Silencing GTSE-1 expression inhibits proliferation and invasion of hepatocellular carcinoma cells

Abstract

G2 and S phase-expressed-1 (GTSE1) was recently reported to upregulate in several types of human cancer, based on negatively regulate p53 expression. However, its expression and functional roles in hepatocellular carcinoma (HCC) remain unknown. In this study, GTSE1 was observed to be highly expressed in HCC specimens and cell lines both at messenger RNA (mRNA) and protein levels. Furthermore, high GTSE1 expression was positively associated with tumor size, venous invasion, advanced tumor stage, and short overall survival. Moreover, we generated stable GTSE1 knockdown HCC cell lines to explore the effects of GTSE1 silencing on the growth and invasion of HCC in vitro. In determining the pathway through which GTSE1 regulated cell proliferation and invasion, GTSE1 silencing was found to inhibit AKT phosphorylation and downregulated cell cycle-related protein. In addition, GTSE1 downregulation decreased the growth of xenografts. In conclusion, these results indicated for the first time that overexpression of GTSE1 was involved in the progress of HCC, enhancing proliferation and promoting cell invasion in HCC cells.

Keywords: GTSE1; HCC; Invasion; Prognosis; Proliferation.

Fig. 1

Upregulation of GTSE1 in HCC. a qRT-PCR analysis of mRNA levels of GTSE1 in 76 paired of HCC tissues and adjacent non-cancerous tissues (NC tissues). b The mRNA level of GTSE1 was quantified in four HCC cells and a non-malignant liver cell (L02). c The protein level of GTSE1 was determined in four HCC cells and a non-malignant liver cell (LO2) by western blot assays. GAPDH was used as an internal control. *P < 0.05, **P < 0.01

Fig. 2

High GTSE1 expression was associated with poor prognosis in HCC. a Immunohistochemistry of GTSE1 protein expression in non-cancerous tissues and HCC specimens. GTSE1-negative staining in normal tissue is shown in the left, whereas strong GTSE1-positive staining in HCC is presented in the right. b Kaplan–Meier survival curve was performed to reveal that patients with high GTSE1 expression suffered the worse outcome

Fig. 3

Silencing of GTSE1 inhibited HCC cell growth. a Western blots were performed to confirm GTSE1 stably downregulated in 97H and LM3 cells. b The CCK-8 assay was used to quantify the relative cell viability at indicated time points. c Representative pictures of colony formation assay in 97H and LM3 cells transfected with or without GTSE1. d The ratio of cells at different cell cycle phases was evaluated by flow cytometric analysis and quantitative analysis of the different cell cycle phases. e Cell apoptosis of 97H and LM3 cells transfected with SCR or GTSE1-SH was assessed by flow cytometric analysis. **P < 0.01

Fig. 4

GTSE1 knockdown suppressed cell invasion and regulated AKT phosphorylation. a Matrigel-uncoated/coated transwell cell invasion assays of 97H cells transfected with SCR or GTSE1-SH. b Matrigel-uncoated/coated transwell cell invasion assays of LM3 cells transfected with SCR or GTSE1-SH. c Western blot detection of GTSE1, ATK, p-AKT, ERK, p-ERK, BCL-2, Bax, cyclin B1, p53, MMP-2, and MMP-9 expression in 97H and LM3 cells transfected with SCR or GTSE1-SH. **P < 0.01

Fig. 5

Knockdown of GTSE1 inhibits the growth of HCC in vivo. a Representative pictures of subcutaneous implantation tumor of LM3 cellsat 5 weeks after injection were shown. b Tumor growth curves of subcutaneous implantation models of LM3 cells at indicated time points. c Tumor weights were quantified. Data were represented as the mean ± SD of three independent experiments. d Representative images of Ki67 in tumor xenograft tissues from subcutaneously inoculated Nu/Nu mice (original magnifications: ×400). **P < 0.01