High expression of LINC00467 promotes proliferation and metastasis of lung adenocarcinoma cells by suppressing autophagy via inhibiting the AMPK/mTOR pathway

doi:10.12122/j.issn.1673-4254.2024.10.08

Abstract

Abstract:

Objective To investigate the regulatory effects of LINC00467 on proliferation and metastasis of lung adenocarcinoma cells and the involvement of autophagy in its regulatory mechanism. Methods LINC00467 expression levels in lung adenocarcinoma tissues and their correlation with the patients' survival outcomes were analyzed using data from TCGA database. LINC00467 expression was also examined using qRT-PCR in human bronchial epithelial cells 16HBE and lung adenocarcinoma cell lines A549 and H1299. In A549 and H1299 cells transfected with a short hairpin RNA targeting LINC00467 (shLINC00467), the effects of 3-methyladenine (3-MA, an autophagy inhibitor) and BML-275 (an AMPK inhibitor) treatment on cell proliferation, migration, and expressions of LC3 and the AMPK/mTOR pathway proteins were tested using colony formation assay, wound-healing and Transwell assays, immunofluorescence staining and Western blotting. GSEA enrichment analysis was conducted to analyze the correlation between LINC00467 and the autophagy pathway. Results The expression level of LINC00467 was significantly higher in lung adenocarcinoma tissues than in the adjacent tissues (P<0.001) and increased progressively with the clinical stage (P<0.05), and its high expression was associated with a poor overall survival (P=0.049) and a high first progression rate (P=0.026) of the patients. LINC00467 expression was also significantly higher in A549 and H1299 cells than in 16HBE cells. In A549 and H1299 cells, LINC00467 knockdown significantly decreased colony-forming, migration and invasion abilities of the cells, lowered p-mTOR/mTOR and p62 expressions, and increased p-AMPK/AMPK expressions and LC3II/I ratio, and these effects were strongly attenuated by application of either 3-MA or BML-275. GSEA analysis suggested an inhibitory effect on LINC00467 on the autophagy pathway (|NES|>1, P<0.05, FDR<0.25). Conclusion High expressions of LINC00467 promote proliferation and metastasis of lung adenocarcinoma cells possibly by inhibiting cell autophagy mediated by the AMPK/mTOR signaling pathway.

Key words: LINC00467, lung adenocarcinoma, autophagy, proliferation, metastasis, AMPK/mTOR signaling pathway

Yonghua LI, Xinran XI, Meng ZHANG, Xun WU, Xianghai WANG. High expression of LINC00467 promotes proliferation and metastasis of lung adenocarcinoma cells by suppressing autophagy via inhibiting the AMPK/mTOR pathway[J]. Journal of Southern Medical University, 2024, 44(10): 1898-1909.

Figures/Tables 14

Fig.1 Expression of LINC00467 in human bronchial epithelial cells 16HBE and lung adenocarcinoma cell lines A549 and H1299. ***P<0.001 vs 16HBE cells.

Fig.2 Expression of LINC00467 in lung adenocarcinoma tissue based on data from TCGA database. ***P<0.001 vs Normal tissue.

Fig.3 Correlation of LINC00467 expression level with survival rates (A) and clinical stages (B) of lung adenocarcinoma patients. *P<0.05 vs Stage I group; **P<0.01 vs Stage II group.

Fig.4 Interference efficiency of Shlinc00467 in A549 and H1299 cells. A: Transfection effect of Shlinc00467 (Original magnification: ×10); B: Quantification of the interference efficiency. **P<0.01 vs shNC group.

Fig.5 Effect of LINC00467 knockdown on proliferation of lung adenocarcinoma cells. A: Colony formation assay for assessing cell proliferation. B: Quantification of the colony number. **P<0.01 vs shNC group.

Fig.6 Effect of LINC00467 knockdown on lung adenocarcinoma cell metastasis. A: Transwell assay for assessing cell migration and invasion (×10). B, C: Numbers of migrating and invading cells. ***P<0.001 vs shNC group.

Fig.7 Effects of LINC00467 knockdown on autophagy and AMPK/mTOR signaling pathways in lung adenocarcinoma cells. A: Immunofluorescence staining for detecting LC3 expression (×40). B, D: Western blotting of AMPK, p-AMPK, mTOR, p-mTOR, and LC3II/I proteins. C, E: Expression levels of p-AMPK/AMPK, p-mTOR/mTOR, LC3II/I, and p62. *P<0.05, **P<0.01 vs shNC group.

Fig.8 GSEA enrichment analysis of the relationship between LINC00467 and autophagy pathway in lung adenocarcinoma tissue.

Fig.9 Effects of the autophagy inhibitor on autophagy of lung adenocarcinoma cells with LINC00467 knockdown. A: Immunofluorescence staining for LC3 expression (×40). B, C: Western blotting for detecting LC3II, LCI and p62 proteins. D-G: Quantification of LC3II/I and p62 protein levels.*P<0.05, **P<0.01, ***P<0.001 vs shNC group; #P<0.05, ##P<0.01 vs shlinc00467 group.

Fig.10 LINC00467 knockdown inhibits proliferation of lung adenocarcinoma cells by suppressing autophagy. A: Colony formation assay for evaluating cell proliferation. B: Quantification of the colony number. ***P<0.001 vs shNC group; ###P<0.01 vs shlinc00467 group.

Fig.11 LINC00467 knockdown suppresses lung adenocarcinoma cell metastasis by regulating autophagy. A, C: Transwell assay for assessing cell migration and invasion (×10). B, D: Numbers of migrating and invading cells. **P<0.01 vs shNC group; #P<0.05, ##P<0.01, ###P<0.001 vs shlinc00467 group.

Fig.12 LINC00467 knockdown suppresses autophagy in lung adenocarcinoma cells by inhibiting the AMPK/mTOR signaling pathway. A,C: Western blotting for detecting AMPK, p-AMPK, mTOR, p-mTOR, LC3II, LC3I, and p62 proteins. B, D: Quantification of p-AMPK/AMPK, p-mTOR/mTOR, LC3II/I, and p62 protein levels. *P<0.05, **P<0.01 vs shNC group; #P<0.05 vs shlinc00467 group.

Fig.13 LINC00467 knockdown suppresses lung adenocarcinoma cell proliferation by inhibiting the AMPK/mTOR signaling pathway. A: Colony formation assay for assessing cell proliferation. B: Quantification of colony formation number. *P<0.05, **P<0.01, ***P<0.001 vs shNC group; #P<0.05, ##P<0.01 vs shlincC00467 group.

Fig.14 LINC00467 knockdown suppresses lung adenocarcinoma cell migration by inhibiting the AMPK/mTOR signaling pathway. A, C: Transwell assay for assessing cell migration and invasion (×10). B, D: Number of migrating and invading cells. *P<0.05, **P<0.01 vs shNC group; ##P<0.01 vs shlinc00467 group.

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