RNA Binding protein KPNA2 promotes the progress of gastric cancer by regulating alternative splicing of related genes [RIP-seq]. RNA Binding protein KPNA2 promotes the progress of gastric cancer by regulating alternative splicing of related genes [RIP-seq]

RNA binding proteins (RBPs) took a critical part in genome regulation. kpna2, as an essential member of the RBP family, was found to be highly expressed and associated with lymph node metastasis in gastric cancer (GC). Transcriptional sequencing data were used to find that KPNA2 primarily regulated alternative 5' splice site (A5SS), alternative 3'splice site (A3SS), exon skipping (ES) and cassette exon. Vitally, KPNA2 was involved in the transcriptional regulation of cancer-related genes. Therefore, we screened potential targets in AGS cells with differential expression and alternative splicing (AS) levels regulated by KPNA2, further identifying that KPNA2 was engaged in biological processes concerning immune response, cell proliferation and apoptosis in GC through transcriptional regulation. Besides, the binding sites of KPNA2 to spliceosomes were identified using improved RNA immunoprecipitation sequencing techniques, and KPNA2 was preferentially bound to the intron region. Notably, KPNA2 regulated the A3SS AS mode of WDR62, which may increase the malignancy of GC. Finally, we also discovered that AS of immune-related molecules can be regulated by KPNA2. Overall, for the first time, our results demonstrated that KPNA2 served as an oncogenic splicing factor in GC regulating AS and differential expression of GC-related genes and may represent a potential target for GC therap Overall design: RIP-seq analysis of KPNA2 IP versus input in AGS cells

RNA结合蛋白（RNA binding proteins, RBPs）在基因组调控中发挥关键作用。KPNA2作为RBP家族的核心成员，被证实在胃癌（gastric cancer, GC）中高表达，且与淋巴结转移密切相关。利用转录测序数据，研究发现KPNA2主要调控可变5'剪接位点（alternative 5' splice site, A5SS）、可变3'剪接位点（alternative 3' splice site, A3SS）、外显子跳跃（exon skipping, ES）以及盒式外显子事件。至关重要的是，KPNA2参与了癌症相关基因的转录调控过程。据此，本研究在AGS细胞中筛选出受KPNA2调控的差异表达基因与可变剪接（alternative splicing, AS）水平异常的潜在靶点，并进一步证实KPNA2可通过转录调控参与胃癌免疫应答、细胞增殖与凋亡相关的生物学进程。此外，通过优化的RNA免疫沉淀测序（RNA immunoprecipitation sequencing, RIP-seq）技术，本研究鉴定了KPNA2与剪接体的结合位点，且发现KPNA2优先结合内含子区域。值得注意的是，KPNA2可调控WDR62的A3SS可变剪接模式，该机制可能增强胃癌的恶性表型。最后，本研究还发现KPNA2能够调控免疫相关分子的可变剪接事件。总体而言，本研究首次证实KPNA2作为胃癌中的致癌性剪接因子，可调控胃癌相关基因的可变剪接与差异表达，有望成为胃癌治疗的潜在靶点。整体实验设计：在AGS细胞中对KPNA2免疫沉淀（IP）样本与输入对照样本进行RIP-seq分析