Activated hepatic stellate cells promote epithelial-to-mesenchymal transition in hepatocellular carcinoma through transglutaminase 2-induced pseudohypoxia

Figures

• 

  Fig. 1. Activated hepatic stellate cells promote EMT in HCC cells in vivo and in vitro. a Morphologic changes in HCC cells (MHCC97H and HepG2) after co-culture with the hepatic stellate cells LX2 cells (MHCC97H-Co, HepG2-Co) observed under phase-contrast microscope (scale bar, 50 μm); b Invasiveness of HCC cells promoted after co-culture with hepatic stellate cells (scale bar, 50 μm); c Resistance to cisplatin in HCC cells promoted after co-culture with hepatic stellate cells (*P < 0.05, **P < 0.01, mean ± SEM); d Lower E-cadherin and higher Vimentin, Zeb1, and Snail expression levels in HCC cells co-cultured with hepatic stellate cells (western blotting); and e Lower E-cadherin and higher Vimentin expression levels in tumours spawned by combined injection of HCC cells and hepatic stellate cells into nude mice. Haematoxylin and eosin (HE)-stained images and immunostaining of E-cadherin and Vimentin are shown (scale bar, 50 μm). Experiments were repeated at least three times in triplicate

• 

  Fig. 2. Activated hepatic stellate cells promote TGM2 upregulation in HCC cells in vivo and in vitro. a Thirty-five differentially expressed proteins identified in relation to EMT in MHCC97H-Co cells and resultant networks, analysed via Ingenuity Pathways Analysis software; b Heightened TGM2 expression in HCC cells after co-culture with LX2 cells (MHCC97H-Co, HepG2-Co) and activated human hepatic stellate cells (MHCC97H-Co’, HepG2-Co’), respectively (western blotting); c Heightened TGM2 expression (red) in MHCC97H-Co and HepG2-Co shown by immunofluorescence, with DAPI (blue) nuclear staining (scale bar, 50 μm); and d Heightened TGM2 expression in tumour cells following combined injection of MHCC97H and LX2 cells into nude mice. HE-stained images and immunostaining of TGM2 are shown (scale bar, 50 μm). Experiments were repeated at least three times in triplicate

• 

  Fig. 3. TGM2 upregulation promotes EMT in HCC cells in vivo and in vitro. a Enhanced invasiveness of HCC cells (MHCC97H and HepG2) due to lentivirus-mediated TGM2 overexpression (MHCC97H-TGM2 OE and HepG2-TGM2 OE), compared with two HCC cell lines and vector control (MHCC97H-Mock and HepG2-Mock) (scale bar, 50 μm); b Increased resistance to cisplatin in MHCC97H-TGM2 OE and HepG2-TGM2 OE cells, compared with MHCC97H-Mock and HepG2-Mock cells, respectively (*P < 0.05, mean ± SEM); c Lower E-cadherin and higher Vimentin, Zeb1, and Snail expression levels in MHCC97H-TGM2 OE and HepG2-TGM2 OE cells, compared with MHCC97H-Mock and HepG2-Mock cells, respectively (western blotting); d Subcutaneous tumours produced by injecting MHCC97H-TGM2 OE cells were heavier, compared with counterpart MHCC97H-Mock cell injections (*P < 0.05, mean ± SEM). Note lower E-cadherin and higher Vimentin expression levels in tumour cells produced by injecting MHCC97H-TGM2 OE cells into nude mice, compared with counterpart MHCC97H-Mock cell injections. Images of immunostained TGM2, E-cadherin, and Vimentin are shown (scale bar, 50 μm). Experiments were repeated at least three times in triplicate

• 

  Fig. 4. TGM2 knockdown inhibits HCC cells EMT promoted by activated hepatic stellate cells in vivo and in vitro. a Diminished invasiveness of HCC cells (MHCC97H and HepG2) after lentivirus-mediated TGM2 knockdown co-culturing with LX2 cells (MHCC97H-shTGM2-Co and HepG2-shTGM2-Co), compared with two HCC cell lines and vector control co-cultured with LX2 cells (MHCC97H-Mock’-Co and HepG2-Mock’-Co), respectively (scale bar, 50 μm); b Less resistance to cisplatin in MHCC97H-shTGM2-Co and HepG2-shTGM2-Co cells, compared with MHCC97H-Mock’-Co and HepG2-Mock’-Co cells, respectively (*P < 0.05, mean ± SEM); c Higher E-cadherin and lower Vimentin expression levels in MHCC97H-shTGM2-Co and HepG2-shTGM2-Co cells, compared with MHCC97H-Mock’-Co and HepG2-Mock’-Co cells, respectively (western blotting); and d Subcutaneous tumours following injection of MHCC97H-shTGM2 cells and LX2 cells were lighter compared with counterpart injections of MHCC97H-Mock’cells and LX2 cells (*P < 0.05, **P < 0.01, mean ± SEM). Note higher E-cadherin and lower Vimentin expression levels in tumour cells produced by injecting MHCC97H-shTGM2 cells and LX2 cells into nude mice, compared with counterpart injection of…

• 

  Fig. 5. TGM2 upregulation results in the accumulation of HIF-1a under normoxic conditions promoting EMT in HCC cells: PGK1, LDHA, ENOG, and HIF-1a expression levels increased in HCC cells (MHCC97H and HepG2) co-cultured with LX2 cells (a) and increased in MHCC97H-TGM2 OE and HepG2-TGM2 OE cells compared with counterpart cells (western blotting). b, c Heightened HIF-1a expression in tumour cells following combined injection of MHCC97H and LX2 cells into nude mice, and following injection of MHCC97H-TGM2 OE cells, compared with counterpart cells injection (Immunohistochemistry, scale bar, 50 μm); d Heightened HIF-1a expression in both cytoplasm and nucleus of MHCC97H-TGM2 OE and HepG2-TGM2 OE cells, compared with MHCC97H-Mock and HepG2-Mock cells, respectively (western blotting); e Higher E-cadherin and lower Vimentin expression levels in HCC cells after plasmid-mediated HIF-1a knockdown co-culturing with LX2 cells (MHCC97H-Mock’-shHIF-1a-Co and HepG2-Mock’-shHIF-1a-Co), compared with two HCC cell lines subjected to plasmid control co-culturing with LX2 cells (MHCC97H-Mock’-Co and HepG2-Mock’-Co), respectively (western blotting). Note higher E-cadherin and lower Vimentin expression levels…

• 

  Fig. 6. Activated hepatic stellate cells promote TGM2 upregulation in HCC cells through inflammatory signals and heightened TGM2 expression in HCC tissue bodies poorly for clinical outcomes: a, b Levels of IL-6 and TGF-β1 released in supernatant of co-cultured HCC cells and hepatic stellate cells proved significantly higher than levels produced by HCC cells alone (enzyme-linked immunosorbent assay) (*P < 0.05, **P < 0.01, mean ± SEM, experiments were repeated three times in triplicate); c, d TGM2 upregulation in HCC cells after IL-6 treatment (0, 50, and 250 pg/mL) and TGF-β1 treatment (0, 300, and 1500 pg/mL), with TGM2 knockdown inhibiting rise in E-cadherin and fall in Vimentin expression levels promoted in HCC cells by IL-6 and TGF-β1 treatment (*P < 0.05, **P < 0.01, mean ± SEM, experiments were repeated three times in triplicate); e Heightened (vs. low-level) postoperative TGM2 expression linked with significantly worse recurrence-free and overall survival in patients with HCC (scale bar, 50 μm). Log-rank P-values are shown; and f Schematic illustration of EMT promotion in HCC cells by activated hepatic stellate cells through TGM2 upregulation