Effects of TRAF3 on the proliferation and migration of lung adenocarcinoma depend partly on pyroptosis

Abstract

Background: Tumor necrosis factor receptor-associated factor 3 (TRAF3) has specific regulatory effects on a wide range of diseases, including tumors. However, the effect and mechanism of TRAF3 on lung adenocarcinoma (LUAD) are still unknown. The aim of the present study was to make clear the role and potential mechanism of TRAF3 in LUAD.

Methods: TIMER2.0 database and western blot were applied to detect the expression of TRAF3 in lung adenocarcinoma tissue. Kaplan-Meier Plotter database was utilized to explore the effect of TRAF3 on the clinical prognosis of lung adenocarcinoma patients. Specific siRNA was used to inhibit the expression of TRAF3 in LUAD cells (A549 and H1299). CCK-8 and EdU assays were performed for assessing LUAD cells proliferation. Wound healing assay and transwell assay were performed for determining cells migration. CCK-8 assay was used to assess the response of the LUAD cells to paclitaxel. TIMER2.0 bioinformatics and western blot were employed to detect the effects of TRAF3 on pyroptosis in LUAD.

Results: TRAF3 was highly expressed in lung adenocarcinoma tissues and cell lines. Patients with TRAF3 hyperexpression had a good prognosis compared to those with lower expression. TRAF3 inhibition notably induced proliferation and migration of LUAD cells. Inhibition of TRAF3 also weakened the sensitivity of LUAD cells to paclitaxel. Moreover, bioinformatics results showed that TRAF3 was positively correlated with the expression of pyroptosis-related genes in LUAD. Western blot assays showed that TRAF3 inhibition visibly decreased the expression of apoptosis-associated speck-like protein (ASC), cleaved caspase-1 and matured- IL-1β.

Conclusions: Inhibition of TRAF3 promotes the proliferation and migration of LUAD cells, and reduces the sensitivity of LUAD cells to paclitaxel. The effects of TRAF3 on LUAD cells were mediated in part by caspase-1-dependent pyroptosis.

Keywords: Lung adenocarcinoma (LUAD); Pyroptosis; Tumor necrosis factor receptor-associated factor 3 (TRAF3).

Fig. 1

TRAF3 is upregulated in LUAD. (A) TIMER2.0 database showed the relative expression of TRAF3 in lung adenocarcinoma and adjacent tissues. *P<0.05, **P<0.01, *** P<0.001 versus normal tissues group. (B) Western blot assays revealed the relative expression of TRAF3 in human bronchial epithelial cells (16HBE) and human LUAD cell lines (A549, H1299). **P<0.01, ***P<0.001 versus 16HBE cell line. The results were presented as the mean ± SD (n = 3). (C) Kaplan-Meier Plotter database showed the relationship between TRAF3 expression levels with lung adenocarcinoma patients’ prognosis

Fig. 2

Downregulation of TRAF3 promotes the proliferation of LUAD cells. (A) Western blot was performed to evaluate the efficiency of si-TRAF3. *P<0.05, **P<0.01 versus control group. (B) CCK-8 assay was used to detect the proliferation activity of LUAD cells after transfection. **P<0.01, ***P<0.001 versus control group. (C) EdU assay was applied to determine the DNA replication ability of LUAD cells after transfection. **P<0.01, *** P<0.001 versus control group. The results were presented as the mean ± SD (n = 3–4)

Fig. 3

Downregulation of TRAF3 promotes the migration of LUAD cells. (A) Scratch test was used to detect the migration and healing of LUAD cells after transfection. (B) Transwell assay was used to detect the migration of LUAD cells after transfection. **P<0.01 versus control group. The results were presented as the mean ± SD (n = 3)

Fig. 4

Downregulation of TRAF3 reduces the sensitivity of LUAD cells to paclitaxel (A) CCK-8 assay was applied to evaluate the effect of different concentrations of paclitaxel on the cell viability of A549 cells after transfection. *P<0.05, ***P<0.001, ****P<0.0001 versus control group. (B) CCK-8 assay was applied to evaluate the effect of different concentrations of paclitaxel on the cell viability of H1299 cells after transfection. **P<0.01, ****P<0.0001 versus control group. The results were presented as the mean ± SD (n = 3–6)

Fig. 5

Downregulation of TRAF3 attenuates the pyroptosis of LUAD cells. (A) TIMER2.0 database predicted the association of TRAF3 with ASC, caspase-1 and IL-1β in lung adenocarcinoma. (B) After LUAD cells were transfected with TRAF3 siRNA, the expression levels of TRAF3, ASC, caspase-1, cleaved caspase-1, IL-1β and matured-IL-1β were analyzed by western blotting. *P<0.05, **P<0.01, ***P<0.001 versus control group. The results were presented as the mean ± SD (n = 3–4)