WTAP-Mediated m6A Modification of TRAIL-DR4 Suppresses MH7A Cell Apoptosis.

Abstract

Background: N6-methyladenosine (m6A) is one of the most conserved internal RNA modifications, which has been implicated in many biological processes, such as apoptosis and proliferation. Wilms tumor 1-associating protein (WTAP), as a key component of m6A methylation, is a nuclear protein that has been associated with the regulation of proliferation and apoptosis. Rheumatoid arthritis (RA), a systemic, infiltrating autoimmune disease, is characterized by synovial hyperplasia. However, little is known about the precise role of WTAP in RA. This study investigated the role of the WTAP-mediated m6A modification of TNF-related apoptosis-inducing ligand death receptor 4 (TRAIL-DR4) in RA.

Method: Methyltransferase WTAP overexpression plasmids and small interfering RNAs were constructed and transfected into MH7A cells. Immunofluorescence (IF) staining, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and Western blot were used to detect changes in the expression of WTAP, the B-cell lymphoma 2 (BCL2) gene family, BCL2-associated X (BAX) and TRAIL-DR4 expression, and the effects of WTAP overexpression on cell viability, cell cycle, apoptosis, and proliferation were assessed by a cell counting kit-8 (CCK-8), flow cytometry, and transmission electron microscopy (TEM). The m6A modification of TRAIL-DR4 was verified by m6A methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) and its stability was assessed by an actinomycin D assay.

Results: Overexpression of WTAP not only increased the levels of WTAP and BCL2, and decreased the levels of BAX and TRAIL-DR4, but also significantly inhibited MH7A cell apoptosis and promoted cell viability and proliferation, while WTAP silencing led to the opposite trend. The SRAMP online database predicted that TRAIL-DR4 has multiple potential methylation-binding sites, and fluorescence in situ hybridization (FISH) combined with IF showed that WTAP and TRAIL-DR4 were mainly expressed in both the nucleus and cytoplasm. MeRIP-qPCR and actinomycin D analysis experiments revealed that WTAP could promote the m6A level of TRAIL-DR4, decrease the stability of TRAIL-DR4 mRNA, and subsequently inhibit apoptosis.

Conclusion: This study suggests that WTAP-mediated m6A modification of TRAIL-DR4 suppresses MH7A cell apoptosis. This discovery offers a new focus and avenue for the clinical treatment of RA, while also extending our understanding of the pathophysiology of RA from the standpoint of m6A alteration.