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Apelin aggravates the migration and invasion of nonsmall cell lung cancer cells via YAP1
doi: 10.2478/fzm-2022-0007
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Abstract:
Background Apelin, an endogenous ligand of G-protein coupled receptor (GPCR), is a secreted peptide involved in the development of various tumors. However, the relationship between apelin and nonsmall cell lung cancer (NSCLC) is not quite clear. This study was designed to investigate the effect and mechanism of apelin on cell proliferation, migration and invasion of NSCLC cells. Methods Twelve NSCLC specimens were collected for hematoxylin-eosin (HE) staining and immunohistochemistry analyses. Cell proliferation was examined by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and cell migration and invasion were assessed using wound-healing and transwell assays. The subcellular location of yes associated protein 1 (YAP1) in A549 cells was determined by immunofluorescence. The mRNA and protein levels in NSCLC tissues and cell lines were measured by qRT-PCR and western blot, respectively. Results Apelin was upregulated in tumor tissues compared with the adjacent tissues. Apelin promoted proliferation, migration, and invasion of A549 and H460 cells, which was reversed by competitive apelin receptor (APJ) antagonist ML221. Additionally, apelin upregulated YAP1 expression, whereas silence of YAP1 by small interfering RNA (siRNA) attenuated apelin-induced cell proliferation, migration and invasion and suppressed epithelial-mesenchymal transition progression. Conclusion Apelin promotes NSCLC cells proliferation, migration, and invasion by modulating YAP1 and might be a potential therapeutic target for NSCLC treatment. -
Figure 1. Exaggerated expression of apelin in NSCLC specimens
(A) HE staining of human NSCLC tissues and matched adjacent tissues. (B) Representative IHC imaging of apelin in NSCLC tissues. Scale bar, 50 μmol/L. (C) qRT-PCR analysis of apelin mRNA levels in human NSCLC tissues and paired adjacent tissues. N = 8, *P < 0.05. (D) Representative western-blot outcomes of apelin in 12 pairs of normal (N) and tumor (T) samples of NSCLC; Scatter diagram on the right panel represents relative apelin expression. N = 12, *P < 0.05. (E) Western-blot assay of apelin protein levels in three NSCLC cell lines was performed.
Figure 2. Effects of different concentrations of apelin on NSCLC
Effect of apelin on cell viability was evaluated by MTT assay in A549 (A) and H460 (B) cells. N = 5, *P < 0.05. (C) The wound-healing assay were used to identify the promigration effect of apelin in A549 cells. N = 3, *P < 0.05.
Figure 3. Apelin promotes A549 and H460 cells proliferation, migration and invasion via APJ
MTT analysis of cell viability in A549 (A) and H460 (B) cells with apelin and APJ antagonist ML221. Wound-healing assays for A549 cells (C) and H460 cells (D) with apelin and ML221 at 0, 24, 48 h after scratching. N = 6, *P < 0.05, **P < 0.01. Transwell assay detected the migration (E) and invasion (F) of A549 and H460 cells with apelin and ML221. Cell counts were for the corresponding assays of at least four random microscope fields. N = 3, **P < 0.01.
Figure 4. Apelin activates YAP1 signaling
(A and B) Immunofluorescence assays showed that the apelin upregulated YAP1 expression in nuclear in A549 and H460 cells. (C and D) Western-blot analysis showing the expression of YAP1 in the presence of apelin treatment in A549 and H460 cells. N = 6, *P < 0.05, **P < 0.01.
Figure 5. Apelin regulates cell migration and invasion through YAP1
Cell viability in A549 (A) and H460 (B) cells with YAP1 silencing. N = 6, *P < 0.05. Wound-healing assays of A549 (C) and H460 (D) cells with YAP1 silencing at 0, 24, 48 h after scratching. N = 6, *P < 0.05. The migration and invasion abilities of A549 (E) and H460 (F) cells after transfection with YAP1 siRNA. Cell counts were for the corresponding assays of at least four random microscope fields. N = 3, *P < 0.05.
Figure 6. Apelin regulates EMT program by modulating YAP1
(A and B) Western-blot analysis showed the expression of EMT-relevant proteins (FN-1, E-cadherin, Vimentin) after knockdown of YAP1 in the presence of apelin treatment. N = 6, *P < 0.05. (C and D) Effect of YAP1 on apelin induced EMT was determined by immunofluorescence in H460 and A549 cells.
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