Upregulation of NDRG1 predicts poor outcome and facilitates disease progression by influencing the EMT process in bladder cancer

Aiwei Li,Yan Qiao,Chanjuan Wang,Jie Zhang,Xi Zhu,Rui Qiao,Shuo Yang

doi:10.1038/s41598-019-41660-w

Abstract

N-myc downstream regulated gene 1 (NDRG1) is an intracellular protein involved in cell differentiation and was recently reported to exert various effects in several cancers. However, its expression and role in bladder cancer remain unclear. Our study enrolled 100 bladder cancer patients to detect NDRG1 expression in tumour tissues by immunohistochemistry. Correlations between NDRG1 expression and clinical factors were analysed. An NDRG1 overexpression plasmid and NDRG1 siRNAs were transfected into bladder cancer cell lines. Cell biological behaviours were assessed by CCK-8, flow cytometry, wound healing and Transwell assays. Additionally, the influence of NDRG1 on epithelial-mesenchymal transition (EMT) was investigated by western blotting and real-time PCR. NDRG1 expression in urine from bladder cancer patients was examined by ELISA. NDRG1 protein levels were significantly increased in bladder cancer patients and correlated with tumour stage (p = 0.025), lymph node metastasis (p = 0.034) and overall survival (p = 0.016). Patients with high NDRG1 expression had poorer outcomes than those with low NDRG1 expression. NDRG1 overexpression was associated with increased cell proliferation, migration, and invasion and decreased apoptotic cell numbers; NDRG1 knockdown resulted in the inverse effects. Moreover, upregulated NDRG1 expression was associated with downregulated Cytokeratin 7 and Claudin-1 expression and upregulated N-cad, β-catenin and slug expression. Downregulated NDRG1 expression was associated with the inverse effects. Urine protein levels could distinguish bladder cancer patients from healthy controls, with an area under the curve of 0.909. NDRG1 promoted EMT in bladder cancer and could be an effective diagnostic and prognostic biomarker in bladder cancer patients.

Highlights

Bladder cancer is the most common tumour of the urinary system worldwide, with an estimated 549,000 new cases in 20181
The results of the real-time PCR and western blot analyses revealed that the N-myc downstream regulated gene 1 (NDRG1) mRNA (p = 0.040) and protein (p = 0.002) expression levels were significantly higher in bladder tumour tissues than in paired tumour-free tissues (Fig. 1A,C)
The area under the curve (AUC) of NDRG1 expression to diagnose bladder cancer was 0.909, with a sensitivity of 84.6% and a specificity of 86.7%

Summary

Introduction

Bladder cancer is the most common tumour of the urinary system worldwide, with an estimated 549,000 new cases in 20181. Some reports have demonstrated that hypoxia-inducible factor 1-alpha (HIF-1α), an indicator of hypoxia, is highly expressed in bladder cancer and that patients with high HIF-1α expression have poor prognoses[7,9]. Reports indicate that NDRG1 is upregulated by HIF-1 and that the NDRG1 protein level could more accurately reflect tumour hypoxia than that of HIF-110,11, suggesting that NDRG1 may play a role in the development of bladder cancer and is a potential biomarker. An event involving the loss of E-cadherin (E-cad) expression and the gain of N-cadherin (N-cad) expression, is a unique pattern of EMT and has been shown to be associated with increased cell invasiveness and poor outcome in patients with bladder cancer[18]. We sought to investigate the expression and function of NDRG1 in bladder cancer and further reveal the ways in which it is involved in EMT

Methods

Results

Conclusion