-97H cells with various treatments were seeded to migrate or invade for 24h or 48h. The number of migratory and invasive cells was quantified. Magnification = 200×, scale bar = 50 µm.

Figure 5

Histones H3 promotes HCC cells proliferation and metastasis in vivo Tumor growth curves (A) and representative H&E staining (B) shown after stable histones H3-expressing cells were engrafted in flanks of BALB/c nude mice. *P<0.05, ****P<0.0001. (C) showed luciferase signal of control cells (the left flank) and stable histones H3-expressing cells (the right flank) in one BALB/c nude mice. Lung metastasis experiments were performed by tail vein injection model. Statistical table of pulmonary metastasis (D), representative luciferase (E) signal images and H&E staining of lung tissues (F) were presented.