SRSF3-mediated regulation of N6-methyladenosine modification-related lncRNA ANRIL splicing in promotion of pancreatic cancer resistance to gemcitabine I

Serine/arginine-rich splicing factor 3 (SRSF3) functions to regulate mRNA alternative splicing, a molecular mechanism to process more than 90% of the protein-coding genes and provides an essential source for the biological versatility and targeting of SRSF3 could be a novel approach for cancer therapy. This study identify that SRSF3 expression was upregulated in pancreatic cancer tissues and associated with drug resistance and poor prognosis. Thus, we found that SRSF3 regulated ANRIL splicing and modified m6A modification of ANRIL in pancreatic cancer cells. More importantly, we demonstrated that the m6A methylation on lncRNA-ANRIL was essential for splicing process. Meanwhile, we also found that the different isoforms of ANRIL were differentially expressed in drug-resistant pancreatic cancer cell lines, and SRSF3 promotes gemcitabine resistance by regulating the expression of ANRIL-208. In addition, ANRIL-208 regulated pancreatic cancer cell chemoresistance by forming a complex with Ring1b and EZH2 and enhanced DNA homologous recombination repair (HR) capacity. In conclusion, the current study first established the link among SRSF3, m6A modification, lncRNA splicing, and DNA HR repair in pancreatic cancer, and first demonstrated that abnormal alternative splicing and m6A modification are closely related to chemotherapy resistance in pancreatic cancer. Overall design: 6 samples; triplicates

富含丝氨酸/精氨酸剪接因子3（Serine/arginine-rich splicing factor 3, SRSF3）的功能是调控mRNA可变剪接（mRNA alternative splicing），这是一种可处理超过90%蛋白编码基因（protein-coding gene）的分子机制；靶向SRSF3或可成为癌症治疗的全新策略，其为SRSF3的生物学多功能性提供了重要依据。本研究发现，SRSF3在胰腺癌（pancreatic cancer）组织中表达上调，且与耐药性（drug resistance）及不良预后（poor prognosis）密切相关。据此，本研究证实，SRSF3可调控胰腺癌细胞中ANRIL的剪接过程，并对ANRIL进行N6-甲基腺嘌呤（m6A）修饰。更为重要的是，本研究证实，长链非编码RNA（long non-coding RNA, lncRNA）ANRIL上的m6A甲基化对其剪接过程至关重要。与此同时，本研究还发现，ANRIL的不同剪接异构体（isoform）在耐药胰腺癌细胞系中存在差异表达，且SRSF3可通过调控ANRIL-208的表达增强吉西他滨（gemcitabine）耐药性。此外，ANRIL-208可通过与Ring1b及EZH2形成复合物，调控胰腺癌细胞的化疗耐药性（chemoresistance），并增强DNA同源重组修复（homologous recombination repair, HR）能力。综上，本研究首次明确了胰腺癌中SRSF3、m6A修饰、长链非编码RNA剪接与DNA同源重组修复之间的关联，并首次证实异常可变剪接与m6A修饰与胰腺癌化疗耐药密切相关。实验整体设计：共6份样本，设置3次重复。