KPNA2 promotes osteosarcoma by regulating the alternative splicing of DDX3X mediated by YBX1

Osteosarcoma (OS) is a rapidly progressive primary malignant bone tumor that usually occurs in adolescents between 15 and 19 years old and adults over 60 years old. Over the past 20 years, limited progress has been made in neoadjuvant chemotherapy and surgery aimed at curing OS. It is well known that alternative splicing (AS) changes caused by abnormal splicing factors contribute to tumor progression. But at present, there is a lack of extensive and in-depth AS research on OS. Gene expression analysis and AS analysis were performed on the sequencing data of 44 patients with osteosarcoma in order to construct a co-expression network among RNA-binding proteins, AS events, and AS genes in the whole genome. The gain or loss of functional osteosarcoma cell model was made, and the osteosarcoma phenotype was proved by in vitro and in vivo experiments. Interactive network analysis and enrichment analysis were carried out to define the internal mechanism.We screened the RBP of Karyopherin Subunit Alpha 2 (KPNA2), which was highly expressed in osteosarcoma cells and negatively correlated with patient survival. KPNA2 transports splicing factor Y-box Binding Protein 1 (YBX1) into the nucleus and promotes the proliferation, migration, and invasion of osteosarcoma. Using RNA-seq, we comprehensively screened and identified multiple AS events affected by YBX1. Specifically, YBX1 accelerates the degradation of ATP-dependent RNA helicase DDX3X (DDX3X) through the Nonsense-mediated decay (NMD) pathway by promoting the intron retention of the DDX3X gene, thus reducing the level of DDX3X protein. The changes in DDX3X in OS will affect significant changes in cell cycle-related proteins, including p53, p21, and AKT1. The KPNA2/YBX1 axis can regulate the stability of DDX3X mRNA and then affect the progress of the cell cycle. YBX1 promotes the proliferation, migration, and invasion of osteosarcoma by regulating the AS event of DDX3X. KPNA2/YBX1 axis activation is an important factor driving abnormal AS in OS. Importantly, we demonstrated that therapeutic targeting of the KPNA2/YBX1/DDX3X axis can inhibit OS proliferation and disease progression. It integrates the AS control of DDX3X into the progression of OS and represents potential prognostic biomarkers and targets of OS therapy.