ARNTL2 is an indicator of poor prognosis, promotes epithelial-to-mesenchymal transition and inhibits ferroptosis in lung adenocarcinoma

Abstract

Objectives: ARNTL2, as a circadian transcription factor, has been recently proposed to play an important role in a variety of tumors. however, the role of ARNTL2 in lung carcinogenesis and progression remains unclear. The purpose of this study was to investigate the effect of ARNTL2 on the clinical characteristics and prognosis of lung adenocarcinoma and to explore the relationship between ARNTL2 and EMT, ferroptosis in lung adenocarcinoma.

Methods: The Cancer Genome Atlas (TCGA) database's multi-omics data were downloaded using the Xena browser. Based on the expression levels of ARNTL2, patients with lung adenocarcinoma from TCGA were divided into two groups: those with high ARNTL2 expression and those with low ARNTL2 expression. ARNTL2 was studied for its effects on lung adenocarcinoma's clinicopathological, genomic, and immunological characteristics. Furthermore, in vivo and in vitro assays were used to confirm the impact of ARNLT2 knockdown on lung adenocarcinoma cells.

Results: We found ARNTL2 is highly expressed in lung adenocarcinoma and was an independent predictor of a poor prognosis in patients with lung adenocarcinoma. In addition, we demonstrated that knockdown of ARNTL2 promoted ferroptosis, inhibited EMT, cell proliferation, migration and invasion in lung adenocarcinoma. In contrast, overexpressing ARNTL2 yielded the opposite results.

Conclusions: ARNTL2 is an independent unfavorable prognostic factor for lung adenocarcinoma. It plays a facilitating role in the development of lung adenocarcinoma, especially in promoting EMT and inhibiting ferroptosis, revealing that ARNTL2 may be a potential biomarker for lung adenocarcinoma.

Keywords: ARNTL2; EMT; Ferroptosis; Lung adenocarcinoma.

Fig. 1

A ARNTL2 expression levels in different tumor types from the TCGA database were determined by TIMER. B Differences of ARNTL2 expression in lung adenocarcinoma tissues and normal tissues. C Survival curves comparing the high and low expression of ARNTL2 in lung adenocarcinoma. D Univariate and multivariate analysis of overall survival in LUAD patients from TCGA database. E Scatterplots of correlations between ARNTL2 expression and TMB. F The comparison of mRNAsi in high, low ARNTL2 expression groups of lung adenocarcinoma tissues versus normal tissues. G Differential mutations and their distributions in the high and low-ARNTL2 expression groups.

Fig. 2

A Differential expressed genes between high and low-ARNTL2 expression groups were shown in a volcano plot. B Dot plot of GO and KEGG pathway analysis to the DEGs. C Scatterplots of correlations between ARNTL2 expression and angiogenesis, EMT, and Metastasis by single-cell analysis of lung adenocarcinoma cells through the CancerSEA database.

Fig. 3

A-B The IHC (A) and immunofluorescence (B) staining of ARNTL2 in LUAD tissue versus adjacent normal tissues: ARNTL2 expression in tumor tissues was significantly higher than that in adjacent normal tissues. C Kaplan-Meier curves of overall survival and progression-free survival according to immunohistochemical staining of the ARNTL2 in patients from our institution. D-E Western blotting analyses verifying the ARNTL2 knockdown and overexpression efficiency, quantification was performed by ImageJ: ARNTL2 knockdown was very efficient. F The effects of ARNTL2 knockdown on cell proliferation in A549 and H1299 cells through CCK8 assays: knockdown of ARNTL2 dramatically inhibited LUAD cells proliferation. G Comparison of colony formation efficiency between ARNTL2-knockdown group and control group: knockdown of ARNTL2 dramatically inhibited colony formation of LUAD cells. H Comparison of Wound-healing assays between ARNTL2-knockdown and control groups: ARNTL2 knockdown decreases cell migration in LUAD cells compared to control cells. I The invasion and migration capability of LUAD cells transfected with NC or sh-ARNTL2 was analyzed by transwell assay: ARNTL2 knockdown diminished the migration and invasion ability of LUAD cells.

Fig. 4

A Correlation of ARNTL2 expression with immune infiltration level in LUAD through QUANTISEQ method. B Differences in some important immune genes' expression are closely related to immune checkpoints between high and low-ARNTL2 groups. C Differences in the expression of important genes associated with ferroptosis between high and low-ARNTL2 groups. D Viability of A549/ H1299 cells upon treatment with ferroptosis inducer, RSL3, at the indicated concentrations for 6 h/ 48 h. E The expression of some key genes related to ferroptosis after knockdown or overexpression of ARNTL2 in A549 and H1299 cells. F Relationship between drug sensitivity to cisplatin in lung adenocarcinoma and ANRTL2 expression were shown in box plot. G Dose-toxicity curves showing the viability of A549 and H1299 cells upon cisplatin treatment at the indicated concentrations for 48 h.

Fig. 5

A-B The effects of ARNTL2 overexpressing on cell proliferation (A), and Comparison of colony formation (B) in A549 and H1299. C The effects of ARNTL2 overexpressing on cell invasion and migration in A549 and H1299 cells. D Expressions of EMT-related markers in A549 and H1299 cells with ARNTL2 knockdown and overexpression by WB assays: indicating a positive correlation between ARNTL2 expression and EMT. E The effects of ARNTL2 knockdown on tumor growth in mouse xenograft model: ARNTL2 knockdown markedly suppressed xenograft tumor growth of A549 cells.