Postexercise downregulation of NUP155 in regulating non-small cell lung cancer progression via the PTEN/AKT signaling pathway

Abstract

Background: Research interest into regulation of gene expression by physical activity and its effect on cancer prognosis has intensified. This study investigated the role of an exercise-related gene, NUP155, in the progression of non-small cell lung cancer (NSCLC) and its potential as therapy target.

Methods: Using the GSE41914 dataset, which includes data related to exercise, and the Cancer Genome Atlas (TCGA)-NSCLC dataset, we identified differentially expressed genes (DEGs) and selected NUP155 as a hub gene for further analysis. NUP155 expression levels were measured in NSCLC cell lines and normal lung cells using in vitro assays. The functional roles of NUP155 were investigated through small interfering RNA (siRNA) knockdown experiments, assessing effects on migration, cell proliferation, invasion, and apoptosis. The involvement of the PTEN/AKT signaling pathway was examined using the PTEN inhibitor SF1670.

Results: NUP155 was downregulated in postexercise samples and upregulated in NSCLC samples, indicating its association with poor prognosis in NSCLC. Knockdown of NUP155 in NSCLC cell lines resulted in reduced cell viability, migration, and invasion, alongside increased apoptosis. Western blotting revealed that NUP155 knockdown upregulated PTEN levels and downregulated phosphorylated AKT (p-AKT), without altering total AKT levels. The addition of SF1670 partially reversed the effects of NUP155 knockdown, indicating the involvement of the signaling pathway PTEN/AKT in NUP155-mediated tumorigenesis.

Conclusions: NUP155 is upregulated in NSCLC, which promotes cell invasion and migration via the PTEN/AKT signaling pathway. Targeting NUP155, potentially influenced by exercise, could be a promising therapy. Combining exercise with targeted treatments may enhance patient outcomes.

Keywords: Exercise; NUP155; PTEN/AKT signaling pathway; non-small cell lung cancer (NSCLC); tumor progression.

Figure 1

Differential expression and prognostic significance of NUP155 in NSCLC-related datasets. (A,B) DEGs screening of the GSE41914 dataset and TCGA-NSCLC dataset. Blue represents the downregulated DEGs, and red represents the upregulated DEGs. (C) Cross-analysis of upregulated and downregulated DEGs in the GSE41914 and TCGA-NSCLC datasets for obtaining the overlapping genes. (D) Boxplot showing the expression level of NUP155 in pre-exercise and postexercise samples in the GSE41914 dataset. (E) Boxplot showing the expression level of NUP155 in NSCLC samples and normal samples in TCGA dataset. (F) Kaplan-Meier survival curve showing the patients’ overall chance of survival from NSCLC. The red line represents high NUP155 expression, and the blue line represents low NUP155 expression. (G) Kaplan-Meier survival curve of first progression probability of patients with NSCLC. The red line represents high NUP155 expression, and the blue line represents low NUP155 expression. **, P<0.01; ****, P<0.0001. NSCLC, non-small cell lung cancer; DEG, differentially expressed gene; TCGA, The Cancer Genome Atlas.

Figure 2

NUP155 was upregulated in NSCLC cells. (A) qRT-PCR measurements of the relative mRNA expression of NUP155 in NSCLC cell lines (A549, H157, H1299, and H1975) and normal lung cells (BEAS-2B). (B,C) WB of NUP155 protein levels in BEAS-2B and NSCLC cell lines. (D) Relative mRNA expression of NUP155 in A549 and H1299 cells after transfection with control siRNA (si-NC) or two different NUP155-targeting siRNAs (si-NUP155-1 and si-NUP155-2). (E,F) WB of NUP155 protein knockdown efficiency in A549 and H1299 cells. *, P<0.05; **, P<0.01. qRT-PCR, quantitative real-time polymerase chain reaction; NSCLC, non-small cell lung cancer; WB, western blotting; si-NC, small interfering RNA negative control; siRNA, small interfering RNA.

Figure 3

Knockdown of NUP155 inhibited the proliferation, migration, and invasion of NSCLC cells. (A,B) CCK8 assay of NSCLC cells (A549 and H1299) at various time points (0, 24, 48, 72, 96, and 120 hours) following NUP155 knockdown. (C-F) Transwell assay for detecting the changes in the invasion and migration ability of NSCLC cells (A549 and H1299) after knockdown of NUP155. Cells were stained using DAPI and imaged with a fluorescence microscope. Scale bar: 50 µm. *, P<0.05; **, P<0.01; ***, P<0.001. OD, optical density; NSCLC, non-small cell lung cancer; CCK8, Cell Counting Kit 8.

Figure 4

Knockdown of NUP155 promoted apoptosis of NSCLC cells. (A-C) Following NUP155 knockdown, apoptosis in NSCLC cells (A549 and H1299) was observed via flow cytometry. Quadrants represent viable cells (Q4), late apoptotic cells (Q2), early apoptotic cells (Q3), and necrotic cells (Q1). (D-F) WB was used to detect the protein expression of apoptotic proteins in NSCLC cells (A549 and H1299) after the knockdown of NUP155. *, P<0.05; **, P<0.01; ***, P<0.001. NSCLC, non-small cell lung cancer; WB, western blotting.

Figure 5

NUP155 activated the PTEN/AKT signaling pathway in NSCLC cells and influenced tumor migration and invasion. (A-C) WB detection of PTEN, p-AKT, and AKT protein levels in NSCLC cells after knockdown of NUP155. *, P<0.05; **, P<0.01; ***, P<0.001. (D-G) Transwell detection of the invasion and migration ability of NSCLC cells after knockdown of NUP155 and addition of PTEN inhibitor SF1670. Cells were stained using DAPI and imaged with a fluorescence microscope. Scale bar: 50 µm. **, P<0.01; ***, P<0.001 vs. si-NC. ^#, P<0.05; ^##, P<0.01 vs. si-NUP155-2. WB, western blotting; NSCLC, non-small cell lung cancer; si-NC, small interfering RNA negative control.