氯化钴对甲状腺乳头状癌NPA细胞上皮间质转化的影响及其机制

目的 研究氯化钻( CoCl2)诱导的低氧对甲状腺乳头状癌NPA细胞上皮间质转化的影响,初步探讨HIF-1α在其中的机制.方法 MTT检测COCl2(50、100、150、200、250 μmol/L)对细胞活力的影响,倒置显微镜观察低氧(150μmoL/L的CoCl2分别作用0、12、24、48 h)对细胞形态的影响,Western blot检测低氧(150 μmol/L的CoCl2分别作用0、3、6、12、24h)对HIF-1α、E-cadherin和Vimentin表达的影响,Transwell法检测低氧(150 μmol/L的CoCl2分别作用0、6h)对细胞侵袭、转移的影响,用免疫荧光对HIF-1α蛋白进行亚细胞定位.结果 低氧模拟剂CoCl2可抑制NPA细胞活力.随着COCl2低氧处理时间的延长,NPA细胞逐渐获得间质细胞的形态特征,HIF-1α、Vimentin表达在0～6h逐渐增加,随后开始下降,E-cadherin表达持续降低,低氧0h与6h相比各蛋白表达均具有统计学差异(P＜0.01).低氧组侵袭、转移细胞数目较对照组明显增加(P＜0.01),同时低氧组细胞HIF-1α发生核转位.结论 CoCl2模拟化学性低氧可促进NPA细胞上皮间质转化及侵袭、转移,其分子机制可能涉及HIF-1α介导的信号通路对E-cadherin、Vimentin基因表达的调控.

CoCl2 improves epithelial-mesenchymal transition of human papillary thyroid carcinoma cells

Objective To determine the effect of CoCl2 induced hypoxia on the epithelial-mesenchymal transition in human papillary thyroid carcinoma and investigate the role of HIF-1α in this process.Methods Cell vitality was evaluated by MTT assay in human papillary thyroid carcinoma NPA cells after the treatment of 50,100,150,200 and 250 μmol/L CoCl2.Morphological changes of NPA cells were observed by inverted microscopy after 150 μmol/L CoCl2 treatment for 0,12,24 and 48 h.The expression levels of HIF-1α,E-cadherin and Vimentin were analyzed by Western blotting in NPA cells after 150 μmol/L CoCl2 treatment for 0,3,6,12,and 24 h.The invasion and metastasis potential in NPA cells after 150 μmol/L CoCl2 treatment for 0 and 6 h was detected by Transwell assay.Immunofluorescence was used to detect the location of HIF-1α in NPA cells.Results CoCl2 treatment resulted in suppression of NPA cell vitality in a dose-dependent manner.The longer CoCl2 was treated,NPA cells gradually acquired the morphological characteristics of mesenchymal cells.The expression of HIF-1α,E-cadherin and Vimentin was gradually enhanced in 0 to 6 h after treatment,and then declined,especially in E-cadherin.Significant differences were found in the expression levels of these proteins at 0 h and 6 h(P<0.01).There were more cells that penetrated through the Transwell membrane in hypoxia group than in normoxic group(P<0.01).HIF-1α translocated to the nucleus in hypoxia group,while it didn’t occur in normoxia group.Conclusion CoCl2 treatment,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.