PLK1 promotes epithelial-mesenchymal transition and metastasis of gastric carcinoma cells

Abstract

Cancer cell epithelial-mesenchymal transition (EMT) is the crucial event for cancer progression and plays a vital role in the metastasis of cancer cells. Activation of Polo-like kinase 1 (PLK1) signaling has been implicated as the critical event in several tumor metastasis and EMT, however, whether PLK1 participates in gastric carcinoma metastasis and EMT still remains unclear. For this study, we elucidated the potential physiological function of PLK1 in the metastasis of gastric tumors, as well its distinct role in cells EMT and subsequently determined the mechanism involved in PLK1 regulated. Immunoblotting assay and Oncomine data mining analysis indicated that PLK1 expression was highly up-regulated in gastric carcinoma. Kaplan-Meier survival analysis for the relationship between survival outcomes and PLK1 expression in gastric carcinoma was performed with an online Kaplan-Meier plotter (http://kmplot.com/analysis/). Over-expression of PLK1 in gastric cancer cells SGC-7901 and MKN-28 significantly promoted cells profound morphological changes and enhanced metastatic ability of tumor cells. On the contrary, silencing of PLK1 induced mesenchymal epithelial transition (MET)-like morphological and inhibited the metastatic process. Furthermore, we found that the metastatic characters promoting effects of PLK1 in gastric carcinoma was related to the activation of protein kinase B (AKT). Our mechanistic investigations revealed that AKT inhibition reversed PLK1-induced EMT, blocked gastric carcinoma cells invasiveness and metastasis. Additionally, over-expression of AKT promoted the migratory and invasion ability of the two cell lines, which was disrupted by PLK1 down-regulation. To conclude, our findings demonstrate that PLK1 accelerates the metastasis and epithelial-mesenchyme transition of gastric cancer cells through regulating the AKT pathway.

Keywords: EMT; PLK1; gastric carcinoma; metastasis.

Figure 1

PLK1 is highly expressed in gastric cancer cells. A. PLK1 protein expression in gastric cancer lines (BGC-823, SGC-7901, MKN-45, MKN-28, and MGC-803) and gastric mucosal epithelial cell line (GES-1) was detected by western blotting analysis. GAPDH was used as an internal standard. B. Total RNAs were extracted from gastric cancer cells. PLK1 mRNA level was determined by means of quantitative real-time PCR and normalized to the level of GAPDH mRNA. The fold changes of mRNA expression of PLK1 gene were compared as a ratio to the GES-1 cells. The data are shown as mean ± SD of triplicates experiments. *P < 0.05, **P < 0.01 compared with the GES-1 cells.

Figure 2

PLK1 expression is over-expressed in gastric cancer and correlates with survival time. A. Box plots derived from gene expression data in Oncomine comparing expression of PLK1 gene in normal tissue (left plot) and gastric cancer tissue (right plot). Oncomine box plots were retrieved from serous gastric carcinoma and normal tissue. B. Kaplan-Meier survival analysis for the relationship between survival time and PLK1 signature in gastric cancer was performed by using the online tool (http://kmplot.com/analysis/).

Figure 3

PLK1 overexpression promotes gastric cancer cell mobility. A. PLK1 was cloned into pIRES2-EGFP vector and then transfected into SGC-7901 and MKN-28 cells. Cells transfected with the empty vector were used as control. The transfection efficiency was evaluated by assayed the green fluorescence protein expression. B. After 24 h post transfections, protein samples were subjected to western blot for measuring PLK1. C. Confluent SGC-7901 and MKN-28 cell monolayers were wounded with 100 µl pipette tip. Cell migration to the wound area was monitored by microscope for 24 h post-wound. The percentage of wound area covered by cells was assessed using the ImageJ program. Bars represent the standard deviation of three independent experiments conducted in triplicate. **P < 0.01 compared with untreated control groups. D. SGC7901 and MKN-28 cells (1 × 10⁵ cells/well) were seeded in the upper chamber, and medium containing 10% FBS was added to the lower chamber. After incubation for 6 h, the cells that invaded the lower membrane of the insert were stained with 0.1% crystal violet and counted by microscopy. The data are expressed as the means of three independent experiments ± standard deviation. **P < 0.01 compared with untreated control groups.

Figure 4

PLK1 silencing decreases the migration and invasion of SGC-7901 and MKN-28 cells. A. Cells transfected with the empty vector were used as control cells. The expression of PLK1 was determined western blot with PLK1 antibody. B. RT-PCR analyses showed effective down-regulation of PLK1 following siRNA transfection. C. PLK1- and control-siRNA transfected SGC-7901 and MKN-28 cells were wounded with pipette and wound closure percentage was quantified 24 h after scratch relative to that at 0 h. *P < 0.05, **P < 0.01 compared to the control cells. D. SGC-7901 and MKN-28 cells transfected with PLK1-siRNA or control siRNA were seeded in the upper chamber. After 6 h, cells invaded through the membrane were stained and counted in five random microscopic fields.

Figure 5

PLK1 regulates EMT in gastric cancer cells. A. Overexpression of PLK1 induced EMT in SGC7901 and MKN-28 cells. SGC7901 and MKN-28 cells were infected with empty vector or PLK1, the morphological change was examined after 10 days. B. SGC7901 and MKN-28 cells were infected with empty vector or PLK1 and the expression of the indicated EMT markers was detected by western blotting. GAPDH was used as an internal standard. C. Control siRNA or siRNA against PLK1 were transfected into SGC7901 and MKN-28 cells. After 48 h post transfections, morphology of the transfected cells were examined. D. SGC7901 and MKN-28 cells were infected with control siRNA or siRNA against PLK1 and the expression of the indicated MET markers was detected by western blotting. GAPDH was used as an internal standard.

Figure 6

PLK1 facilities invasion of gastric cancer cells through AKT. A. Western blot result shown that the phosphorylation of AKT at Ser-473 was elevated in cells transfected with pIRES2-EGFP-PLK1. Total AKT expression was used as a loading control. B. The phosphorylation of AKT at Ser-473 was perturbed in cells transfected with PLK1 siRNA plasmid. Total AKT expression was used as a loading control. C. Expression of D2AKT was confirmed by western blot with antibody against AKT, and GAPDH was used as loading control. D. Transwell assay was performed to determine the invasion of cells co-transfected with PLK1 silencing plasmid and D2AKT plasmid. E. In the presence of GSK690693, cells were incubated for 6 h, protein extracts were analyzed by western blot with antibodies against phosphorylated AKT (S473) or AKT. F. In the presence of GSK690693 (2 μM), transwell invasion assay was conducted to evaluate the cell invasiveness after transfection. Representative pictures were taken after staining with crystal violet.