【Abstract】
MiR-324-5p is overexpressed in papillary thyroid carcinoma (PTC) with lymph node metastasis and promotes malignant phenotypes of KTC-1 cell line. However, the detailed regulatory mechanism remains unknown. Tumor microenvironment plays a key role in tumor progression. CCAAT enhancer-binding protein delta (CEBPD) is important in immune and inflammatory responses. In this study, we investigated the interaction between miR-324-5p/PTPRD/CEBPD axis and tumor microenvironment in PTC progression. K1 and KTC-1 were transfected by lenti-CEBPD or CEBPD-sh vectors. Supernatant from different groups was harvested and added into culture media of human macrophages and HUVEC. Cell viability, colony formation, invasive and migrated cell number, and gap closure rate were elevated in lenti-CEBPD group. Compared with the control, supernatant from lenti-CEBPD group contained more abundant levels of VEGF and IL-4/IL-13, which, respectively, induced higher HUVEC invasion/migration rates and more obvious M2 marker (CD206) and genes (PPAR-γ and MRC-1) expression in macrophages. By means of luciferase reporter assay and gene manipulation, we identified that CEBPD was negatively regulated in PTC by protein tyrosine phosphatase receptor delta (PTPRD) which was the target of miR-324-5p. CEBPD-shRNA was also proved to reverse the effect of PTPRD-sh1 or miR-324-5p mimic on IL-4/IL-13 expression and HUVEC invasion. These results suggested that miR-324-5p/PTPRD/CEBPD axis was involved in the progression of PTC by inducing HUVEC invasion/migration and macrophage M2 polarization via VEGF and IL4/IL13, respectively.
【中文摘要】
MiR-324-5p在伴有淋巴結轉移的甲狀腺乳頭狀癌(papillary thyroid carcinoma, PTC)中表達上調，並能促進KTC-1細胞的惡性表型。然而，其具體調控機制仍不清楚。基於腫瘤微環境在腫瘤進展中的關鍵作用，以及CCAAT增強子結合蛋白(CCAAT enhancer-binding protein delta, CEBPD)在免疫和炎症反應中的重要作用。本課題組採用以下方法研究了在PTC進展過程中miR-324-5p/PTPRD/CEBPD軸與腫瘤微環境的相互作用。用lenti-CEBPD或CEBPD-sh載體轉染K1和KTC-1細胞，收集不同組別細胞的培養上清，加入人巨噬細胞和HUVEC培養基中。lenti-CEBPD組的細胞活力、克隆形成數、侵襲和遷移細胞數量均高於對照組。與對照組相比，lenti-CEBPD組上清液中VEGF和IL-4/IL-13含量更豐富，前者促進HUVEC侵襲與遷移，後者促使巨噬細胞M2型轉換。螢光素酶報告實驗證明酪氨酸磷酸酶受體δ(protein tyrosine phosphatase receptor δ, PTPRD) 是miR-324-5p的靶基因；細胞內基因操作結果表明CEBPD與PTPRD負相關。回復實驗表明，在PTC細胞中敲減CEBPD可以逆轉PTPRD-sh1或miR-324-5p mimic對IL-4/IL-13表達和HUVEC侵襲能力的影響。以上結果表明miR-324-5p/PTPRD/CEBPD軸分別通過VEGF和IL4/IL13誘導HUVEC的侵襲/遷移和巨噬細胞M2極化參與PTC的進展。