NSUN6-mediated m5C RNA methylation aggravate osteosarcoma progression through promoting PKP2 mRNA stability and expression.

Osteosarcoma (OS) is a highly aggressive malignant bone tumor with poor prognosis. Both Plakophilin 2 (PKP2) and NOP2/Sun RNA methyltransferase 6 (NSUN6) were reported to be upregulated in OS, and NSUN6 is one of the important RNA methyltransferases catalyzing 5-methylcytosine (m5C) formation and participates in many critical bioprocesses in various tumors. However, the roles and underlying molecular mechanisms of NSUN6-mediated m5C modification in OS remain unclear. Differentially expressed genes in OS and the expression of PKP2 were analyzed using the GSE126209 dataset of the GEO database. The mRNA and protein levels of PKP2 as well as NSUN6 in OS tissues and cells were then measured by RT-qPCR and western blot. Then, the relationship between the expression of PKP2 and the survival rate of OS patients was presented by the Kaplan-Meier (KM) survival curve. Subsequently, OS cell proliferation, migration, and apoptosis were assessed by colony formation, Transwell, and flow cytometry. Meanwhile, the mitochondrial membrane potential, the levels of MDA, ROS, and Fe2+ was determined by the corresponding kits. Besides, the binding of PKP2 and NSUN6 was verified by RIP assay. Dot blot and Me-RIP assay were used to evaluate the 5-methylcytosine (m5C) modification level of PKP2. Finally, a xenograft model was constructed to investigate the role of NSUN6/PKP2 axis in vivo. PKP2 was highly expressed in OS tissues and cells. PKP2 knockdown could effectively inhibit the proliferation and migration of OS cells, and promote their apoptosis, oxidative stress and ferroptosis. NSUN6 stabilized PKP2 expression through m5C methylation modification. Moreover, overexpression of PKP2 effectively reversed the effects of NSUN6 knockdown on OS cells and xenograft tumors in mice. NSUN6 regulated the m5C methylation modification of PKP2 to stabilize its expression, thereby driving the malignant progression of OS cells, providing a promising targeted therapeutic strategy for OS.