Down-regulation of the tumour suppressor κ-opioid receptor predicts poor prognosis in hepatocellular carcinoma patients

Abstract

Background: Opioid receptors have become increasingly implicated in cancer progression and long-term patient outcomes. However, the expression and significance of the κ-opioid receptor (KOR) in hepatocellular carcinoma (HCC) remain unclear.

Methods: In this study, KOR mRNA expression was analysed by real-time quantitative PCR in 64 pairs of HCC tumour tissues and adjacent non-tumour tissues, and KOR protein expression was analysed by immunohistochemistry in 174 HCC patients. We investigated the correlation between KOR expression and clinicopathological parameters to illustrate the potential prognostic significance of KOR expression in HCC.

Results: KOR mRNA expression was significantly down-regulated in 79.69% (51 of 64) of the HCC tumour samples, and KOR expression in tumour tissue was significantly lower than that in adjacent non-tumour tissues (P < 0.001). ROC curve analysis showed that KOR mRNA expression yielded AUC of 0.745, for the detection of HCC patients. Low KOR mRNA expression in HCC was correlated with aggressive clinicopathological parameters, such as tumour size (P = 0.015), differentiation grade (P = 0.011), and TNM stage (P = 0.021). Moreover, down-regulation of KOR protein expression in HCC tissues was detected in 174 HCC patients. Similarly, negative KOR protein expression was significantly correlated with aggressive clinicopathological features, such as tumour size (P = 0.002), vascular invasion (P = 0.003), differentiation grade (P = 0.026), and TNM stage (P = 0.030). Furthermore, Kaplan-Meier survival analysis demonstrated that down-regulation of KOR in HCC indicated poor prognosis. KOR deficiency (KOR^{T < N}) was correlated to a shorter survival rate and an increased recurrence (both P < 0.001). In univariate and multivariate survival analyses, KOR was identified as a promising independent risk factor for both overall survival (OS, both P < 0.001) and recurrence-free survival (RFS, both P < 0.001).

Conclusions: Down-regulation of KOR in HCC tumour tissues has a strong association with poor prognosis and KOR might be a potential tumour suppressor.

Keywords: Hepatocellular carcinoma; Prognosis; Tumour suppressor; κ-opioid receptor.

Fig. 1

Down-regulation of KOR mRNA in HCC. a KOR mRNA expression in 64 paired HCC tumour tissues (T) and corresponding non-tumour tissues (N). The data revealed that down-regulation of KOR was detected in 79.69% (51 of 64) of HCC samples. b Relative expression levels of KOR in tumour tissues are significantly lower than in corresponding non-tumour tissues (P < 0.001). c ROC curve was constructed according to KOR mRNA expression in HCC and adjacent non-tumour tissues

Fig. 2

Down-regulation of KOR protein in HCC. a Representative immunohistochemical staining of KOR protein expression in 174 HCC tissue samples (magnification ×200). Absent or weak staining was defined as negative expression, while moderate or strong staining was defined as positive expression. b The presence of KOR staining in HCC tissues and corresponding non-tumour tissues. c, d Kaplan-Meier analysis for overall survival (OS) and recurrence-free survival (RFS) of 174 HCC patients in correlation with KOR expression. The OS and RFS rates were significantly decreased in KOR-negative HCC patients compared with those in the KOR-positive group (both P < 0.001). The duplicated images in Figs. 2, 3 and Additional file 1: Figure S1 represent the same experiment

Fig. 3

Down-regulation of KOR correlates with poor prognosis in HCC patients. a Representative immunostaining images of KOR loss/gain/retain cases in HCC tumour tissues and adjacent non-tumour tissues (magnification ×200). b Kaplan-Meier curves for OS and RFS according to KOR expression and KOR loss/gain/retain in the validation cohort. The duplicated images in Figs. 2, 3 and Additional file 1: Figure S1 represent the same experiment

Fig. 4

KOR-positive HCC patients in different subgroups showed a better OS than did KOR-negative HCC patients. a The cohort was classified into 2 groups based on AFP levels with a cut-off point of 20 ng/ml: AFP ≤ 20 ng/ml and AFP > 20 ng/ml; b The cohort was classified into 2 groups based on tumour size with a cut-off point of 5 cm: Tumour size ≤5 cm and Tumour size >5 cm; c The cohort was classified into 2 groups based on differentiation grade: Differentiation grade I/II and Differentiation grade III/IV; d The cohort was classified into 2 groups based on TNM stage: TNM stage I and TNM stage II/III

Fig. 5

KOR-positive HCC patients in different subgroups showed a better RFS than the KOR-negative HCC patients. a The cohort was classified into 2 groups based on AFP levels with a cut-off point of 20 ng/ml: AFP ≤ 20 ng/ml and AFP > 20 ng/ml; b The cohort was classified into 2 groups based on tumour size with a cut-off point of 5 cm: Tumour size ≤5 cm and Tumour size >5 cm; c The cohort was classified into 2 groups based on differentiation grade: Differentiation grade I/II and Differentiation grade III/IV; d The cohort was classified into 2 groups based on TNM stage: TNM stage I and TNM stage II/III