Downregulation of EVI1 Expression Inhibits Cell Proliferation and Induces Apoptosis in Hilar Cholangiocarcinoma via the PTEN/AKT Signalling Pathway

Abstract

Aims: Hilar cholangiocarcinoma (HCCA) is a tumour with high malignancy, low surgical resection potential, and a poor prognosis. Ecotropic Viral Integration site 1 (EVI1) is a transcriptional regulator that has been proven to be associated with tumourigenesis and progression in many human solid tumours. However, the expression of EVI1 and its role in HCCA progression remain unclear. The aim of this study was to clarify the association between EVI1 expression and clinical outcomes in patients with HCCA. Methods: The expression of EVI1 in HCCA tissue samples and cell lines was examined by quantitative real-time PCR (qRT-PCR), Western blotting, and immunohistochemistry (IHC). Kaplan-Meier analysis was used for survival analysis. A log-rank test was performed for univariate analysis of survival, and a Cox regression model was utilized for multivariate analysis of survival. Cell proliferation was measured by cell counting kit-8 (CCK-8), colony formation, and 5-ethynyl-2'-deoxyuridine (EdU) assays. The cell cycle was evaluated by flow cytometry. Cell apoptosis was detected by flow cytometry and a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) assay. In vivo tumour growth was observed for xenografts in nude mice. Results: EVI1 expression was upregulated in HCCA tissue samples and correlated with a poor prognosis. In clinical specimens, the expression of EVI1 correlated with tumour histological grade and tumour size. Knocking down EVI1 expression reduced HCCA cell proliferation, blocked cell cycle progression, and promoted apoptosis in vitro and in vivo. Furthermore, we found that EVI1 could regulate the AKT signalling pathway by regulating PTEN levels in HCCA. Conclusion: Our data revealed that EVI1 played important roles in HCCA tumourigenesis and development. Our findings suggest that EVI1 may be a potentially useful therapeutic target in HCCA.

Keywords: Cell proliferation; EVI1; Hilar cholangiocarcinoma; PTEN.

Figure 1

Expression level of EVI1 in HCCA tissue samples and the relationship between EVI1 and overall survival. (A) EVI1 expression in 30 pairs of HCCA tissue and adjacent normal bile duct tissue. (B) Protein expression of EVI1 in HCCA tissue samples detected by Western blotting. (C) Representative immunohistochemical staining for EVI1 in 114 HCC samples. (D) Kaplan-Meier survival analysis of patients with HCCA stratified by EVI1 expression. Overexpression of EVI1 was associated with decreased overall survival in the patients with HCCA. Data are presented as the mean±SEM and were analysed with a paired-sample t test. **P<0.01, N=normal, T=tumour.

Figure 2

EVI1 promotes the growth of HCCA cells. (A) Quantitative real-time PCR (qRT-PCR) and Western blot analyses of EVI1 expression in CCA cell lines and human intrahepatic biliary epithelial cells (HIBEpiC). (B) Knocked down EVI1 expression in QBC939 cells detected by qRT-PCR and Western blotting. (C) FRH0201 cells transduced with an EVI1-overexpression plasmid. The overexpression efficiency was detected by qRT-PCR and Western blotting. (D, F and H) CCK-8 assays, colony formation assays and EdU assays to detect the proliferation of QBC939 cells following shRNA transfection. (E, G and I) FRH0201 cell proliferation analysed by CCK-8, colony formation and EdU assays following transfection with the EVI1-overexpression plasmid. Data are presented as the mean±SEM. **P<0.01, ***P<0.001, Ctrl=without transfection, NC=transfected with a noncoding shRNA, shEVI1= transfected with an EVI1-specific shRNA, and EVI1= transfected with the EVI1-over-expression plasmid.

Figure 3

Knocking down EVI1 expression affects cell cycle progression and apoptosis regulation in QBC939 cells. EVI1 suppresses the protein expression of PTEN and thus modulates the PI3K/AKT pathway. (A) Flow cytometry analysis of the cell cycle was performed to determine the cell cycle distribution after transfection. (B) Western blot analysis of p21, CDK2 and Cyclin A expression in transfected QBC939 cells was performed. (C) Flow cytometry analysis of apoptosis was performed to evaluate cell apoptosis after transfection. (D) A TUNEL assay was carried out to assess apoptosis. (E) BCL2, Bax and Caspase 3 expression was measured by Western blotting. Data are presented as the mean±SEM. **P<0.001, Ctrl=without transfection, NC= transfected with a noncoding shRNA, shEVI1= transfected with an EVI1-specific shRNA.

Figure 4

PTEN expression is inversely correlated with EVI1 expression in human HCCA tissue. (A) PTEN expression was low in EVI1-positive tissue samples and high in EVI 1-negative tissue samples. (B) The correlation between EVI1 and PTEN expression in TMA clinical samples was determined. (C) The correlation between relative EVI1 mRNA expression (the data in Figure 1A) and relative PTEN mRNA expression was determined in 30 pairs of HCCA tissue and adjacent normal bile duct tissue. (D) Comparisons of PTEN and AKT signalling pathways were analysed by Western blotting. (E) QBC939 cells transfected with NC or shEVI1 were treated with siPTEN. After 48 h of treatment, cell lysates were harvested for Western blotting. Western blot analysis showed that the repression of pAKT expression mediated by shEVI1 could be partially rescued by siPTEN. Ctrl=without transfection, NC= transfected with a noncoding shRNA, shEVI1= transfected with an EVI1-specific shRNA, siPTEN= transfected with a PTEN-specific siRNA.

Figure 5

Knocking down EVI1 expression reduces the growth of HCCA tumour xenografts. (A) The tumourigenicity of EVI1 knockdown cells and their counterparts in nude mice was determined. Nude mice were subcutaneously transplanted with cells transfected with NC or shEVI1. Beginning on the third day, tumour volumes were measured every 3 days, and tumour growth curves were plotted until the mice were euthanized on day 21. (B) All animals were sacrificed on the 21st day after tumour cell injection, and the tumours were photographed. (C) Tumours were weighed when the mice were sacrificed. (D) QRT-PCR was used to analyse EVI1 and PTEN expression in the xenografts. (E) EVI1, PTEN, AKT and pAKT expression in the nude mouse tumours was measured by Western blotting. (F) The in vivo relationship between EVI1 and PTEN was shown by immunohistochemical staining of the transplanted tumours from nude mice. Data are presented as the mean±SEM and were analysed with an independent-sample t test. **P<0.01, NC= transfected with a noncoding shRNA, shEVI1= transfected with an EVI1-specific shRNA.