Effect And Mechanism Of Hypoxia On Epithelial-Mesenchmal Transition Of Human Throid Carcinoma Cells

Objective To determine the effect of hypoxia (induced by CoCl2) on the epithelial-mesenchymal transition in human thyroid carcinoma, investigate the role of HIF-1α and metallothioneins in this process.Methods NPA cells were divided into3groups, normoxia (21%O2), hypoxia (5%O2) and hypoxia with MTs-siRNA. The expression levels of MTs, E-cadherin and Vimentin in the3groups of NPA cells were analyzed by Western blotting and RT-PCR. The invasion and metastasis potential of these groups were detected by Transwell/matrigel assay. Cell vitality was evaluated by MTT assay in human thyroid carcinoma cells NPA, KAT18and WRO after the treatment of50,100,150,200and250μmol/L CoCl2. Morphological changes of NPA, KAT18and WRO cells were observed by inverted microscope after150μmol/L CoCl2treatment for0,12,24and48h. The expression levels of HIF-la, E-cadherin and Vimentin were analyzed by Western blotting in NPA, KAT18and WRO cells after the treatment of150μmol/L CoCl2for various hours. The invasion and metastasis potential in NPA cells after150μmol/L CoCl2treatment for0and6h were detected by Transwell/matrigel assay. Immunofluorescence was used to detect the location of HIF-la in NPA cells.Results Hypoxia treatment led to inducible expression of MTs, down-regulated of E-cadherin and up-regulated Vimentin at the same time, which were marker molecules of epithelial-mesenchymal transition in NPA cells. Meanwhile, invasive cell population of hypoxia group was increased to40.3±5.1from15.6±2.8of normoxia group (P<0.01). Migrating cell population was increased to48.7±7.6from25.1±5.6of normoxia group (P<0.01). However, the inducible expression of MTs was effectively inhibited by transfection of MTs-siRNA vector in NPA cells, followed by increased expression of E-cadherin and decreased expression of Vimentin. Moreover, invasive and migrating cell population of the hypoxia group with MTs-siRNA were only increased to25.7±4.2and35.5±5.4(P<0.05). CoCl2treatment resulted in suppression of vitality in NPA, KAT18and WRO cells in a dose-dependent manner. The longer CoCl2was treated, the NPA, KAT18and WRO cells gradually acquired the morphological characteristics of mesenchymal cells. The expression of HIF-1α and Vimentin was gradually enhanced in0to6h after treatment,and then declined. The expression of E-cadherin was down-regulated in0to24h after treatment. Significant differences were found in the expression levels of thses proteins at0h and6h (P<0.01). There were more cells that penetrated through the Transwell membrane in hypoxia group than in normoxia group (P<0.01). HIF-1α translocated to the nucleus in hypoxia group, while it didn’t occur in normoxia group.Conclusion The expression of metallothioneins induced by hypoxia can promote epithelial-mesenchymal transition in human thyroid carcinoma NPA cells. siRNA targeting metallothioneins can partially inhibit epithelial-mesenchymal transition promoted by hypoxia. CoCl2treatment, which mimics chemical hypoxia improves the epithelial-mesenchymal transition, invasion and migration of NPA cells, which might be due to that HIF-1α mediated signal pathway regulates E-cadherin and Vimentin. CoCl2induced hypoxia could promote pithelial-mesenchymal transition in human differentiated thyroid carcinoma KAT18and WRO cells through the down-regulation of E-cadherin and the up-regulation of Vimenti...