Periostin involved in the activated hepatic stellate cells-induced progression of residual hepatocellular carcinoma after sublethal heat treatment: its role and potential for therapeutic inhibition

Figures

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  Fig. 1. Activated HSCs or HSC-CM promoted the proliferation, migration, invasion, colony formation, and decreased the apoptosis of heat-exposed residual HCC cells. a When heat-treated residual HCC cells (Huh7, MHCC97H, Hep3B and HepG2) were co-cultured with activated HSCs LX2, proliferation was significantly higher than the cells cultured alone, as analyzed by WST-1 assay. b, c Proliferation of heat-treated residual HCC cells cultured in HSC-CM from LX2 cells was significantly higher than the cells cultured with control medium, as indicated by the WST-1 assay, BrdU-ELISA and growth curves. d PCNA and cyclinD1 mRNA expression of heat-treated residual HCC cells cultured in HSC-CM from LX2 cells were significantly higher than the cells cultured with control medium, as measured by qRT-PCR. e PCNA protein expression was analyzed by western blot. f Heat-treated residual HCC cells displayed distinct spindle-like appearance when cultured with HSC-CM from LX2 cells. g HSC-CM from LX2 cells promoted the in vitro migration and invasion of heat-treated residual HCC cells. The number of migrated and invaded cells was counted. h Colony formation of heat-treated residual HCC cells cultured in HSC-CM…

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  Fig. 2. POSTN in HSC-CM mediated the increased proliferation, migration, and invasion of heat-exposed residual HCC cells. a, b The alterations of Snail, Vimentin, E-cadherin, N-cadherin, PCNA protein expression in heat-exposed residual HCC cells cultured with POSTN-depleting HSC-CM from LX2 cells or pHSC cells. c Distinct spindle-like appearance of heat-treated residual HCC cells (MHCC97H and HepG2) induced by exogenous POSTN (100 ng/mL). d After treated with exogenous POSTN, heat-exposed residual HepG2 cells showed significantly higher levels of Ki-67, cyclinD1, PCNA, Snail mRNA expression in a dose-dependent manner, as measured by qRT-PCR. e, f After treated with exogenous POSTN, heat-exposed residual HCC cells (Huh7, MHCC97H, HepG2 and Hep3B) showed significantly higher levels of cyclinD1, Snail, PCNA, N-cadherin, Vimentin and markedly lower level of E-cadherin compared to the control, as detected by qRT-PCR and western blot. pHSC primary hepatic stellate cells. **P < 0.01; *P < 0.05

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  Fig. 3. POSTN induced the Shc-ERK activation of heat-exposed residual HCC cells through integrin β1. a The mRNA expression profile of heat-treated residual MHCC97H cells in response to POSTN was illustrated as a heatmap. Red, green represent high and low mRNA expression. b With POSTN treatment, the phosphorylated of p52Shc and ERK1/2 in heat-exposed residual HCC cells (MCHCC97H and HepG2) were significantly increased in a time-dependent manner. c PPI network analysis of the differentially expressed genes identified Shc as a gene of biological importance in POSTN-mediated signaling networks and a diagram illustrated the interaction of Shc with the molecules (e.g., ITGB1 and MAPK1). d When heat-exposed residual HCC cells (MCHCC97H and HepG2) were treated with POSTN, the levels of PCNA, N-cadherin and ERK1/2 phosphorylation were significantly increased. ERK1/2 inhibitor (U0126, 25 μM) reversed the above POSTN-induced increase. e With the stimulation of exogenous POSTN, the levels of Ki-67, PCNA and Snail mRNA expression were significantly decreased in heat-exposed residual integrin β1-knockdown MHCC97H cells. f Expression of POSTN in HCC tissues (n = 374) than that of adjacent non-tumor…

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  Fig. 4. Activated HSCs promoted the in vivo tumor progression of heat-treated residual HCC cells. a HSCs cells were found in the tumors at 2 weeks after the inoculation of heat-treated residual MHCC97H cells with CFSE-labelled pHSCs. b, c Up-regulation of Ki-67, PCNA, cyclin D1 and Snail mRNA expression was found in the tumors formed from heat-exposed residual MHCC97H and pHSCs compared with the tumors from heat-exposed residual MHCC97H alone, as detected by qRT-PCR. The protein expression of PCNA and E-Cadherin was shown by immunohistochemical analysis. d Xenograft tumorigenicity assay. Heat-exposed residual MHCC97H (2 × 104 cells) with 100 ng/mL POSTN subcutaneously inoculated into NOD/SCID mice developed more and larger tumors compared to the cells injected alone. Red arrows on the left flank represent the tumors formed by heat-exposed residual MHCC97H cells with 100 ng/mL POSTN while black arrows on the right flank represent the tumors formed by heat-exposed residual MHCC97H cells alone. The number and images of tumors developed in mice after 8 weeks are shown. The two groups were compared by using Fisher exact test. e The mRNA expression of NANOG, CD133, EpCAM was significantly…

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  Fig. 5. VDR regulates the expression of POSTN in presence of calcipotriol. a Vitamin D receptor (VDR) expression in activated HSCs (LX2 and pHSC) and HCC cell lines (MHCC97H, Hep3B, HepG2 and Huh7) was detected by Western blot. b The markers and activities of activated HSCs (α-SMA, COL1A1, COL1A2, Ki-67, and POSTN) were down-regulated in LX2 cells and pHSC cells by calcipotriol treatment, as analyzed using qRT-PCR. c Protein expression of α-SMA, POSTN and collagen-1 were reduced in LX2 cells and pHSC cells by calcipotriol treatment, as measured by western blot. d Results of Luciferase reporter assay for POSTN promoter constructs. A ~ 2-kb-long POSTN promoter and its serial deletion constructs were measured in HEK 293T cells and results of the firefly luciferase were normalized to the Renilla luciferase activity. e Luciferase activity of mutant reporter plasmids that the two putative VDR transcription factor binding sites were altered by site-directed mutagenesis in the full-length POSTN promoter. Calcipotriol, Cal. VDR, VDR-expressing plasmid. **P < 0.01; *P < 0.05

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  Fig. 6. Calcipotriol suppressed the activated HSCs-induced tumor progression of heat-treated residual HCC cells in vivo. a, b The changes of Snail, Vimentin, E-cadherin and N-cadherin, PCNA expression in heat-exposed residual HCC cells (MHCC97H, Hep3B, HepG2 and Huh7) cultured with conditioned medium (CM) from HSCs or calcipotriol-treated HSCs were compared. HSCs cells (LX2 and pHSC) were stimulated by the pre-treatment with TGF-β1. c The changes of Snail, Vimentin, E-cadherin and N-cadherin, PCNA expression in heat-treated residual HCC cells were reversed when CM from calcipotriol-treated LX2 cells was supplemented with 100 ng/mL POSTN. d Calcipotriol inhibited tumor growth compared with the control group. The combination therapy of calcipotriol and cisplatin significantly reduced tumor growth. e The expression of POSTN, α-SMA, PCNA, E-Cadherin and cleaved-Caspase-3 was detected by immunohistochemical analysis. Calcipotriol, Cal. **P < 0.01; *P < 0.05

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  Fig. 7. A proposed graphical diagram that activated HSCs promote tumor progression of heat-treated residual HCC cells via POSTN secretion which could be inhibited by calcipotriol