The expression of EMT-related genes in various HCC tissues and cell lines
To elucidate the conjunction between EMT and MVI formation, we first concurrently evaluated the expression profiles of EMT-promoting TFs (Snail, Slug, Twist, Goosecoid, Zeb1, Zeb2, FOXC1, FOXC2) and EMT-regulated genes (E-cadherin, N-cadherin, and Vimentin) in HCC tissues. We found that the tissue with MVI expressed higher levels of FOXC1, Snail, Vimentin and lower levels of E-cadherin when compared with the tissue without MVI (all P < 0.05, Fig.A). Especially, among these up-regulated TFs, the FOXC1 discrepancy was the most significant, increasing by more than 2.5 folds. Thus, we hypothesized that FOXC1 might act as a major modulator of EMT in HCC tissue and a candidate mediator of MVI formation.
Figure 1 Expression profile of EMT-related genes in HCC tissue samples and cell lines. (A) Expression of EMT-related TFs and markers were measured by qRT-PCR in two groups of HCC samples with or without MVI. (B) Expression of FOXC1 and snail proteins was measured (more ...)
We further tested the expression of FOXC1 in 9 HCC cells lines with different metastatic abilities. FOXC1 expression increased approximately in parallel with the metastatic potential of HCC cell lines. Compared with no-metastatic cells (Huh7, HepG2 and Hep-3B), the other cancer cell lines (Bel-7402, SK-Hep1, SMMC-7721, MHCC-97L, MHCC-97H, and MHCC-LM3) consistently expressed higher levels of FOXC1 (Fig.B).
Expression of FOXC1 in HCC tissue and its relationship to clinicopathological parameters
We evaluated the FOXC1 expression using qRT-PCR on 50 paired HCC surgical tissues (tumor tissues and matched adjacent non-tumor liver tissues). Our result showed that nearly 54% of primary HCC tumors expressed higher level of FOXC1 compared with the matched adjacent non-tumor liver tissues (Fig. C), however, the difference was not significant (P=0.668).We next evaluated the relationship between FOXC1 expression and various clinicopathological parameters. FOXC1 expression was significantly correlated with MVI incidence and histological differentiation (P < 0.05, Table ). There was no statistically significant difference in FOXC1 expression by age, gender, liver cirrhosis, HBV, CK19, CK34, serum AFP, and Microsatellite lesion (Table ). To investigate whether FOXC1 was also over-expressed at the protein level in tumor tissue with MVI, western-blot was performed on 15 HCC clinical samples with MVI. As shown in Figure D, FOXC1 protein expression was higher in the tumors, consistent with the results of real-time quantitative PCR.
the relationship between FOXC1 expression and clinicopathological features of 50 patients with hcc.
FOXC1 Knockdown Depressed Mesenchymal Differentiation
To determine the pathological role of FOXC1 in EMT processes of HCC cells, we used knockdown strategy with shRNA in Bel-7402 cell and SK-Hep1 cells, which express high level of FOXC1. FOXC1 expression in the transfected cells was confirmed by qRT-PCR and western-blot (Fig. A, B). We proceeded to test some phenotypic markers with western-blot and found decreased expression of FOXC1 was correlated with repressed expression of mesenchymal markers such as Vimentin and N-cadherin. The expression of epithelial cell-specific protein, however, was only partially affected. Concretely, the expression of E-cadherin, a central marker of epithelial cell phenotype, was not consistently reduced with the down-expression of FOXC1 (Fig. C), Meanwhile, western-blot examination showed that some other epithelial cell-specific proteins such as ZO-1 and claudin-1 were significantly elicited. Some above results were confirmed by confocal microscopy examination in Bel-7402 cells. Moreover, the redistribution of β-catenin without significant change of content was also observed (Fig. C, D), reflecting the reorganization of intercellular junctions
Figure 2 Effect of FOXC1 knockdown on the expression of epithelial and mesenchymal markers. (A) Knockdown of FOXC1 mRNA expression in Bel-7402 and SK-Hep1 through shRNA plasmid transfection was confirmed by qRT-PCR (B) Knockdown of FOXC1 protein in Bel-7402 and (more ...)
Interaction between FOXC1 and EMT-inducer
To further understand the function of FOXC1 in inducing EMT, we tested how down-expression of FOXC1 affected the expression of other TFs. We used western-blot to test the expression level of snail, twist, slug in Bel-7402 and SK-Hep1 cell after FOXC1 knockdown and confirmed none of these TFs was consistently depressed in respond to inhibition of FOXC1 (Fig. A). We further studied whether other TFs modulated the expression of FOXC1. pcDNA3.1(-)-snail, pcDNA3.1(-)-twist or control vector were transfected into Huh7 cells, which expresses low endogenous FOXC1 level, and western-blot was used to confirm the validity of these transfections (Fig. B). We observed that ectopic expression of both snail and twist could induce the expression of FOXC1 (Fig. C). Moreover, the expression level of FOXC1 in Huh7 cells transfected with pcDNA3.1(-)-snail was relatively higher.
Figure 3 Interaction between FOXC1 and other EMT-regulator. (A) Expression of snail, slug and twist protein in Bel-7402 after transfected with control-shRNA or FOXC1-shRNA was examined by western-blot. (B) Over-expression of snail and twist in Huh7 cells after (more ...)
TGF-β1 is one of the most potent EMT-inducer present in tumor microenvironment. Indeed, treatment certain cell with TGF-β1 for a period 12 day can induce an EMT and the expression of some EMT-inducing TFs 18
. Here, we tested the expression of FOXC1 by qRT-PCR in Huh7 cell after treatment with TGF-β1 for 14 days. The elevated expression of FOXC1 was firstly detected on day 8 after TGF-β1 treatment, and increased by more than 5 folds on day 14 (Fig. D). In addition, we detected the expression of TGF-β1, β2, β3 mRNA in Bel-7402 cell after FOXC1 knockdown to determine whether FOXC1 affected, in turn, the expression TGF-β. We found none of these isomers expression was altered in this condition (Fig. E), suggesting that FOXC1 may locate in the TGF-β1-induced downstream.
FOXC1 Knockdown Inhibited the Viability of Bel-7402 Cell
To explore the effect of FOXC1 knockdown on tumor cell growth, we used CCK-8 test to measure cellular viability within 7 days after FOXC1 knockdown and found FOXC1 down-regulation sufficiently decreased the cell viability (Fig. A). Next, we observed a significant reduction in colony formation ability in cells transfected with FOXC1-shRNA (P < 0.05, Fig. B). These observations collectively suggest that FOXC1 depression may regulate cell cycle or apoptosis. Then, we proceeded to use flow cytometer to investigate apoptosis and cell cycle, and found that FOXC1 knockdown induced G1 phase arrest (P < 0.05, Fig. C). In contrast, no significant change of apoptosis rate was observed (P>0.05, Fig. D), suggesting that FOXC1 knockdown inhibited the viability of Bel-7402 cell through inducing G1 phase arrest rather than apoptosis.
Figure 4 Effect of FOXC1 knockdown on HCC cell viability. (A) CCK-8 analysed the growth curve of Bel-7402 cell transfected with control-shRNA or FOXC1-shRNA. (B) Colony formation assay evaluated the cell growth after tranfection. (C) The cell cycle of Bel-7402 (more ...)
FOXC1 Silencing Suppressed Migration and Invasiveness in Bel-7402 Cell
We determined whether down-regulation of FOXC1 rendered altered ability of migration and invasiveness using wound healing assay and transwell assay. In transwell assay, FOXC1-shRNA Bel-7402 cells, migrated into the lower compartment of the migration chamber, were significantly less than control-shRNA Bel-7402 cells (P < 0.05, Fig. A). In wound healing assay, microscopic examination at 12 and 24h revealed a significant delay in the wound closure of Bel-7402 cells after FOXC1 silencing (P < 0.05, Fig. B).
Figure 5 Effect of FOXC1 knockdown on cellular ability of migration and invasion. (A) Transwell invasion assays of Bel-7402 cells after transfected with control-shRNA or FOXC1-shRNA. Images are shown on the left (magnification: ×100), and the quantification (more ...)
Actin cytoskeleton organization and polymerization have a well described role in cell migration. In present study, we used Atto 655-Phalloidin to mark distribution and dynamic changes of the actin cytoskeleton in cells. The decreased fluorescence intensity, which reflecting F-actin content, was observed in FOXC1-shRNA cells (Fig. C). Additionally, inhibition of FOXC1 expression led the stress fibers to be disassembled and disrupted into short fragments with irregular pattern, accompanied by cell rounding (Fig. C).
FOXC1 Silencing Depressed the Expression of Certain MMPs Proteins and VEGF-A
Various MMPs proteins seen to be capable of prompting cancer cell migration ability, we accordingly determined the effect of FOXC1 knockdown on certain MMPs expressions. Our western-blot results confirmed that the expression of MMP1, MMP2, MMP7 and MMP9 were consistently reduced in respond to FOXC1 silencing, suggesting that these four MMPs could be involved in FOXC1-related elevation of migratory potency. In contrast, MMP3, MMP13 were not down-regulated by FOXC1 knockdown (Fig. D). In addition, we also evaluated the alternation of VEGF-A expression, whose predictive role in angiogenesis has been widely understood, and revealed that VEGF-A expression level was significantly decreased after FOXC1 silencing (Fig. D), indicating a possible contribution of FOXC1 to tumor angiogenesis.