Molecular mechanism of fibulin-5 expression in the regulation of angiogenesis in thyroid cancer
Objective To investigate the molecular mechanism of Fibulin-5(FBLN5)in the regulation of an-giogenesis and vascular permeability in the tumor microenvironment of thyroid cancer.Methods Ten matched samples of papillary thyroid carcinoma(PTC)tissues and adjacent normal thyroid tissues diagnosed from June 2022 to December 2022 at Jiamusi Central Hospital were analyzed.Protein immunoblotting and RT-qPCR were used to assess FBLN5 ex-pression in endothelial cells(ECs).Co-culture experiments with human dermal microvascular ECs(HDMECs)and thy-roid cancer cell line KTC-1 were performed to investigate changes in FBLN5 expression in HDMECs.In order to study the function of FBLN5 in regulating the angiogenesis of ECs,HDMECs were divided into Vector group,FBLN5 overex-pression plasmid group,random sequence(sh-NC)group and FBLN5 knock-down lentivirus(sh-FBLN5)group.Overexpression plasmids or shRNA lentivirus targeting FBLN5 were used to upregulate or downregulate FBLN5 in HDMECs.Cell proliferation,tube formation,and migration were assessed using WST-1,tube formation,and Transwell assays,respectively.A xenograft tumor model was established in NOD-SCID mice by injecting PTC cells mixed with HDMECs transfected with Vector or FBLN5.Immunohistochemistry was performed to assess microvessel density in tumor tissues.Results Compared to normal ECs,FBLN5 mRNA and protein expression in tumor-associated ECs of PTC pa-tients were significantly downregulated(P＜0.05).FBLN5 expression in HDMECs co-cultured with and without KTC-1 cells was comparable.Compared to the Vector group,the cell viability of FBLN5-transfected HDMECs was significant-ly reduced at 48 h and 72 h(P＜0.05).Compared to the sh-NC group,the cell viability of sh-FBLN5-transfected HDMECs was significantly increased at 48 h and 72 h(P＜0.05).The cell migration(P＜0.001)and tube formation(P＜0.001)of FBLN5-transfected HDMECs were significantly decreased compared to the Vector group;compared to the sh-NC group,the cell migration(P=0.005)and tube formation(P＜0.001)of sh-FBLN5-transfected HDMECs were significantly increased.FBLN5-transfected HDMECs inhibited the growth of PTC tumors on days 3,6,10,and 14.The CD31-labeled microvessel density in the FBLN5 group was significantly lower than that in the Vector group(P=0.005),and FBLN5 protein expression was significantly higher than that in the Vector group(P＜0.001).Conclusion Hypoxia-induced upregulation of HIF1α in endothelial cells promotes angiogenesis in PTC by inhibiting FBLN5 expres-sion.
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