Knockdown of ANLN inhibits the progression of lung adenocarcinoma via pyroptosis activation

Abstract

Significant advancements have been achieved in the area of molecular targeted therapy for lung adenocarcinoma (LUAD). However, the complex molecular patterns and high heterogeneity of LUAD confine the efficacy of these therapies to a specific subset of patients; therefore, it is necessary to explore novel targets for LUAD treatment. The expression levels of anillin (ANLN) in LUAD were analyzed using the Gene Expression Profiling Interactive Analysis database. Furthermore, the association between ANLN gene expression and patient survival outcomes was evaluated using the Kaplan‑Meier Plotter. Subsequently, small interfering RNA (siRNA) transfection was performed to knock down ANLN in A549 and H1299 cell lines, after which, TUNEL, colony formation and Transwell assays were conducted to assess cell death, colony formation and migration, respectively. Additionally, western blot analysis was performed to analyze the expression levels of caspase‑1, interleukin (IL)‑18 (IL‑18), IL‑1β, NLR family pyrin domain‑containing 3 (NLRP3), apoptosis‑associated speck‑like protein containing a CARD domain (ASC) and cleaved gasdermin D (GSDMD) following ANLN knockdown. The results revealed that ANLN mRNA expression was significantly increased in LUAD tissues compared with adjacent normal samples. Furthermore, the expression levels of ANLN displayed an increasing trend with advancing clinical stage. Furthermore, patients with high ANLN expression levels exhibited poor overall survival rates compared with those with low ANLN expression levels. Subsequent ANLN knockdown experiments indicated elevated cell death rate, and reduced colony formation and migration in both A549 and H1299 cells. Additionally, ANLN knockdown resulted in increased protein expression levels of pyroptosis‑associated molecules, including caspase‑1, NLRP3, cleaved‑GSDMD, IL‑1β, ASC and IL‑18 in both A549 and H1299 cells. In conclusion, ANLN represents an important gene and a promising therapeutic target for LUAD. Its potential as a therapeutic target makes it an interesting candidate for further exploration in the development of novel treatment strategies for LUAD.

Keywords: anillin; lung adenocarcinoma; pyroptosis; tumor progression.

Figure 1.

ANLN is upregulated and serves as a prognostic factor in LUAD. (A) ANLN mRNA expression in LUAD tissues and normal tissues in The Cancer Genome Atlas database. *P<0.05. (B) ANLN mRNA expression increased with TNM stage (P=6.17×10⁻⁵). (C) Kaplan-Meier analysis of the overall survival of patients with LUAD based on ANLN expression (P=4.1×10⁻⁶). ANLN, anillin; LUAD, lung adenocarcinoma; HR, hazard ratio.

Figure 2.

Successful knockdown of ANLN in A549 and H1299 cells. (A) Reverse transcription-quantitative PCR confirming successful siRNA-induced knockdown of ANLN in A549 and H1299 cells. (B) Protein expression levels of ANLN in A549 and H1299 cells were determined by western blotting. Grayscale analysis was performed using ImageJ software. Data are presented as the mean ± SD (n=3). **P<0.01, ***P<0.001 vs. NC. ANLN, anillin; NC, negative control; si, small interfering.

Figure 3.

ANLN promotes cell death, and suppresses the colony formation and migration of A549 and H1299 cells. (A) A549 and H1299 cell death was detected by TUNEL assay (scale bars, 25 µm). (B) Colony-forming ability of A549 and H1299 cells was detected by colony formation assay. (C) Migration of A549 and H1299 cells was evaluated by Transwell assay (magnification, ×100). Data are presented as the mean ± SD (n=3). ***P<0.001 vs. NC. ANLN, anillin; NC, negative control; si, small interfering.

Figure 4.

ANLN knockdown induces pyroptosis in A549 and H1299 cells. (A) Protein expression levels of pro-caspase-1, caspase-1, NLRP3, cleaved-GSDMD, IL-1β, ASC and IL-18 protein level in A549 and H1299 cells were detected by western blot analysis. (B) Grayscale analysis was performed by ImageJ software. Data are presented as the mean ± SD (n=3). ***P<0.001 vs. NC. ANLN, anillin; ASC, apoptosis-associated speck-like protein containing a CARD domain; GSDMD, gasdermin D; IL, interleukin; NC, negative control; NRP3, NLR family pyrin domain-containing 3; si, small interfering.