TAp63α Is Involved in Tobacco Smoke-Induced Lung Cancer EMT and the Anti-cancer Activity of Curcumin via miR-19 Transcriptional Suppression

Abstract

As a key risk factor for lung cancer, tobacco smoke (TS) influences several cellular processes, including epithelial-mesenchymal transition (EMT). TAp63α is a crucial transcription factor involved in tumor progression. The present study was designed to investigate the potential role and underlying mechanisms of TAp63α in TS-induced lung cancer EMT. We found that compared to normal tissues, the tumor tissues collected from lung cancer patients showed a lower level of TAp63α expression, along with downregulated E-cadherin expression and upregulated Vimentin expression. Results of treatment with TAp63α and TAp63α siRNA as well as with tumor growth factor-β (TGF-β) showed that TAp63α acted as a tumor suppressor gene, and its upregulated expression suppressed lung cancer EMT. Significantly, TS exposure altered expression of EMT-related markers, enhanced cell migratory and invasive capacities, and decreased the TAp63α expression level in lung cancer cells. Overexpression of TAp63α significantly alleviated TS-stimulated lung cancer EMT. Mechanistically, TAp63α expression transcriptionally reduced the miR-19 level, which resulted in the suppression of lung cancer EMT. Additionally, as a natural compound possessing anti-cancer effects, curcumin inhibited TS-induced lung cancer EMT by increasing TAp63α expression and reducing miR-19 expression. Collectively, our results indicate that TAp63α inhibits TS-induced lung cancer EMT via transcriptionally suppressing miR-19 and the inhibitory effect of TAp63α on miR-19 mediates the anti-cancer action of curcumin. These findings provide new insights into novel targets for lung cancer prevention.

Keywords: EMT; TAp63α; curcumin; lung cancer; miR-19; tobacco smoke.

FIGURE 1

TAp63α is correlated with lung cancer EMT. (A) Immunohistochemistry analysis (200×) was used to determine TAp63α, E-cadherin, and N-cadherin protein levels in lung cancer tissues and normal pneumocyte tissues. Scale bar, 100 μm. (B) The expression of EMT-related markers, including E-cadherin and Vimentin was measured using western blotting. (C) Densitometry results are shown as fold change compared with the control group after normalization to β-actin (n = 32). Results are shown as mean ± SD. *P < 0.05, ***P < 0.001 compared with the normal group.

FIGURE 2

Inhibition of TAp63α promotes lung cancer EMT process. (A) A549 cells were treated with TAp63α transfection plasmids (2 ng), and the EMT-related biomarkers were analyzed using western blotting. (B) H1299 cells were transfected with TAp63α siRNA (75 μM) for 48 h, and the expression of the indicated proteins was measured using western blot in H1299 cells following transfection with TAp63α siRNA. (C,D) The cell abilities of migration and invasion were detected using a Transwell assay and the number of migrated and invaded H1299 cells was determined (100 ×). (E,F) A549 and H1299 cells were treated with TGF-β (2 ng/mL) for 2 days, western blotting analysis was used to measure the levels of TAp63α, ZO-1, E-cadherin, N-cadherin, and Vimentin. (G) H1299 cells were exposed to TGF-β in the presence or absence of TAp63α plasmids (2 ng) for 2 days, and the indicated gene expression was detected. Results are shown as mean ± SD of at least three independent experiments. **P < 0.01, ***P < 0.001 compared with the control group.

FIGURE 3

TAp63α affects the TS-induced lung cancer EMT process. (A) H1299 cells were treated with various concentrations of CSE (0–1%) for 2 days, and the expression of TAp63α and EMT-associated markers was analyzed using western blotting. (B) H1299 cells were treated with 1% CSE in the presence or absence of TAp63α plasmid transfection for 48 h, and western blotting analysis was used to detect the expression of the indicated proteins. (C) The cell migration and invasion abilities were tested using Transwell assays (100×). (D) Migrated and invaded cell numbers. Results are shown as the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, compared with the control group. ^#P < 0.05, ^##P < 0.01, compared with the 1% CSE treatment group. CSE: cigarette smoke extract.

FIGURE 4

TAp63α transcriptionally suppresses miR-19 to inhibit TS-induced EMT in lung cancer. (A) RT-qPCR was used to detect the levels of miR-19a and miR-19b in H1299 cells exposed to 1% CSE for 2 days. (B,C) H1299 cells were transfected with TAp63α siRNA (75 μM) or TAp63α plasmids (2 ng) for 48 h, and RT-qPCR was used to detect the expression of miR-19a and miR-19b in H1299 cells. (D) Bioinformatics analysis of the binding site of the miR-17-92 promoter and TP63α. (E) H1299 cells were transfected with wild-type miR-17-92 promoter (wt-miR-17-92 promoter), TAp63α plasmids, or TAp63α siRNA, and the luciferase activity was measured. (F) A dual-luciferase reporter assay was used to detect the luciferase activity in H1299 cells transfected with mutant type miR-17-92 promoter (mut-miR-17-92 promoter) and TAp63α plasmids or TAp63α siRNA. (G,H) The expression of E-cadherin and Vimentin was analyzed in miR-19 overexpression and miR-19 knockdown H1299 cells. Results are shown as the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group. ^##P < 0.01, compared with the control siRNA group.

FIGURE 5

Curcumin blocks the TS-triggered lung cancer EMT process by targeting TAp63α and miR-19. (A,B) A549 and H1299 cells were exposed to various concentrations (0, 2.5, 5, and 7.5 μM) of curcumin for 2 days, and the levels of TAp63α and EMT-related markers, including ZO-1, E-cadherin, N-cadherin, and Vimentin were detected using western blot. (C) H1299 cells were co-treated with curcumin (7.5 μM) and 1% CSE for 2 days, and the indicated gene expression was analyzed. (D) A Transwell assay was used to measure the cell migration and invasion capacities (100×). (E) The migrated and invaded cell numbers. (F) RT-qPCR was used to detect the levels of miR-19a and miR-19b in those cells. Results are shown as mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group. ^#P < 0.05, ^##P < 0.01, compared with the 1% CSE treatment group. CSE: cigarette smoke extract.

FIGURE 6

Schematic illustration of the regulatory effect of TAp63α on TS-induced lung cancer EMT and the curcumin anti-cancer effects. TGF-β treatment and tobacco smoke exposure decreased TAp63α expression; TAp63α transcriptionally reduces the expression of miR-19a/miR-19b, thereby inhibiting the lung cancer cell EMT process. Additionally, curcumin alleviates TS-stimulated lung cancer EMT via TAp63α upregulation and a miR-19 level decrease.