ABSTRACT

Fibrotic microenvironment has been reported to have a pro-metastasis effect on tumor cells, but the mechanism remains unclear. The current study aimed to explore the underlying mechanism by which the fibrotic microenvironment affects tumor cells. A tumor metastasis model was established by injecting tumor cells containing GFP into mice with pulmonary fibrosis. Lung tissues and fibroblasts were harvested, and conditioned medium (CM) were collected from fibrotic lungs and fibroblasts. Hematoxylin & eosin staining and immunohistochemistry were used to detect pulmonary metastasis and FSP1 expression, respectively. Bioinformatics and dual-luciferase reporter assay proved that the target genes of ZEB1-AS1 and miR-200b-3p were miR-200b-3p and ZEB1, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expressions of GFP, ZEB1-AS1, and miR-200b-3p. Transwell assay, Annexin V/PI assay, and colorimetry were performed to examine the effects of CM, ZEB1-AS1, miR-200b-3p, and ZEB1 on cell invasion, apoptosis, and the activity level of caspase-3/-9. Pulmonary metastasis was promoted and the expressions of FSP1 and GFP were increased in mice with pulmonary fibrosis. CM enhanced the invasion and inhibited the apoptosis of tumor cells. SiZEB1-AS1 and siZEB1 inhibited the invasion and apoptosis of tumor cells, while miR-200b-3p inhibitor had the opposite effect of SiZEB1-AS1 and siZEB1, and further reversed the effect of siZEB1 on tumor cell invasion and apoptosis. SiZEB1-AS1 reversed the effects of both miR-200b-3p inhibitor and miR-200b-3p inhibitor+siZEB1 on tumor cell invasion and apoptosis. Fibrotic microenvironment promoted the metastatic seeding of tumor cells into the lungs via mediating the ZEB1-AS1/miR-200b-3p/ZEB1 signaling.

摘要

据报道，纤维化微环境对肿瘤细胞具有促转移作用，但机制尚不清楚。目前的研究旨在探索纤维化微环境影响肿瘤细胞的潜在机制。将含有GFP的肿瘤细胞注射到肺纤维化小鼠体内，建立肿瘤转移模型。收获肺组织和成纤维细胞，并从纤维化肺和成纤维细胞中收集条件培养基（CM）。苏木精和伊红染色和免疫组织化学分别用于检测肺转移和FSP1的表达。生物信息学和双荧光素酶报告基因分析证明ZEB1-AS1和miR-200b-3p的靶基因分别是miR-200b-3p和ZEB1。进行定量实时聚合酶链反应 (qRT-PCR) 以检测 GFP、ZEB1-AS1 和 miR-200b-3p 的表达。采用 Transwell 试验、Annexin V/PI 试验和比色法检测 CM、ZEB1-AS1、miR-200b-3p 和 ZEB1 对细胞侵袭、凋亡和 caspase-3/-9 活性水平的影响。肺纤维化小鼠促进肺转移，FSP1和GFP表达增加。CM增强肿瘤细胞的侵袭能力并抑制其凋亡。SiZEB1-AS1和siZEB1抑制肿瘤细胞的侵袭和凋亡，而miR-200b-3p抑制剂的作用与SiZEB1-AS1和siZEB1相反，进一步逆转了siZEB1对肿瘤细胞侵袭和凋亡的影响。SiZEB1-AS1逆转了miR-200b-3p抑制剂和miR-200b-3p抑制剂+siZEB1对肿瘤细胞侵袭和凋亡的影响。纤维化微环境通过介导 ZEB1-AS1/miR-200b-3p/ZEB1 信号传导促进肿瘤细胞转移到肺部。