Meibomian Gland Carcinoma (MGC) is a highly malignant eyelid tumor with a poor prognosis. This study investigates the molecular mechanisms underlying MGC, focusing on the abnormal expression of E2F transcription factor 2 (E2F2), often observed in tumors and potentially linked to DNA methylation.E2F2 expression was measured in MGC cells and tissues. Tissue samples from 3 normal meibomian glands (MG) and 36 MGC patients were used to construct a tissue microarray. Functional assays were performed by modifying E2F2 expression, including CCK8, wound healing, Transwell, and analysis of epithelial-mesenchymal transition (EMT)-related markers. Flow cytometry was used to assess cell apoptosis and cell cycle. RNA sequencing was conducted to identify differential genes after treating MGC cells with the methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-2-dc), to explore the relationship between E2F2 downregulation in MGC and methylation.E2F2 expression was significantly lower in MGC cells compared to normal MG cells. Immunohistochemical analysis showed low E2F2 expression in MGC. Specifically, immunohistochemical staining results have revealed a negative correlation trend between E2F2 and Ki-67 expression, as well as a positive correlation trend between E2F2 and P21, P27 expression. E2F2 knockdown increased MGC cell proliferation, migration, and invasion. Flow cytometry revealed that E2F2 knockdown reduced apoptosis, decreased the G0/G1 phase, and increased the S phase, while E2F2 overexpression produced opposite effects. RNA sequencing revealed that a total of 87 genes were differentially expressed in the 5-aza-2-dc experimental group compared to the control group, with 72 mRNAs showing upregulated expression and 15 mRNAs showing downregulated expression. Bioinformatics analysis results indicated that the functions of these differentially expressed genes were concentrated, and the biological processes mainly involved DNA replication, among others. The signaling pathways associated with these genes primarily included DNA replication and the cell cycle. RNA sequencing identified differential gene expression after methylation inhibition in MGC cells with 5-aza-2-dc, demonstrating that demethylation significantly upregulated E2F2. MSP assays confirmed reduced methylation levels. Additionally, inhibiting gene methylation in MGC cells suppressed proliferation, migration, and invasion.E2F2 presents a promising therapeutic target for MGC. Overexpression of E2F2 and methylation inhibition in MGC cells may reverse E2F2 gene silencing, inhibiting malignant progression. These findings provide new perspectives for targeted therapies and precise, individualized treatment in MGC.© 2025. The Author(s).