Exosomal miR-92b-5p regulates N4BP1 to enhance PTEN mono-ubiquitination in doxorubicin-resistant AML

Aim: Doxorubicin, pivotal for acute myeloid leukemia (AML) treatment, often succumbs to resistance, impeding therapeutic success. Although exosomal transfer is linked to chemoresistance, the detailed role of exosomal miRNAs in doxorubicin resistance remains incompletely understood. Methods: We employed miRNA sequencing to delineate the profile of exosomal miRNAs in doxorubicin-resistant K562/DOX cells and AML patients. Subsequently, qPCR was utilized to scrutinize the expression of exosomal miR-92b-5p in these resistant cells and AML patients. A dual-luciferase reporter assay was conducted to elucidate the direct binding of miR-92b-5p to NEDD4 binding protein 1 (N4BP1). Furthermore, interactions between N4BP1 and NEDD4, as well as between NEDD4 and PTEN, were investigated by co-immunoprecipitation (Co-IP). Meanwhile, the ubiquitination of PTEN was also examined by Co-IP. Western blot analysis was applied to assess the expression levels of N4BP1, NEDD4, PTEN, RAD51, and proteins associated with the PI3K-AKT-mTOR pathway. Gain- and loss-of-function studies were conducted to ascertain the functional role of miR-92b-5p in doxorubicin resistance by using miR-92b-5p-mimic and miR-92b-5p-inhibitor transfections. Results: Our study found exosomal miR-92b-5p was upregulated both in doxorubicin-resistant cells and AML patients. Moreover, miR-92b-5p targets N4BP1, promoting NEDD4-mediated mono-ubiquitination of PTEN. This alters PTEN's subcellular localization, promoting nuclear PTEN and reducing cytoplasmic PTEN, which in turn leads to increased RAD51 for DNA repair and activation of the PI3K-AKT-mTOR pathway for cell proliferation, contributing to doxorubicin resistance. Conclusion: Our study reveals a novel mechanism of doxorubicin resistance mediated by exosomal miR-92b-5p and provides potential therapeutic targets for overcoming drug resistance in AML.