RBM39 promotes hepatocarcinogenesis by regulating RFX1's alternative splicing and subsequent activation of integrin signaling pathway

There is increasing evidence that alternative splicing (AS) is crucial for tumor cells because it can produce various protein isoforms that may play different or even opposing roles in tumor growth and metastasis. Despite its significance, the role of AS and related splicing factors, particularly splicing-related messenger ribonucleoproteins (mRNPs), in hepatocarcinogenesis is poorly understood. High-throughput transcriptome sequencing of HCC patients revealed that the spliceosome pathway may play an important role in HCC development. Through the combined analysis of the three gene clusters consisting of differentially expressed genes (DEGs), genes encoding RNA-binding proteins (RBPs) and genes related to splicing-related mRNPs, the splicing factor RBM39 was identified, which was highly expressed in HCC tumor tissues with prognostic value. Functional studies showed that RBM39 silencing inhibited cell proliferation, migration and invasion via the integrin pathway. By performing RNA immunoprecipitation sequencing (RIP-seq), we found that RBM39 combined with RFX1 pre-mRNA and regulated alternative splicing of its exon 2. Mechanistically, the skipping of exon 2 in RFX1, influenced by high RBM39 expression in HCC cells, led to production of an N-terminal truncated RFX1, which lost the ability to transcriptionally repress oncogenic collagen genes. This process further activated the downstream FAK/PI3K/AKT signaling pathway. Furthermore, we further validated these findings in the hydrodynamic tail vein injection (HTVI) mouse model and human HCC tissues. Therefore, we hypothesized that high RBM39 expression enhances the malignant capabilities of HCC cells by regulating the alternative splicing of RFX1 and subsequently activating the FAK/PI3K/AKT signaling pathway.