TRIM11 overexpression promotes proliferation, migration and invasion of lung cancer cells

Abstract

Background: Tripartite Motif Containing 11 (TRIM11), a member of TRIM proteins, is overexpressed in high-grade gliomas and plays an oncogenic function in glioma biology. However, little is known about the role of TRIM11 in lung cancer.

Methods: We analyzed TRIM11 mRNA expression in lung cancer tissues and adjacent non-neoplastic tissues by real-time PCR. We then explored the function of TRIM11 in lung cancer cells by small interfering RNA-mediated downregulation of this protein followed by analyses of cell proliferation, migration and invasion.

Results: TRIM11 was highly expressed in lung cancer tissues and lung cancer cell lines. The higher expression of TRIM11 was correlated with the poorer prognosis of patients. Suppressing of TRIM11 expression in lung cancer cells with higher expression of TRIM11 (A549 and NCI-H446 cells) significantly reduced cell growth, motility and invasiveness. We further demonstrated that knockdown of TRIM11 affected the expression of cell proliferation-related proteins (Cyclin D1 and PCNA), and epithelial-mesenchymal transformation-related proteins (VEGF, MMP-2, MMP-9, Twist1, Snail and E-cadherin). The activity of ERK and PI3K/AKT was also suppressed in TRIM11 knocked down cells. Further experiments in lung cells with lower expression of TRIM11 (NCI-H460 and NCI-H1975 cells) with AKT inhibitor suggested that TRIM11 may promote cell motility and invasiveness through AKT pathway.

Conclusions: Our results indicate that TRIM11 acts as an oncogene in lung cancer through promoting cell growth, migration and invasion. Our findings may have important implication for the detection and treatment of lung cancer.

Keywords: Invasion; Lung cancer; Migration; PI3K/AKT; TRIM11.

Fig. 1

TRIM11 expression in lung cancer tissues a Analysis of TRIM11 expression in lung cancer tissues (n = 488) and normal tissues (n = 58) in TCGA dataset. b TRIM11 mRNA levels in lung cancer and normal tissues from patients admitted to Department of Thoracic Surgery, Northern Jiangsu People’s Hospital were determined by real-time PCR, with GAPDH as a control. c Kaplan-Meier survival analysis of 120 lung cancer patients revealed the correlation between TRIM11 expression and prognosis

Fig. 2

TRIM11 was highly expressed in lung cancer cells and its expression was suppressed by TRIM11 siRNA transfection. a Real-time PCR analysis for the TRIM11 mRNA expression level in 5 lung cancer cell lines (A549, NCI-H446, NCI-H1975, NCI-H460 and PC-9) and 1 human embryo lung fibroblast cell line (MRC-5). **P < 0.01 and ***P < 0.001 versus MRC-5 cells. b TRIM11 protein expression was determined by Western blot. Quantification (right panel) based on at least 3 independent experiments was made by normalizing against GAPDH levels. **P < 0.01 and ***P < 0.001 versus MRC-5 cells. c Real-time PCR analysis showed the efficiency of TRIM11 knockdown in A549 and NCI-H446 cells. WT: wild-type cells. NC: control siRNA-transfected cells. siRNA1, 2 and 3: TRIM11 siRNA1, 2 or 3-transfected cells. ***P < 0.001 versus NC. d The protein levels of TRIM11 were determined by Western blot and GAPDH was used as an internal control. Quantification (right panel) was shown. ***P < 0.001 versus NC

Fig. 3

Knockdown of TRIM11 reduced the proliferation of lung cancer cells. a, b CCK-8 assay results of A549 and NCI- H446 cells at 0 h, 24 h, 48 h and 72 h after TRIM11 expression was inhibited by siRNA3 transfection. c, d Expression of PCNA and Cyclin D1 was evaluated by Western blot. Quantification (bottom panel) based on at least 3 independent experiments was shown. ***P < 0.001 versus NC

Fig. 4

Knockdown of TRIM11 inhibited the motility and invasiveness of lung cancer cells. a Migration assay was performed on wild-type (WT) cells, cells transfected with control siRNA (NC) and cells transfected with TRIM11 siRNA3 (siRNA3). Representative images (left panel) and quantitative results (right panel) were shown. b Invasion assay was carried out. c, d Expression of metastasis-related proteins was evaluated by Western blot. e *P < 0.05, **P < 0.01 and ***P < 0.001 versus NC

Fig. 5

Knockdown of TRIM11 inhibited the activation of PI3K/AKT and ERK pathways in lung cancer cells. Total protein and phosphorylation of PI3K, AKT and ERK was evaluated by western blot in A549 a and NCI-H446 cells b. **P < 0.01 and ***P < 0.001 versus NC

Fig. 6

Ectopic expression of TRIM11 impaired the effects of AKT inhibitor on the migration and invasion of lung cancer cells. a, b NCI-H460 and NCI-1975 cells were infected with control virus (Vector) or TRIM11 virus, and migration and invasion assay were performed in the present of either DMSO or AKT inhibitor (MK-2206, 2 μM). MK-2206 was dissolved in DMSO as a 10 mM stock solution. Thus, 0.02 % DMSO was used as negative control. ***P < 0.001 versus Vector + DMSO; ###P < 0.001 versus TRIM11 + DMSO; $$$P < 0.001 versus Vector + MK-2206

Fig. 7

Schematic representation of the biologic role of TRIM11 in lung cancer. TRIM11 may act as an oncogene lung cancer by promoting cell proliferation, migration and invasion. TRIM11 may modulate PI3K/AKT pathway