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目的研究氯化钴(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、250μmol/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(150μmol/L的CoCl2分别作用0、3、6、12、24 h)对HIF-1α、E-cadherin和Vimentin表达的影响,Transwell法检测低氧(150μmol/L的CoCl2分别作用0、6 h)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0～6 h逐渐增加,随后开始下降,E-cadherin表达持续降低,低氧0 h与6 h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。
Objective To investigate the effect of cobalt chloride (CoCl2) -induced hypoxia on epithelial-mesenchymal transition of papillary thyroid carcinoma NPA cells and to explore the mechanism of HIF-1α in it. Methods The effect of CoCl2 (50, 100, 150, 200 and 250μmol / L) on cell viability was examined by MTT assay. The effect of hypoxia (0, 12, 24, 48 h, 150μmol / L CoCl2) The expression of HIF-1α, E-cadherin and Vimentin in hypoxia (150μmol / L CoCl2, 0,3,6,12,24 h respectively) were detected by Western blot. Respectively, 0,6 h) on cell invasion and metastasis, immunofluorescence HIF-1α protein subcellular localization. Results The hypoxic mock CoCl2 inhibited NPA cell viability. With the prolonged hypoxic treatment of CoCl2, NPA cells gradually obtained the morphological characteristics of interstitial cells. The expression of HIF-1α and Vimentin gradually increased from 0 to 6 h, then began to decrease, the expression of E-cadherin decreased continuously, Compared with 6 h, the expression of each protein was statistically significant (P <0.01). Hypoxia group invasion, the number of metastatic cells was significantly increased compared with the control group (P <0.01), while hypoxia group HIF-1α nuclear translocation. Conclusions CoCl2 can promote epithelial-mesenchymal transition, invasion and metastasis of NPA cells induced by chemical hypoxia, and its molecular mechanism may involve the regulation of E-cadherin and Vimentin gene expression by HIF-1α-mediated signaling pathway.