Data Sheet 1_HPV-driven transcriptome and splicing rewiring under SRPK1 inhibition in cervical cancer.xlsx

BackgroundSerine/arginine protein kinase 1 phosphorylates serine-arginine-rich (SR) proteins to regulate splice-site selection during alternative splicing. While its role in general RNA regulation is established, its contribution to the HPV-dependent transcriptome and splicing stratification in cervical cancer remains unclear. Therefore, we sought to determine how SRPK1 inhibition differentially remodels gene expression and alternative splicing in HPV+ versus HPV- cervical cancer cells.
MethodsHPV16+ SiHa and HPV- C33A cervical cancer cells were treated with the SRPK1 inhibitor, SPHINX31. RNA profiling was performed, and differentially expressed genes were defined as |log2FC| ≥ 1.5. AS events were classified by SUPPA as exon skipping (SE), intron retention (RI), mutually exclusive exons (MXE), alternative 3′ splice site (A3SS), and alternative 5′ splice site (A5SS). Pathway enrichment was assessed using Gene Ontology/KEGG, STRING protein-protein interaction (PPI) networks, and Molecular Complex Detection (MCODE) was used to identify protein hubs. To determine computational prediction of docking, SPHINX31 was docked into SRPK1 (PDB 5MY8) using SP/XP docking and MM-GBSA rescoring.
ResultsSRPK1 inhibition was associated with distinct responses that were HPV-related. In C33A cells, upregulated genes were enriched for translation, RNA processing, and glycosylation, with KEGG highlighting ribosome and metabolic modules. Ribosomal hubs dominated the PPI/MCODE, suggesting possible translational and metabolic adjustments. In contrast, SiHa cells exhibited transcriptomic changes consistent with reduced expression of genes linked to Hippo, Wnt, PI3K-AKT, ERK1/2 signaling, migration, angiogenesis, and growth factor cytokine networks. Targets of YAP/TAZ (e.g., CCND1, BIRC5, SNAI2, SERPINE1) and their regulators (RASSF1, CSNK1E) were suppressed. At the splicing level, SiHa cells displayed fewer total AS events but with larger effect sizes, particularly in A3SS/A5SS. C33A cells showed abundant SE (59,234 events; small median ΔPSI) and RI (1,770 events, often binary), including complete shifts in HLA-DRB1/PLIN2 (+1.00) and KLF4 (-1.00). Notable A5SS switches included LEF1 (+1.00) and CDK6 (-1.00) in C33A, and DLX1/MRPL14/THAP5 (-1.00) in SiHa. Docking computationally predicted the low-energy poses of SPHINX31 in the SRPK1 ATP pocket. While not definitive, this evidence may potentially support the transcriptomic and splicing findings.
ConclusionSRPK1 inhibition may remodel the cervical cancer transcriptome in an HPV-linked manner, with SiHa cells exhibiting changes consistent with suppression of oncogenic signaling, whereas C33A cells adapt through translational and metabolic reprogramming.

背景
丝氨酸/精氨酸蛋白激酶1（Serine/arginine protein kinase 1, SRPK1）可磷酸化富含丝氨酸-精氨酸（serine-arginine-rich, SR）的蛋白，在可变剪接过程中调控剪接位点的选择。尽管其在通用RNA调控中的作用已得到证实，但它在人乳头瘤病毒（human papillomavirus, HPV）依赖的转录组及宫颈癌剪接分层中的贡献仍不明确。因此，本研究旨在探究SRPK1抑制如何在HPV阳性（HPV+）与HPV阴性（HPV-）宫颈癌细胞中差异性重塑基因表达与可变剪接。

方法
将HPV16阳性的SiHa细胞与HPV阴性的C33A宫颈癌细胞用SRPK1抑制剂SPHINX31处理后，开展RNA表达谱分析。将|log₂FC|≥1.5的基因定义为差异表达基因。利用SUPPA工具对可变剪接（alternative splicing, AS）事件进行分类，包括外显子跳跃（exon skipping, SE）、内含子保留（intron retention, RI）、互斥外显子（mutually exclusive exons, MXE）、可变3'剪接位点（alternative 3' splice site, A3SS）以及可变5'剪接位点（alternative 5' splice site, A5SS）。采用基因本体论（Gene Ontology, GO）/京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）进行通路富集分析，借助STRING数据库构建蛋白质-蛋白质相互作用（protein-protein interaction, PPI）网络，并通过分子复杂检测（Molecular Complex Detection, MCODE）工具识别蛋白枢纽。为实现分子对接的计算预测，采用SP/XP对接与MM-GBSA重打分方法，将SPHINX31对接至SRPK1蛋白结构（PDB编号5MY8）中。

结果
SRPK1抑制引发的细胞应答呈现HPV相关性特征。在C33A细胞中，上调基因富集于翻译、RNA加工及糖基化通路，KEGG分析显示其显著富集于核糖体与代谢模块；PPI/MCODE分析显示核糖体枢纽占据主导地位，提示细胞可能发生翻译与代谢重编程。与之相反，SiHa细胞的转录组变化与致癌信号通路的表达下调高度一致，涉及的通路包括Hippo、Wnt、PI3K-AKT、ERK1/2信号通路，以及细胞迁移、血管生成与生长因子细胞因子网络。YAP/TAZ的靶基因（如CCND1、BIRC5、SNAI2、SERPINE1）及其调控因子（RASSF1、CSNK1E）的表达均被抑制。在剪接层面，SiHa细胞的总可变剪接事件更少，但效应量更大，尤其体现在A3SS/A5SS事件中。C33A细胞则存在大量的SE事件（59234个事件，ΔPSI中位数较小）与RI事件（1770个事件，多为二元变化），包括HLA-DRB1/PLIN2（ΔPSI=+1.00）与KLF4（ΔPSI=-1.00）的完全剪接转换。值得关注的A5SS转换事件包括C33A细胞中的LEF1（+1.00）与CDK6（-1.00），以及SiHa细胞中的DLX1/MRPL14/THAP5（-1.00）。分子对接计算预测了SPHINX31在SRPK1 ATP结合口袋中的低能构象。尽管并非确定性证据，但该发现或可为转录组与剪接分析结果提供辅助支撑。

结论
SRPK1抑制可通过HPV依赖的方式重塑宫颈癌转录组：SiHa细胞的转录变化与致癌信号通路的抑制相一致，而C33A细胞则通过翻译与代谢重编程产生适应性改变。