SIRT1 silencing ameliorates malignancy of non-small cell lung cancer via activating FOXO1

Abstract

Non-small cell lung cancer (NSCLC), being the most prevalent and lethal malignancy affecting the lungs, poses a significant threat to human health. This research aims at illustrating the precise role and related mechanisms of silent information regulator type-1 (SIRT1) in NSCLC progression. The expression pattern of SIRT1 in NSCLC cell lines was examined using quantitative real-time polymerase chain reaction and western blotting. Functional assays in NSCLC cell lines validated the biological capabilities of SIRT1 on malignant phenotypes, and its impact on tumorigenicity was further evaluated in vivo. In addition, the FOXO1 inhibitor AS1842856 was applied to verify the role of SIRT1 on FOXO pathway in vitro. SIRT1 expression was prominently elevated in NSCLC cell lines. The depletion of SIRT1 retarded the capabilities of proliferation, migration and invasion, while enhancing apoptosis in NSCLC cells. Furthermore, SIRT1 silencing restricted the tumorigenesis of NSCLC in vivo. Additionally, AS1842856 treatment ameliorated the inhibitory effect of SIRT1 deficiency on malignant phenotypes in NSCLC cells. SIRT1 deletion exerted an anti-oncogenic role in NSCLC via activation of FOXO1.

Keywords: FOXO; Non-small cell lung cancer; SIRT1.

Fig. 1

SIRT1 silencing suppresses proliferation and promotes apoptosis of NSCLC cells. (A) The expression of SIRT1 at mRNA and protein levels in A549, H1299, and Calu-1 cells. (B) The silencing efficiency of SIRT1 was determined using qRT‐PCR and western blot technique. (C) The CCK-8 assay was used to measure the viability of A549 and H1299 cells in the si-NC and si-SIRT1s groups. (D) The proliferation of A549 and H1299 cells in the si-NC and si-SIRT1s groups was evaluated using the EdU assay. (E) Apoptosis of NSCLC cells in each group was detected by flow cytometry. ^**p < 0.01 vs. si-NC.

Fig. 2

SIRT1 knockdown blocks the migration and invasion of NSCLC cells. (A) Wound‐healing assay was conducted to detect cell migration in NSCLC cell lines characterized by knockdown of SIRT1. Scale bar: 100 μm. (B) To assess the effect of SIRT1 silencing on NSCLC cell invasion, the transwell assay was conducted. Scale bar: 100 μm. ^**p < 0.01 vs. si-NC.

Fig. 3

Downregulation of SIRT1 attenuates tumorigenicity of NSCLC in vivo. (A) Representative image of the tumors harvested from the lv-NC and lv-SIRT1 group (n = 6). (B) Tumor weight was compared between two groups. The tumor volume growth curves of each group were determined. (C) HE staining was used to detect the pathological changes in tumors. Amplification: 400 × , scale bar: 20 μm. (D) IHC images of Ki‐67 in each group. Amplification: 400 × , scale bar: 20 μm. (E) IHC staining of SIRT1 expression in tumor tissues of lv-NC and lv-SIRT1 groups. Amplification: 400 × , scale bar: 20 μm. (F) IHC staining of FOXO1expression in tumor tissues of lv-NC and lv-SIRT1 groups. Amplification: 400 × , scale bar: 20 μm. ^**p < 0.01 vs. lv-NC.

Fig. 4

Inhibition of SIRT1 is correlated with FOXO pathway activation in NSCLC cells. (A) Venn diagram of SIRT1 co-expressed genes and NSCLC-associated genes. (B) KEGG functional enrichment analysis of intersected genes. (C) FOXO1 protein expression of each group in A549 and H1299 cells were detected by western blot. (D) FOXO3 mRNA expression of each group in A549 and H1299 cells were determined. ^**p < 0.01 vs. si-NC.

Fig. 5

SIRT1 deletion restrains the malignant phenotypes of NSCLC cells via FOXO pathway. (A) The expression levels of FOXO1 in H1299 cells after SIRT1 interference and subsequent AS1842856 treatment were checked by qRT-PCR. (B) The viability of H1299 cells after different treatment was measured by CCK-8. (C) EDU assay for the assessment of the cell proliferation in H1299 cells after different treatment. (D) The apoptosis of NSCLC cells in each group was assessed through flow cytometry. (E) The migration of NSCLC cells in each group was measured using the wound healing assay. Scale bar: 100 μm. (F) The invasion ability of NSCLC cells stably in each group was detected by transwell assay. Scale bar: 100 μm. ^**p < 0.01 vs. si-NC; ^##p < 0.01 vs. si-SIRT1.