MiR-548t-5p regulates pancreatic ductal adenocarcinoma metastasis through an IL-33-dependent crosstalk between cancer cells and M2 macrophages

IL-33 has been associated with pro- and anticancer functions in cancer. However, its role in pancreatic cancer metastasis remains unknown. This study aimed to explore the role of miR-548t-5p/IL-33 axis in the metastasis of pancreatic cancer. Luciferase activity assay, qRT-PCR, Western blot and ELISA were performed to prove whether IL-33 is the target of miR-548t-5p. In vivo metastasis assay and cellular transwell assay were performed to explore the role of miR-548t-5p/IL-33 axis in the invasion and metastasis of pancreatic cancer. Co-culture experiments and immunohistochemistry were performed to observe whether IL-33 affects cell invasion and metastasis dependent on the involvement of M2 macrophages. THP-1 cell induction experiment and flow cytometry were performed to explore the effect of IL-33 on macrophage polarization. CCK-8, colony formation, cell apoptosis, cell cycle, cell wound healing and transwell assay were performed to investigate the effect of IL-33 induced M2 macrophages on cell malignant biological behavior by coculturing pancreatic cancer cells with the conditioned medium (CM) from macrophages. We found that miR-548t-5p regulated the expression and secretion of IL-33 in pancreatic cancer cells by directly targeting IL-33 mRNA. IL-33 secreted by cancer cells promoted the recruitment and activation of macrophages to a M2-like phenotype. In turn, IL-33 induced M2 macrophages promoted the migration and invasion of cancer cells. Moreover, IL-33 affected pancreatic cancer cell invasion dependent on the involvement of M2 macrophages in the co-culture system. Thus, our study suggested that manipulation of this IL-33-dependent crosstalk has a therapeutic potential for the treatment of pancreatic cancer metastasis.

IL-33在癌症中已被证实兼具促癌与抑癌功能，但其在胰腺癌转移中的作用仍不明晰。本研究旨在探讨miR-548t-5p/IL-33轴在胰腺癌转移中的调控作用。采用荧光素酶活性检测（Luciferase activity assay）、实时定量荧光PCR（qRT-PCR）、蛋白质免疫印迹（Western Blot）与酶联免疫吸附测定（ELISA），验证IL-33是否为miR-548t-5p的靶基因。通过体内转移实验与Transwell细胞侵袭实验，探究miR-548t-5p/IL-33轴对胰腺癌侵袭与转移的影响。采用共培养实验与免疫组织化学（Immunohistochemistry），观察IL-33是否通过M2型巨噬细胞的参与调控肿瘤细胞的侵袭与转移。通过THP-1细胞诱导实验与流式细胞术（Flow Cytometry），分析IL-33对巨噬细胞极化的作用。采用CCK-8实验、集落形成实验、细胞凋亡检测、细胞周期检测、细胞划痕愈合实验与Transwell实验，将胰腺癌细胞与巨噬细胞条件培养基（CM）共培养，以探究IL-33诱导的M2型巨噬细胞对肿瘤细胞恶性生物学行为的影响。本研究发现，miR-548t-5p可通过直接靶向IL-33 mRNA，调控胰腺癌细胞中IL-33的表达与分泌。肿瘤细胞分泌的IL-33可促进巨噬细胞招募并极化为M2样表型。反之，IL-33诱导的M2型巨噬细胞可促进肿瘤细胞的迁移与侵袭。此外，在共培养体系中，IL-33对胰腺癌细胞侵袭的调控作用依赖于M2型巨噬细胞的参与。综上，本研究表明，靶向调控这种IL-33依赖的细胞串扰，有望为胰腺癌转移的临床治疗提供潜在治疗策略。