Transcription factor 3 enhances hepatocellular carcinoma metastasis by upregulating matrix metalloproteinase-11

Figure 1 Elevated transcription factor 3 Levels predict poor prognostic outcomes in hepatocellular carcinoma patients. A: Transcription factor 3 (TCF3) mRNA expression levels in different tumor types and matched non-carcinoma samples from FireBrowse; B: Immunohistochemical staining for TCF3 in hepatocellular carcinoma (HCC) tissues and normal tissues obtain from The Human Protein Atlas. Scale bars: 200 μm; C: TCF3 mRNA expression in 76 paired freshly obtained HCC samples; D: Immunohistochemical staining for TCF3 in HCC tissues. Scale bars: 50 μm (lower panel) and 100 μm (upper panel). The University of ALabama at Birmingham CANcer data analysis Portal analysis for association between TCF3 mRNA levels and clinic-pathological features of liver cancer; E: Tumor grade; F: Nodal metastasis status; G: Tumor stage; H: Kaplan-Meier survival curves revealed the correlation between TCF3 Levels and overall survival (OS) outcomes in HCC patients; I: Kaplan-Meier analysis revealed the correlation between TCF3 mRNA levels and OS for patients with liver HCC in The Cancer Genome Atlas dataset; J and K: Reverse transcription-quantitative polymerase chain reaction and western blot analyses of TCF3 Levels in HCC cell lines. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. HCC: Hepatocellular carcinoma; LIHC: Liver hepatocellular carcinoma; TCF3: Transcription factor 3; TCGA: The Cancer Genome Atlas.

Figure 2 Transcription factor 3 promotes hepatocellular carcinoma cell invasion as well as metastasis. A: Western blotting to verify transcription factor 3 (TCF3) overexpression in Hep3B and TCF3 knockdown in HCCLM3 cells; B: Migration as well as invasions of hepatocellular carcinoma cells as determined by Transwell assays (magnification: × 10); C: Overall survival outcomes for mice in each group; D: Demonstrative hematoxylin and eosin staining images for lung metastases in mice with transfected cells. Scale bars: 100 μm (high magnification) and 1 mm (low magnification); E: Counts of lung metastatic nodules in groups. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. HCC: Hepatocellular carcinoma; Sh: Short hairpin; TCF3: Transcription factor 3.

Figure 3 Matrix metalloproteinase-11 is a direct transcriptional target of transcription factor 3. A: Differential gene expressions (DEGs) between Hep3B-transcription factor 3 (TCF3) and Hep3B-control through RT2 profiler polymerase chain reaction arrays test. The ordinate is a multiple of differences and threshold for DEGs was set as |log2 foldchange (FC)| > 3; B: Protein expression levels of matrix metalloproteinase-11 (MMP11) in treated Hep3B and HCCLM3 cells; C: Luciferase activities of reporter gene driven by the MMP11 promoter in hepatocellular carcinoma cells; D: The TCF3 binding motif logo obtain from JASPAR; E: Luciferase activities of the reporter gene driven by the serially truncated/mutated MMP11 promoter indicates TCF3 binding sites in Hep3B cells; F: Chip assay for binding of TCF3 to MMP11 promoter in Hep3B cells. Y-axis denotes enrichments with anti-TCF3 antibodies vs the immunoglobulin G control. ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. HCC: Hepatocellular carcinoma; IgG: Immunoglobulin G; MMP11: Matrix metalloproteinase-11; Sh: Short hairpin; TCF3: Transcription factor 3.

Figure 4 Matrix metalloproteinase-11 is involved in transcription factor 3 associated hepatocellular carcinoma migration as well as metastasis. A: Western blotting for matrix metalloproteinase-11 Levels in cells; B and C: Transwell assays for cells (magnification: × 10); D and E: Overall survival outcomes for mice in every group; F: Demonstrative hematoxylin and eosin stained lung tissue images. Scale bars: 100 μm (high magnification) and 1 mm (low magnification); G: Counts of lung metastatic nodules in several groups. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. HCC: Hepatocellular carcinoma; MMP11: Matrix metalloproteinase-11; Sh: Short hairpin; TCF3: Transcription factor 3.

Figure 5 Transcription factor 3 expression levels are positively correlated with matrix metalloproteinase-11 Levels in hepatocellular carcinoma. A: Heatmap of transcription factor 3 (TCF3) and matrix metalloproteinase-11 (MMP11) mRNA expression in liver hepatocellular carcinoma (HCC) cases obtained from The Cancer Genome Atlas; B: Correlation between the expression of TCF3 and MMP11 at mRNA levels extracted from cbioportal database; C: Demonstrative immunohistochemical staining images for TCF3 and MMP11 in human HCC tissues. Scale bars: 50 μm (high magnification) and 200 μm (low magnification). HCC: Hepatocellular carcinoma; MMP11: Matrix metalloproteinase-11; TCF3: Transcription factor 3.