Down-regulation of EGFL8 regulates migration, invasion and apoptosis of hepatocellular carcinoma through activating Notch signaling pathway

Abstract

Abstract Background Our previous studies have reported the down-regulation of EGFL8 correlates to the development and prognosis of colorectal and gastric cancer. The present study is carried out to explore the expression pattern and role of EGFL8 in hepatocellular carcinoma (HCC). Methods and materials EGFL8 expression in 102 cases of HCC tissues matched with adjacent non-tumorous liver tissues, a normal liver cell line and three liver cancer cell lines with different metastatic capacity was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot. Moreover, the clinicopathological features and prognosis of HCC patients were correlated with expression of EGFL8. Subsequently, the gain-and loss-of-function experiments were carried out to investigate the biological function of EGFL8 in HCC. We also used N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-(S)- phenylglycine t-butyl ester (DAPT), an inhibitor for Notch signaling pathway, in these experiments to verify the involvement of Notch signaling pathway in the effects of EGFL8. Additionally, a mouse model was established to investigate the effect of EGFL8 on metastasis of HCC cells. The expression of Notch signaling pathway in HCC cells and xenograft mouse tumors were detected by Western blot and immunohistochemistory. Results The expression of EGFL8 was significantly decreased in HCC tissues and cell lines and EGFL8 down-regulation correlated to multiple nodules, vein invasion, high TNM stage and poor prognosis of HCC. Interestingly, the expression levels of EGFL8 in three liver cancer cell lines were negatively associated with their metastatic capacity. In vitro and in vivo experiments indicated that EGFL8 obviously suppressed metastasis and invasion of HCC cells but slightly promoted apoptosis. Meanwhile, the expression of Notch signaling pathway was obviously suppressed in EGFL8 overexpressed HCCLM3 cells and xenograft mouse tumors generated from these cells but markedly elevated in EGFL8 depleted Hep3B cells. Furthermore, the up-regulated expression of Notch signaling pathway and effects induced by EGFL8 knockdown in Hep3B cells could be counteracted by DAPT treatment. Conclusion The down-regulation of EGFL8 was correlated to progression and poor prognosis of HCC and regulates HCC cell migration, invasion and apoptosis through activating the Notch signaling pathway, suggesting EGFL8 as a novel therapeutic target and a potential prognostic marker for HCC.

Keywords

• EGFL8
• Hepatocellular carcinoma
• Migration
• Invasion
• Apoptosis
• Notch signaling pathway