去甲斑蝥素对人未分化甲状腺癌FRO细胞迁移、侵袭影响及机制研究

Study on the effects of norcantharidin on the migration and invasion of human undifferentiated thyroid carcinoma FRO cells and its mechanism

目的探讨去甲斑蝥素对人未分化甲状腺癌FRO细胞迁移、侵袭能力及机制研究。方法取对数生长期的人未分化甲状腺癌FRO细胞,加入不同浓度的去甲斑蝥素(2.5,5,10,20,40,80μg/mL)处理12 h、24 h和48 h,采用CCK-8法检测细胞增殖抑制情况。取对数生长期的人未分化甲状腺癌FRO细胞,分为空白组、10μg/mL去甲斑蝥素组、20μg/mL去甲斑蝥素组、40μg/mL去甲斑蝥素组,各组培养24 h后,采用划痕实验和Transwell实验检测FRO细胞的迁移和侵袭能力,流式细胞术检测细胞周期,TUNEL法检测细胞凋亡情况,Western blot法检测转移相关蛋白E-钙黏蛋白(E-cadherin)、基质金属蛋白酶-9(MMP-9)表达水平。结果2.5,5,10,20,40,80μg/mL的去甲斑蝥素对FRO细胞均有明显的增殖抑制作用,呈剂量时间依赖性。10μg/mL去甲斑蝥素组、20μg/mL去甲斑蝥素组、40μg/mL去甲斑蝥素组FRO细胞的迁移和侵袭能力均逐渐减弱,G2/M期细胞逐渐增多,细胞凋亡率逐渐增高,E-cadherin表达量逐渐增高,MMP-9表达量逐渐降低,各组间两两比较差异均有统计学意义(P均<0.05)。结论去甲斑蝥素能够抑制人未分化甲状腺癌FRO细胞的增殖、迁移和侵袭,并可诱导细胞周期阻滞,促进细胞凋亡,其机制可能与上调E-cadherin和下调MMP-9蛋白表达有关。

Objective It is to explore the effects of norcantharidin on the migration and invasion of human undifferentiated thyroid carcinoma FRO cells and its mechanism.Methods The human undifferentiated thyroid cancer FRO cells in the logarithmic growth phase were taken and cultured with different concentrations of norcantharidin(2.5,5,10,20,40,80μg/mL)for 12 h,24 h and 48 h,the cell proliferation inhibition was detected by CCK-8 method.The human undifferentiated thyroid cancer FRO cells in the logarithmic growth phase were taken and divided into blank group,10μg/mL norcantharidin group,20μg/mL norcantharidin group,40μg/mL norcantharidin group,each group was cultured for 24 h,the migration and invasion ability of FRO cells were detected by the scratch test and the Transwell test,the cell cycle was detected by flow cytometry,the apoptosis was detected by the TUNEL method,and the expression levels of transfer-associated protein E-cadherin(E-cadherin),matrix metalloproteinase-9(MMP-9)was detected by Western blot.Results 2.5,5,10,20,40,80μg/mL norcantharidin had a significant proliferation inhibitory effect on FRO cells,which was dose-time dependent.The migration and invasion ability of FRO cells in the 10μg/mL norcantharidin group,20μg/mL norcantharidin group,and 40μg/mL norcantharidin group gradually weakened,and the number of cells in the G2/M phase gradually increased,and the apoptosis rate gradually increased,the expression of E-cadherin gradually increased,and the expression of MMP-9 gradually decreased,there were statistically significant differences between every two groups(all P<0.05).Conclusion Norcantharidin can inhibit the proliferation,migration and invasion of human undifferentiated thyroid cancer FRO cells,and can induce cell cycle arrest and promote cell apoptosis.The mechanism may be related to the up-regulation of E-cadherin and down-regulation of MMP-9 protein expression.