[Proteomic analysis of meibomian gland carcinoma cells after overexpression of thrombospondin 1].

Objective: To screen and validate key proteins involved in the progression of meibomian gland carcinoma (MGC) influenced by thrombospondin 1 (THBS1) overexpression through proteomic analysis. Methods: It was an experimental study conducted from February 2023 to June 2024. After lentiviral transfection, MGC cells were divided into the THBS1 overexpression group and the control group. Proteins were extracted from both groups for 4D label-free quantitative proteomic analysis. Functional annotation of differentially expressed proteins (DEPs) and their regulated signaling pathways was performed via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to validate the mRNA and protein expression levels of the top 5 DEPs. Results: Following successful THBS1 gene overexpression in MGC cells, 666 proteins were screened based on the criteria of |log₂(fold change)|>1.5 and P<0.05. GO analysis showed that DEPs were mainly localized in the cytosolic matrix and exosomes (cellular component), involved in RNA binding and cadherin binding (molecular function), and associated with translation and intracellular protein transport (biological process). KEGG pathway analysis indicated significant enrichment in DNA replication and cell cycle pathways. RT-qPCR results showed mRNA expression levels of asparagine-linked glycosylation 1 homolog (ALG1), AP2-associated protein kinase 1 (AAK1), Aladin WD repeat nucleoporin (AAAS), SUMO-specific peptidase 3 (SENP3), and Serrate RNA effector molecule homolog (SRRT) in the THBS1 overexpression group were 0.48±0.05, 0.83±0.04, 0.90±0.01, 0.73±0.06, and 0.92±0.02, respectively, significantly lower than those in the control group (1.00±0.03, 1.00±0.01, 1.00±0.03, 1.00±0.03, and 1.00±0.02; all P<0.05). Western blotting confirmed protein expression levels of ALG1, AAK1, AAAS, SENP3, and SRRT in the THBS1 overexpression group were 0.53±0.04, 0.86±0.04, 0.40±0.11, 0.59±0.01, and 0.63±0.05, respectively, significantly reduced compared with the control group (1.00±0.01, 1.00±0.03, 1.00±0.19, 1.00±0.14, and 1.00±0.01; all P<0.05). Conclusion: Among the key proteins regulated by THBS1 overexpression in the MGC progression, the top 5 DEPs were ALG1, AAK1, AAAS, SENP3, and SRRT.