NKILA, a prognostic indicator, inhibits tumor metastasis by suppressing NF-κB/Slug mediated epithelial-mesenchymal transition in hepatocellular carcinoma

Abstract

The metastasis of hepatocellular carcinoma (HCC) is one of the major obstacles hindering its therapeutic efficacy, leading to low surgical resection rate, high mortality and poor prognosis. Accumulating evidence has shown that both long noncoding RNA (lncRNA) and NF-κB play vital roles in the regulation of cancer metastasis. However, the clinical significance and biological function of NKILA (NF-κB interacting lncRNA) and its interaction with NF-κB in HCC remain unknown. In this study, we demonstrated that NKILA was down-regulated in HCC tissues and cell lines, and decreased NKILA expression was significantly associated with larger tumor size and positive vascular invasion in HCC patients. NKILA reduction was an independent risk factor of HCC patients' poor prognosis, and the 5-year overall survival (OS) rates of patients with low and high NKILA expression were 15.6% and 60.0%, respectively. Moreover, NKILA inhibits migration and invasion of HCC cells both in vitro and in vivo. Mechanistically, NKILA prevents Slug/epithelial to mesenchymal transition (EMT) pathway via suppressing phosphorylation of IκBα, p65 nuclear translocation and NF-κB activation. In conclusion, these results indicate that NKILA might serve as an effective prognostic biomarker and a promising therapeutic target against HCC metastasis.

Keywords: Epithelial to mesenchymal transition; Hepatocellular carcinoma; LncRNA-NKILA; Metastasis; NF-κB.

Figure 1

NKILA is down-regulated in HCC and acts as an independent predictor of HCC patients' prognosis. (A) The expression of NKILA in 139 pairs of HCC tissues and corresponding adjacent normal tissues was detected by qRT-PCR. (B) The expression of NKILA in HCC tissues was normalized to that of corresponding noncancerous tissues. The data was shown as log₂(Fold change) = log₂(T_NKILA/N_NKILA). (C) NKILA expression in human immortalized normal hepatocytes L-02 and four human HCC cell lines was detected by qRT-PCR. (D) Kaplan-Meier overall survival curves of 90 HCC patients with low and high NKILA levels. The data was presented as mean ± SD of three independent experiments. ***P < 0.001.

Figure 2

NKILA inhibits migration and invasion of HCC cells in vitro. (A-B) The expression of NKILA in SMMC-7721 and HCC-LM3 cells transfected with lentiviral vector expressing NKILA (overexpression group) or empty vector (negative control group) was detected by qRT-PCR. (C-D) The migration and invasion abilities of SMMC-7721 and HCC-LM3 cells in NKILA overexpression and negative control groups were detected by transwell assay, and images were obtained at 400× magnification. The data was presented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. OE, overexpression; NC, negative control.

Figure 3

NKILA suppresses metastasis of HCC in vivo. (A) Representative images of lung sections stained by H&E in each group of SMMC-7721 cells. (B) The numbers of metastatic lesions in the lungs at 6 weeks after tail vein injection. **P < 0.01. OE, NKILA overexpression; NC, negative control.

Figure 4

NKILA prevents EMT via the inhibition of NF-κB/Slug pathway in HCC. (A-B) EMT and NF-κB pathway related proteins of SMMC-7721 and HCC-LM3 cells in NKILA overexpression and negative control groups were detected by western blot. Relative semi‑quantitative analysis results were presented below. (C) Nuclear p65 of SMMC-7721 cells in NKILA overexpression and negative control groups treated with or without TNF-α or CAPE was detected by western blot. β-actin and Lamin-A were the loading control for cytoplasm and nuclear, respectively. Relative semi‑quantitative analysis results were presented (right panel). n.s., P > 0.05; *P < 0.05, **P < 0.01, ***P < 0.001. OE, overexpression; NC, negative control.

Figure 5

Schematic model of the underlying molecular mechanism of NKILA on HCC metastasis. NKILA inhibits HCC cells migration and invasion by hindering IKK induced IκB phosphorylation, p65 nuclear translocation and NF-κB activation, and subsequently suppressing Slug regulated EMT.