Bhandari Kritisha, Kong Jeng Shi, Ho Wang-Ting Tina, Bourne Philip C, Mooers Blaine H M, Ding Wei-Qun
Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma, USA.
FASEB J. 2025 Jan 15;39(1):e70287. doi: 10.1096/fj.202401811RR.
Small extracellular vesicles (sEVs) are enriched in certain miRNAs, impacting the progression of pancreatic ductal adenocarcinoma (PDAC). The mechanisms involved in the selective sEV miRNA enrichment remain to be elucidated. We recently reported that Serine/Arginine-rich splicing factor 1 (SRSF1) regulates selective sEV miRNA enrichment in PDAC cells. SRSF1 is an onco-protein that is overexpressed in PDAC, and its function is dictated by posttranslational modifications such as phosphorylation and arginine methylation. The objective of this study was to examine the role of phosphorylation and arginine methylation in SRSF1-mediated sEV miRNA enrichment in PDAC cells. Treatment of PDAC cells with the protein arginine methyltransferase inhibitors AMI-5 and EPZ015666, but not with the phosphorylation inhibitor SRPIN340, selectively enhanced the level of sEV miR-1246, a miRNA known to be highly enriched in PDAC sEVs. Consistently, overexpression of the mutant SRSF1 with the three arginine residues R93, R97, and R109 being replaced with lysinaugmented sEV miR-1246 levels in both wild-type and SRSF1-knockdown PANC-1 cells. Interestingly, the binding of SRSF1 to miR-1246 was significantly reduced in PDAC cells overexpressing the mutant SRSF1, which was further confirmed using purified wild-type and the mutant SRSF1 proteins. We demonstrate that arginine demethylation of SRSF1 reduces SRSF1-miRNA binding in PDAC cells and enhances selective sEV miRNA enrichment, providing novel insight into SRSF1-mediated sEV miRNA enrichment in PDAC cells and opening up new avenues of investigation on the biology and function of extracellular vesicles in PDAC.
小细胞外囊泡（sEVs）富含某些微小RNA（miRNAs），影响胰腺导管腺癌（PDAC）的进展。选择性sEV miRNA富集所涉及的机制仍有待阐明。我们最近报道，富含丝氨酸/精氨酸的剪接因子1（SRSF1）调节PDAC细胞中选择性sEV miRNA的富集。SRSF1是一种癌蛋白，在PDAC中过表达，其功能由磷酸化和精氨酸甲基化等翻译后修饰决定。本研究的目的是探讨磷酸化和精氨酸甲基化在SRSF1介导的PDAC细胞sEV miRNA富集中的作用。用蛋白质精氨酸甲基转移酶抑制剂AMI-5和EPZ015666处理PDAC细胞，但不用磷酸化抑制剂SRPIN340处理，可选择性地提高sEV miR-1246的水平，miR-1246是一种已知在PDAC sEVs中高度富集的miRNA。一致地，在野生型和SRSF1敲低的PANC-1细胞中，将三个精氨酸残基R93、R97和R109替换为赖氨酸的突变型SRSF1的过表达增加了sEV miR-1246的水平。有趣的是，在过表达突变型SRSF1的PDAC细胞中，SRSF1与miR-1246的结合显著减少，使用纯化的野生型和突变型SRSF1蛋白进一步证实了这一点。我们证明，SRSF1的精氨酸去甲基化降低了PDAC细胞中SRSF1与miRNA的结合，并增强了选择性sEV miRNA的富集，为SRSF1介导的PDAC细胞sEV miRNA富集提供了新的见解，并为PDAC细胞外囊泡的生物学和功能研究开辟了新的途径。
