GATA5 inhibits hepatocellular carcinoma cells malignant behaviours by blocking expression of reprogramming genes

Abstract

Evidence indicated that GATA5 may suppress hepatocellular carcinoma (HCC) cell malignant transformation, but the mechanism of how GATA5 affects cancer cell reprogramming to inhibit HCC malignant behaviour is still unclear. In this study, we report that the expression of β-catenin and reprogramming genes p-Oct4, Nanog, Klf4, c-myc and EpCAM was significantly higher in HCC tissues compared to normal liver tissues. In contrast, the expression of GATA5 was significantly lower in HCC tissues compared to normal liver tissues. Transfection of CDH-GATA5 vectors into HCC cells (HLE, Bel 7402 and PLC/PRF/5 cells) increased the GATA5 expression and decreased the expression of β-catenin and reprogramming genes p-Oct4, Nanog, Klf4, c-myc and EpCAM. Increased GATA5 expression by transfection with its expression vectors was also able to inhibit the cell growth, colony formation and capability of migration, invasion, while promoting apoptosis in HCC cells. Results revealed that GATA5 co-localization with β-catenin in the cytoplasm, preventing β-catenin from entering the nucleus. Treatment with the specific Wnt/β-catenin pathway inhibitor salinomycin was able to reduce the expression of β-catenin and reprogramming genes. Salinomycin exerted a similar influence as GATA5, and siRNA-GATA5 restored β-catenin and reprogramming gene expression. This study demonstrates that an increase in the expression of GATA5 inhibits the expression of β-catenin and reprogramming genes and suppresses tumour growth, colony formation, metastasis and invasion, while promoting apoptosis in HCC cells. The mechanism of GATA5 inhibiting the malignant behaviours of HCC cells may involve in the disruption of the Wnt/β-catenin pathway and the reduction of reprogramming gene expression.

Keywords: GATA5 expression; HCC; Wnt/β-catenin; reprogramming genes; stemness marker.

Figure 1

The expression of GATA5, β‐catenin and reprogramming genes in human HCC tissues and cells. A, Immunohistochemistry analysis of the expression of β‐catenin and reprogramming genes p‐Oct4, Nanog, Klf4, c‐myc and EpCAM in HCC tissues and normal human liver tissues. Brown staining represents positive expression. B, Western blot analysis of the expression of β‐catenin and reprogramming genes p‐Oct4, Nanog, Klf4, c‐myc and EpCAM in HCC tissues and normal human liver tissues, the right image indicated the expression difference of proteins, *P < 0.05, **P < 0.01 vs control groups, N = 3. C, HLE, Bel 7402 and PLC/PRF/5 cell were transfected with CDH‐GATA5 for 0, 7 and 14 d, and Western blot analysis was performed for the expression of β‐catenin and reprogramming genes p‐Oct4, Nanog, Klf4, c‐myc and EpCAM. Images are representative of three independent experiments

Figure 2

Enhancing GATA5 expression inhibits growth and colony formation in HCC cells. A, Semi‐quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) was applied to analyse the effect of the expression of mRNA of GATA5 while transfected with siRNA‐GATA5 vectors in HCC cells. B, HLE, Bel 7402 and PLC/PRF/5 cell were transfected with CDH empty vector, CDH‑GATA5, siRNA‐GATA5 or siRNA‐scramble for 72 h, and the growth of the cells was measured by MTT assays. C, HLE, Bel 7402 and PLC/PRF/5 cell were transfected with CDH‐GATA5, siRNA‐GATA5 or no transfect (control cells) for 72 h, and colony formation was observed by optical microscopy (100×). P < 0.05 indicates statistical significance

Figure 3

Influence of GATA5 on scratch repair and migration in HCC cells. A, HLE, Bel 7402 and PLC/PRF/5 cells were transfected with CDH empty vector, CDH‐GATA5, siRNA‐GATA5 or siRNA‐scramble for 48 h. Scratch repair was observed by microscopy. The low columnar graph shows repair ratio of the cells, P < 0.05 indicates statistical significance. B, HLE, Bel 7402 and PLC/PRF/5 cells were transfected with CDH‐GATA5, siRNA‐GATA5 or no transfect (control cell) for 72 h. Migratory cells were stained with 0.1% crystal violet and observed by microscopy. The low columnar graph indicates the quantity of migratory cells. P < 0.05 indicates statistical significance

Figure 4

Influence of GATA5 on the expression of MMP2 and MMP9 in HCC cells. A, HLE, Bel 7402 and PLC/PRF/5 cells were transfected with CDH empty vector, CDH‐GATA5 or no transfect (control cells) for 48 h. Western blot analysis of the expression of MMP2 and MMP9 in HLE, Bel 7402 and PLC/PRF/5 cells. B and C, HLE, Bel 7402 and PLC/PRF/5 cell were transfected with pcDNA3.1‐GATA5 or no transfect (control cells) and were then cultured at 37°C in a humidified atmosphere of 5% CO₂. Localization of MMP2 and MMP9 was visualized, and images were captured under laser confocal microscopy. Nuclei are stained with DAPI (blue). MMP2 and MMP9 are labelled with FRITC (red). D, The invasion of the cells was detected while transfected with GATA5 expressed vectors or siRNA vectors in HCC cells. Three independent experiments were performed for these data

Figure 5

Influence of GATA5 and paclitaxel on the growth and apoptosis in HCC cells. A, HLE, Bel 7402 and PLC/PRF/5 cells were transfected with CDH empty vector and CDH‐GATA5 followed by treatment with paclitaxel (20 μg/mL) for 24 h. HCC growth was assessed by MTT P < 0.05 indicates statistical significance, N = 6. B, HLE, Bel 7402 and PLC/PRF/5 cells were transfected with CDH empty vector and CDH‐GATA5 followed by treatment with paclitaxel (20 μg/mL) for 24 h. Apoptosis in HLE cells was assessed by flow cytometry. The low columnar picture is the statistical analysis of the apoptosis ratios. P < 0.05 indicates statistical significance

Figure 6

Localization of β‐catenin and co‐localization of GATA5 and β‐catenin in HCC cells. A, HLE, Bel 7402 and PLC/PRF/5 cells were transfected with pcDNA3.1‐GATA5 or no transfect (control cells), followed by culturing at 37°C in a humidified atmosphere of 5% CO₂. Localization of β‐catenin was visualized, and images were captured under laser confocal microscopy. Nuclei are stained with DAPI (blue). β‐Catenin is labelled with FRITC (red). B, HCC cell lines transfected with pcDNA3.1‐GATA5 were cultured at 37°C in a humidified atmosphere of 5% CO₂. Localization of GATA5 and β‐catenin was visualized. Images were captured under laser confocal microscopy. Nuclei are stained with DAPI (blue). GATA5 and β‐catenin are labelled with Alex488 (green) and Alex647 (red) respectively. C, Co‐IP was applied to analyse GATA5 interact with β‐catenin in HCC cells. Three independent experiments were performed for these data

Figure 7

Influence of GATA5 on the expression of β‐catenin and reprogramming genes in HCC cells. HLE, Bel 7402 and PLC/PRF/5 cells were transfected with CDH‐GATA5, siRNA‐GATA5 or were treated with salinomycin (Sali) for 72 h followed by Western blot analysis of the expression of GATA5, β‐catenin, and reprogramming genes p‐Oct4, Nanog, Klf4, c‐myc and EpCAM. Images are representative of three independent experiments

Figure 8

A schematic showing the role of GATA5 in suppressing the expression of reprogramming genes in HCC cells through blocking the Wnt/β‐catenin signalling. GATA5 decreases expression levels of β‐catenin and reduces its entry into the nucleus, leading to a decline in transcription of reprogramming genes which play critical roles in HCC cell proliferation, metastasis, invasion and drug resistance