PO.ET01.04 · 实验与分子治疗
SRPK1抑制对宫颈癌HPV⁺和HPV⁻的差异效应：转录组和剪接重塑
Differential effects of SRPK1 inhibition on HPV⁺ and HPV⁻ in cervical cancer: Transcriptome and splicing rewiring
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摘要 Abstract
中文摘要
背景：宫颈癌（CCa）仍是全球癌症相关死亡的主要原因之一，很大程度上由高危型人乳头瘤病毒（hrHPV）（如HPV16和HPV18）的持续感染所驱动。病毒E6和E7癌蛋白破坏p53和Rb抑癌通路，并重编程宿主RNA加工，导致产生促肿瘤的剪接异构体。丝氨酸/精氨酸蛋白激酶1（SRPK1）是剪接因子磷酸化的关键调节因子，已成为一个潜在的治疗靶点。本研究旨在鉴定和评估宫颈癌对SRPK1抑制的剪接模式反应。
材料与方法：用SRPK1抑制剂SPHINX31（0.3-10 μM）处理HeLa（HPV18⁺）、SiHa（HPV16⁺）和C33A（HPV⁻）宫颈癌细胞系。使用Alamar Blue实验评估细胞活力，同时通过流式细胞术采用荧光激活细胞分选（FACS）和Annexin V/碘化丙啶染色评估细胞周期进程和凋亡。通过RNA测序进行转录组分析，以鉴定差异表达基因和可变剪接（AS）事件，随后进行通路富集和蛋白-蛋白相互作用（PPI）/MCODE网络分析。采用分子对接评估SPHINX31在SRPK1 ATP口袋内的结合。
结果与讨论：用SPHINX31处理宫颈癌细胞导致无显著的细胞反应，对细胞活力、细胞周期进程或凋亡诱导均无影响。在HPV阴性的C33A细胞中，SRPK1抑制上调了参与翻译、RNA加工和糖基化的基因，揭示了核糖体网络枢纽，提示可能的翻译和代谢适应。C33A还表现出外显子跳跃（SE）和内含子保留（RI）事件以及可变5'剪接位点（A5SS）改变。相比之下，HPV16⁺的SiHa细胞表现出致癌信号通路的下调，包括Hippo、Wnt、PI3K-AKT和ERK1/2。SiHa表现出较少的整体AS事件，但效应量更大。分子对接分析支持了计算预测的结合。
结论：本研究提供了关于SRPK1抑制对细胞反应、剪接模式和转录组图谱影响的见解。总体而言，SRPK1抑制在CCa中可能诱导因HPV状态而异的变化，HPV⁺细胞表现出易感性，而HPV⁻细胞可能表现出可能的代谢适应。
查看英文原文 English abstract
Background: Cervical cancer (CCa) remains a major cause of cancer-related mortality worldwide, largely driven by persistent infection with high-risk human papillomavirus (hrHPV) types such as HPV16 and HPV18. The viral E6 and E7 oncoproteins disrupt the p53 and Rb tumour suppressor pathways and reprogram host RNA processing, resulting in the production of tumour-promoting splice isoforms. Serine/arginine protein kinase 1 (SRPK1), a key regulator of splicing factor phosphorylation, has emerged as a potential therapeutic target. This study aimed to identify and evaluate splicing patterns in cervical cancer in response to SRPK1 inhibition.
Materials and Methods: HeLa (HPV18⁺), SiHa (HPV16⁺), and C33A (HPV⁻) cervical cancer cell lines were treated with the SRPK1 inhibitor SPHINX31 (0.3-10 μM). Cell viability was assessed using the Alamar Blue assay, while cell cycle progression and apoptosis were evaluated by flow cytometry using fluorescence-activated cell sorting (FACS) and Annexin V/propidium iodide staining. Transcriptomic profiling was performed by RNA sequencing to identify differentially expressed genes and alternative splicing (AS) events, followed by pathway enrichment and protein-protein interaction (PPI)/MCODE network analyses. Molecular docking was used to assess the binding of SPHINX31 within the SRPK1 ATP pocket.
Results and Discussion: Treatment of cervical cancer cells with SPHINX31 resulted in a non-significant cellular response, with no effect on cell viability, cell cycle progression, or induction of apoptosis. In HPV-negative C33A cells, SRPK1 inhibition upregulated genes involved in translation, RNA processing, and glycosylation, which revealed ribosomal network hubs suggesting possible translational and metabolic adaptation. C33A also displayed skipped exon (SE) and retained intron (RI) events along with alternative 5' splice site (A5SS) alterations. In contrast, HPV16⁺ SiHa cells exhibited downregulation of oncogenic signalling pathways, including Hippo, Wnt, PI3K-AKT, and ERK1/2. SiHa exhibited fewer overall AS events but with greater effect sizes. Molecular docking analyses supported a computationally predicted binding.
Conclusion: This study provides insight into the effects of SRPK1 inhibition on cellular response, splicing patterns, and the transcriptomic landscape. Overall, SRPK1 inhibition in CCa may induce changes that differ according to HPV status, with HPV⁺ cells exhibiting vulnerability, while HPV⁻ cells may display a possible metabolic adaptation.
利益披露 Disclosure
A. T. Basera, None..
M. Alaouna, None..
J. Duvanhage, None..
D. Bates, None..
Z. Dlamini, None..
R. Marima, None.