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Proteomic analysis of BEAS-2B lung epithelial cells after the introduction of the BRAF V600E mutation using CRISPR/Cas9

ABSTRACT: To mimic the naturally occurring BRAF V600E mutation in lung cancer, a BRAF V600E knock-in BEAS-2B cell model was established using CRISPR/Cas9. The proteomics analysis was carried out to evaluate the underlying changes at the proteome level. Proteomics analysis revealed significant changes in the proteins involved in the biological processes including epithelial-mesenchymal transition (EMT), extracellular matrix (ECM)-receptor interaction, cell adhesion, focal adhesion, and cell metabolism upon BRAF V600E mutation.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Non-small Cell Lung Carcinoma

SUBMITTER: Lingyun Dai

LAB HEAD: Lingyun Dai

PROVIDER: PXD058027 | Pride | 2025-04-22

REPOSITORIES: Pride

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			Action	DRS
	20230116_BEAS_2B_VE_1.raw	Raw
	20230116_BEAS_2B_VE_1.raw.quant	Raw
	20230116_BEAS_2B_VE_2.raw	Raw
	20230116_BEAS_2B_VE_2.raw.quant	Raw
	20230116_BEAS_2B_VE_3.raw	Raw

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Project description:Background: In classrooms high concentrations of particulate matter PM10 were measured. It is unknown whether the hazard of indoor particles is similar to that of the better studied outdoor particles. This study therefore analyzed adverse biological effects of classroom in comparison to outdoor PM10. Methods: Samples were taken from six schools during teaching hours. Genome-wide gene expression in human bronchial BEAS-2B epithelial cells was analyzed, and regulated genes were verified by quantitative PCR. Polycyclic aromatic hydrocarbons (PAH), endotoxin, and cat allergen Fel d 1 were analyzed with standard methods. Enhancement of allergic reactivity by PM10 was confirmed with CD63 upregulation in human primary basophils. Acceleration of human blood coagulation was determined with supernatants of PM10-exposed human peripheral blood monocytes. Results: Indoor PM10 induced SERPINB2 (involved in blood coagulation) and inflammatory genes (like CXCL6, CXCL1, IL6, IL8, all p<0.001). Outdoor PM10 induced xenobiotic metabolizing enzymes (CYP1A1, CYP1B1, TIPARP, all p<0.001). The induction of inflammatory genes by indoor PM10 could be explained by endotoxin (indoor 128.5M-BM-142.2EU/mg versus outdoor 13.4M-BM-121.5EU/mg, p<0.001), the induction of CYP by outdoor PAH (indoor 8.3M-BM-14.9ng/mg versus outdoor 16.7M-BM-115.2ng/mg, p<0.01). The induction of SERPINB2 was confirmed by a more rapid human blood coagulation (p<0.05). Indoor PM10 had no effect on the allergic reactivity from human primary basophils, except in cat allergic individuals. This was explained by varying Fel d 1 concentrations in indoor PM10 (p<0.001). Conclusions: Indoor PM10, compared to outdoor PM10, was 6 times higher, had a different composition, and on an equal weight basis induced more inflammatory and allergenic reactions, and accelerated blood coagulation. Outdoor PM10 had significantly lower effects, but induced detoxifying enzymes. Therefore, preliminary interventions for the reduction of classroom PM10 seem reasonable, perhaps by intensified ventilation. For genome-wide gene expression analysis, BEAS-2B cells (passage 41) were incubated with 10M-BM-5g/ml PM10 (school 4 indoor and outdoor) for 4, 10 or 24h, all in triplicate. experiment type : time course

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Proteomic analysis of BEAS-2B lung epithelial cells after the introduction of the BRAF V600E mutation using CRISPR/Cas9

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