Objective: To investigate the effect of serine/arginine-rich splicing factor 2 (SRSF2) on ferroptosis and its possible mechanism in glioblastoma cells. Methods: The online database of gene expression profiling interactive analysis 2 (GEPIA 2) and Chinese Glioma Genome Atlas were used to analyze the expression of SRSF2 in glioblastoma tissue and its association with patients prognosis. To validate the findings of the online databases, the pathological sections of glioblastoma and non-tumor brain tissues from Tianjin Medical University General Hospital, Tianjin, China were collected and analyzed by using immunohistochemistry. Silencing SRSF2 gene expression in glioblastoma cells by siRNA was analyzed with Western blot. The proliferation index was detected by using CCK8 assay. The rescued experiment was conducted by using expression plasmid of pcDNA3.1(+)-SRSF2. The activity of ferroptosis was assessed by using the levels of iron ions and malondialdehyde in glioblastoma cells and the changes in the ratio of glutathione to oxidized glutathione. The changes of gene expression and differential pre-mRNA alternative splicing (PMAS) induced by SRSF2 were monitored by using the third-generation sequencing technology analysis, namely Oxford nanopore technologies (ONT) sequencing analysis. Results: SRSF2 expression was higher in glioblastoma tissues than non-tumor brain tissues. Immunohistochemistry also showed a positive rate of 88.48%±4.60% in glioblastoma tissue which was much higher than the 9.97%±4.57% in non-tumor brain tissue. The expression of SRSF2 was inversely correlated with overall and disease-free disease survivals (P<0.01). The proliferation index of glioblastoma cells was significantly reduced by silencing with SRSF2 siRNA (P<0.01) and could be reversed with transfection of exogenous SRSF2. The levels of intracellulariron ions and malondialdehyde increased (P<0.05), but the glutathione/oxidized glutathione ratio and the expression of key proteins in the glutathione pathway remained unchanged (P>0.05). ONT sequencing results showed that silencing SRSF2 in glioblastoma cells could induce a significant alternative 3' splice site change on ferroptosis suppressor protein 1 (FSP1). Conclusion: SRSF2 inhibits the ferroptosis in glioblastoma cells and promotes their proliferation, which may be achieved by regulating FSP1 PMAS.
目的： 探讨富含丝氨酸/精氨酸剪接因子2（serine/arginine-rich splicing factor 2，SRSF2）对胶质母细胞瘤细胞铁死亡的影响及其可能的作用机制。 方法： 结合基因表达谱交互分析版本2（gene expression profiling interactive analysis 2，GEPIA2）与中国脑胶质瘤基因组图谱计划（Chinese Glioma Genome Atlas，CGGA）在线数据库，对收集的天津医科大学总医院胶质母细胞瘤组织和非肿瘤脑组织病理切片进行免疫组织化学染色，分析SRSF2在胶质母细胞瘤组织中的表达情况及其与患者预后的关系；运用小干扰RNA（siRNA）技术靶向沉默胶质母细胞瘤细胞中的SRSF2基因，经蛋白免疫印迹（Western blot）和CCK8法检测其沉默SRSF2的效果及其对细胞增殖能力的影响；利用表达质粒pcDNA3.1（+）-SRSF2进行挽救实验；通过检测胶质母细胞瘤细胞中铁离子和丙二醛的水平，以及谷胱甘肽和氧化型谷胱甘肽的比值变化研究铁死亡活性；牛津纳米孔技术（Oxford nanopore technologies，ONT）3代全长转录组测序分析SRSF2沉默后诱导的差异基因表达水平和差异选择性剪接（pre-mRNA alternative splicing，PMAS）变化。 结果： 与非肿瘤脑组织相比，SRSF2在胶质母细胞瘤组织中呈高表达，免疫组织化学结果阳性细胞率达88.48%±4.60%，远超非肿瘤脑组织中的9.97%±4.57%；高表达与患者总生存期和无病生存期呈负相关（P<0.01）；经siRNA靶向沉默SRSF2的胶质母细胞瘤细胞增殖能力明显降低（P<0.01），引入外源性SRSF2后增殖能力恢复；SRSF2 siRNA处理的胶质母细胞瘤细胞内铁离子和丙二醛水平升高（P<0.05），谷胱甘肽/氧化型谷胱甘肽比值及谷胱甘肽途径中关键蛋白的表达均无明显变化（P>0.05）；转录组测序结果显示SRSF2沉默后胶质母细胞瘤细胞中铁死亡抑制蛋白1（ferroptosis suppressor protein 1，FSP1）发生显著的3′端PMAS改变。 结论： 胶质母细胞瘤细胞中SRSF2抑制铁死亡、促进增殖可能是通过调控FSP1 PMAS实现的。.