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.

MiR-324-5p在具有淋巴结转移的乳头状甲状腺癌（PTC）中过表达，并促进KTC-1细胞系的恶性表型。但是，详细的监管机制仍然未知。肿瘤微环境在肿瘤进展中起关键作用。CCAAT增强子结合蛋白δ（CEBPD）在免疫和炎症反应中很重要。在这项研究中，我们研究了miR-324-5p / PTPRD / CEBPD轴与PTC进展中的肿瘤微环境之间的相互作用。K1和KTC-1被慢速CEBPD或CEBPD-sh载体转染。收获来自不同组的上清液，并将其加入人巨噬细胞和HUVEC的培养基中。lenti-CEBPD组细胞活力，集落形成，侵袭和迁移细胞数量以及缺口闭合率均升高。与对照相比，lenti-CEBPD组的上清液含有更丰富的VEGF和IL-4 / IL-13水平，分别诱导更高的HUVEC侵袭/迁移速率和更明显的M2标记（CD206）和基因（PPAR-γ和MRC-1 ）在巨噬细胞中的表达。通过萤光素酶报告基因检测和基因操作，我们确定PTC中的CEBPD被miR-324-5p的靶蛋白酪氨酸磷酸酶受体δ（PTPRD）负调控。还证明了CEBPD-shRNA可以逆转PTPRD-sh1或miR-324-5p模拟物对IL-4 / IL-13表达和HUVEC侵袭的影响。这些结果表明，miR-324-5p / PTPRD / CEBPD轴通过分别通过VEGF和IL4 / IL13诱导HUVEC侵袭/迁移和巨噬细胞M2极化而参与了PTC的进展。诱导的HUVEC侵袭/迁移速率更高，巨噬细胞中M2标记（CD206）和基因（PPAR-γ和MRC-1）的表达更明显。通过萤光素酶报告基因检测和基因操作，我们确定PTC中的CEBPD被miR-324-5p的靶蛋白酪氨酸磷酸酶受体δ（PTPRD）负调控。还证明了CEBPD-shRNA可以逆转PTPRD-sh1或miR-324-5p模拟物对IL-4 / IL-13表达和HUVEC侵袭的影响。这些结果表明，miR-324-5p / PTPRD / CEBPD轴通过分别通过VEGF和IL4 / IL13诱导HUVEC侵袭/迁移和巨噬细胞M2极化而参与了PTC的进展。诱导的HUVEC侵袭/迁移速率更高，巨噬细胞中M2标记（CD206）和基因（PPAR-γ和MRC-1）的表达更加明显。通过萤光素酶报告基因检测和基因操作，我们确定PTC中的CEBPD被miR-324-5p的靶蛋白酪氨酸磷酸酶受体δ（PTPRD）负调控。还证明了CEBPD-shRNA可以逆转PTPRD-sh1或miR-324-5p模拟物对IL-4 / IL-13表达和HUVEC侵袭的影响。这些结果表明，miR-324-5p / PTPRD / CEBPD轴通过分别通过VEGF和IL4 / IL13诱导HUVEC侵袭/迁移和巨噬细胞M2极化而参与了PTC的进展。通过萤光素酶报告基因检测和基因操作，我们确定PTC中的CEBPD被miR-324-5p的靶蛋白酪氨酸磷酸酶受体δ（PTPRD）负调控。还证明了CEBPD-shRNA可以逆转PTPRD-sh1或miR-324-5p模拟物对IL-4 / IL-13表达和HUVEC侵袭的影响。这些结果表明，miR-324-5p / PTPRD / CEBPD轴通过分别通过VEGF和IL4 / IL13诱导HUVEC侵袭/迁移和巨噬细胞M2极化而参与了PTC的进展。通过萤光素酶报告基因检测和基因操作，我们确定PTC中的CEBPD被miR-324-5p的靶蛋白酪氨酸磷酸酶受体δ（PTPRD）负调控。还证明了CEBPD-shRNA可以逆转PTPRD-sh1或miR-324-5p模拟物对IL-4 / IL-13表达和HUVEC侵袭的影响。这些结果表明，miR-324-5p / PTPRD / CEBPD轴通过分别通过VEGF和IL4 / IL13诱导HUVEC侵袭/迁移和巨噬细胞M2极化而参与了PTC的进展。