Supplementary Material for: DYRK2 regulates epithelial-mesenchymal transition restriction in pancreatic cancer liver metastasis by inhibiting Twist

Objectives: To explore the underlying variables and molecular pathways leading to pancreatic cancer liver metastasis.
Methods: Hs766T and Hs766T-L3 cells were used to create in vitro and in vivo pancreatic cancer liver metastasis models. DYRK2 involvement in pancreatic cancer metastasis was investigated using cell adhesion assays, wound healing assays, and migration and invasion assays. To examine the link between DYRK2 expression and epithelial-mesenchymal transition, Western blot, quantitative real-time PCR, immunofluorescence assays, and immunoprecipitation (IP) were utilized. We found that mice with DYRK2 overexpression had a lower incidence of liver metastasis compared to controls.
Results: DYRK2 expression decreased pancreatic cancer tumorigenic activities, including proliferation, migration, and invasion. By analyzing the expression levels of epithelial-mesenchymal transition markers and IP results after overexpressing DYRK2, we found that DYRK2 decreased Twist levels by increasing Twist ubiquitination, thereby inhibiting epithelial-mesenchymal transition.
Conclusions: Our findings provide theoretical and experimental support for the ongoing development of DYRK2-based targeted therapies for pancreatic cancer liver metastases.

研究目的：本研究旨在探讨胰腺癌肝转移的潜在致病变量及分子通路。
方法：本研究采用Hs766T与Hs766T-L3细胞构建体外及体内胰腺癌肝转移模型。通过细胞黏附实验、划痕愈合实验、迁移与侵袭实验，探究双特异性酪氨酸磷酸化调节激酶2（DYRK2）在胰腺癌转移中的作用。为检测DYRK2表达与上皮间质转化（epithelial-mesenchymal transition, EMT）的关联，本研究采用蛋白质印迹法（Western blot）、定量实时聚合酶链反应（quantitative real-time PCR, qRT-PCR）、免疫荧光实验及免疫沉淀（immunoprecipitation, IP）开展相关检测。研究发现，过表达DYRK2的小鼠肝转移发生率较对照组更低。
结果：DYRK2可抑制胰腺癌细胞的致瘤活性，包括增殖、迁移及侵袭能力。通过分析DYRK2过表达后上皮间质转化标志物的表达水平及免疫沉淀实验结果，本研究发现DYRK2可通过促进Twist蛋白的泛素化修饰降低其表达水平，进而抑制上皮间质转化进程。
结论：本研究结果为基于DYRK2的胰腺癌肝转移靶向治疗的后续研发提供了理论与实验依据。