Shengli Clinical Medical College of Fujian Medical University, Fujian Medical University, No. 134, East Street, Fuzhou, 350001, Fujian Province, People's Republic of China.
Department of Hepatobiliary Pancreatic Surgery, Fujian Provincial Hospital, Fuzhou, 350001, People's Republic of China.
J Hematol Oncol. 2021 Apr 13;14(1):60. doi: 10.1186/s13045-021-01072-8.
Both aberrant alternative splicing and m6A methylation play complicated roles in the development of pancreatic cancer (PC), while the relationship between these two RNA modifications remains unclear.
RNA sequencing (RNA-seq) was performed using 15 pairs of pancreatic ductal adenocarcinoma (PDAC) tissues and corresponding normal tissues, and Cdc2-like kinases 1 (CLK1) was identified as a significantly upregulated alternative splicing related gene. Real-time quantitative PCR (qPCR) and western blotting were applied to determine the CLK1 levels. The prognostic value of CLK1 was elucidated by Immunohistochemistry (IHC) analyses in two independent PDAC cohorts. The functional characterizations and mechanistic insights of CLK1 in PDAC growth and metastasis were evaluated with PDAC cell lines and nude mice. SR-like splicing factors5 (SRSF5) was identified as an important target phosphorylation site by phosphorylation mass spectrometry. Through transcriptome sequencing, Methyltransferase-like 14 (METTL14) and Cyclin L2 skipping were identified as key alternative splicing events regulated by the CLK1-SRSF5 axis. RIP assays, RNA-pulldown and CLIP-qPCR were performed to confirm molecular interactions and the precise binding sites. The roles of the shift of METTL14 and Cyclin L2 skipping were surveyed.
CLK1 expression was significantly increased in PDAC tissues at both the mRNA and protein levels. High CLK1 expression was associated with poor prognosis. Elevated CLK1 expression promoted growth and metastasis of PC cells in vitro and in vivo. Mechanistically, CLK1 enhanced phosphorylation on SRSF5, which inhibited METTL14 skipping while promoted Cyclin L2 skipping. In addition, aberrant METTL14 skipping enhanced the N6-methyladenosine modification level and metastasis, while aberrant Cyclin L2 promoted proliferation of PDAC cells.
The CLK1/SRSF5 pathway induces aberrant exon skipping of METTL14 and Cyclin L2, which promotes growth and metastasis and regulates m6A methylation of PDAC cells. This study suggests the potential prognostic value and therapeutic targeting of this pathway in PDAC patients.
异常的可变剪接和 m6A 甲基化在胰腺癌（PC）的发展中都起着复杂的作用，而这两种 RNA 修饰之间的关系尚不清楚。
使用 15 对胰腺导管腺癌（PDAC）组织和相应的正常组织进行 RNA 测序（RNA-seq），并鉴定出 Cdc2 样激酶 1（CLK1）为显著上调的可变剪接相关基因。实时定量 PCR（qPCR）和 Western blot 用于确定 CLK1 水平。免疫组织化学（IHC）分析在两个独立的 PDAC 队列中阐明了 CLK1 的预后价值。使用 PDAC 细胞系和裸鼠评估 CLK1 在 PDAC 生长和转移中的功能特征和机制见解。通过磷酸化质谱鉴定出 SR 样剪接因子 5（SRSF5）作为重要的磷酸化靶标。通过转录组测序，确定了甲基转移酶样 14（METTL14）和细胞周期蛋白 L2 跳跃作为 CLK1-SRSF5 轴调控的关键可变剪接事件。进行 RIP 测定、RNA 下拉和 CLIP-qPCR 以确认分子相互作用和精确结合位点。调查了 METTL14 和细胞周期蛋白 L2 跳跃移位的作用。
CLK1 在 PDAC 组织中的 mRNA 和蛋白水平均显著升高。高 CLK1 表达与预后不良相关。升高的 CLK1 表达促进了 PC 细胞在体外和体内的生长和转移。机制上，CLK1 增强了 SRSF5 的磷酸化，从而抑制了 METTL14 跳跃，同时促进了细胞周期蛋白 L2 跳跃。此外，异常的 METTL14 跳跃增强了 N6-甲基腺苷修饰水平和转移，而异常的细胞周期蛋白 L2 促进了 PDAC 细胞的增殖。
CLK1/SRSF5 通路诱导 METTL14 和细胞周期蛋白 L2 的异常外显子跳跃，促进生长和转移，并调节 PDAC 细胞的 m6A 甲基化。这项研究表明该通路在 PDAC 患者中具有潜在的预后价值和治疗靶向性。
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