WTAP Mediated the N6-methyladenosine Modification of PDK4 to Regulate the Malignant Behaviors of Colorectal Cancer Cells In Vitro and In Vivo.

Abstract

The role of WT1-associated protein (WTAP) in mediating the N6-methyladenosine (m6A) modification of pyruvate dehydrogenase kinase 4 (PDK4) in colorectal cancer (CRC) has been previously reported. This research manages to unveil the function and mechanism of WTAP mediating the m6A modification in CRC. Expressions of PDK4 and WTAP in CRC were assessed by bioinformatics analysis and verified by Western blot. After the transfection with short hairpin RNAs (shRNAs) for WTAP (shWTAP) and PDK4 (shPDK4) to manipulate the expressions of PDK4 and WTAP, the viability, proliferation, migration, invasion, and levels of m6A, PDK4 and WTAP in CRC cells were determined by cell counting kit-8 (CCK-8), colony formation, transwell, Western blot, or M6A-RNA immunoprecipitation (MeRIP)-qPCR assays. M6A binding sites in PDK4 were additionally predicted through bioinformatics analysis, and the interaction of PDK4 and WTAP was confirmed using an RNA pull-down assay. Tumor volume and weight in the constructed xenograft-tumor mouse model were recorded. PDK4 expression was low, yet WTAP and m6A expressions were high in CRC cells. WTAP bound with the m6A binding sites in PDK4. PDK4 silencing facilitated the viability, proliferation, migration and invasion, inhibited the expression of PDK4 in CRC cells, and accelerated the growth of xenografts in vivo. However, the depletion of WTAP4 exerted the opposite effects and further offset the impact of PDK4 silencing. WTAP mediates the m6A modification of PDK4 to regulate the malignant behaviors of CRC cells in vitro and in vivo.