摘要
目的:探讨青藤碱(SME)对缺氧微环境诱导的甲状腺乳头状癌细胞“干性”的影响及机制。方法:采用不同浓度(0.25、0.5和1 mmol/L)的SME处理缺氧条件下的人甲状腺乳头状癌TPC-1细胞或采用1 mmol/L SME处理缺氧条件下小泛素相关修饰蛋白(SUMO)特异性蛋白酶1(SENP-1)基因过表达的TPC-1细胞。流式细胞术检测肿瘤干细胞亚群(CD44^(+)CD24^(-))比例;qRT-PCR检测肿瘤干细胞标志物Nanog、八聚体结合转录因子4(OCT4)和SRY盒转录因子2(SOX2)的mRNA表达水平;肿瘤细胞成球实验观察细胞的干性特征;Western blot检测细胞中SENP-1、SUMO1和低氧诱导因子1α(HIF-1α)的蛋白表达水平;试剂盒法检测细胞中HIF-1α蛋白SUMO化水平;免疫荧光染色检测细胞中HIF-1α蛋白分布情况。结果:SME可呈浓度依赖性降低缺氧微环境下TPC-1细胞中干细胞亚群(CD44^(+)CD24^(-))比例,下调肿瘤干细胞标志物Nanog、OCT4和SOX2的mRNA表达水平,降低肿瘤细胞成球数量和成球体积,同时促进HIF-1α蛋白SUMO化,下调SENP-1和HIF-1α蛋白表达水平,上调SUMO1蛋白表达水平,并抑制HIF-1α蛋白向细胞核转位。然而,过表达SENP-1可逆转缺氧微环境下SME对TPC-1细胞“干性”的抑制作用,上调HIF-1α蛋白表达水平,并降低HIF-1α蛋白SUMO化水平。结论:SME通过促进缺氧条件下HIF-1α蛋白的SUMO化修饰,降低HIF-1α蛋白的稳定性和转录活性,进而抑制TPC-1细胞“干性”。
AIM:To investigate the effect of sinomenine(SME)on the stemness of papillary thyroid carcinoma cells induced by hypoxic microenvironment,and to explore its mechanism.METHODS:Human papillary thyroid carcinoma TPC-1 cells were treated with different concentrations(0.25,0.5 and 1 mmol/L)of SME under hypoxic condition,while those with small ubiquitin-related modifier(SUMO)-specific protease-1(SENP-1)overexpression under hypoxia were treated with 1 mmol/L SME.The proportion of cancer stem cell subset(CD44^(+)CD24^(-))was determined by flow cytometry.The mRNA expression levels of cancer stem cell markers Nanog,octamer-binding transcription factor 4(OCT4)and SRY-box transcription factor 2(SOX2)were detected by qRT-PCR.Tumor cell sphere-forming assay was conducted to observe the stemness of tumor cells.The protein expression levels of SENP1,SUMO1 and hypoxia-inducible factor-1α(HIF-1α)were detected by Western blot.The SUMO level of HIF-1αprotein was detected using a kit,and the distribution of HIF-1αprotein was assessed by immunofluorescence staining.RESULTS:Treatment with SME decreased the proportion of CD44^(+)CD24^(-)cell subset in TPC-1 cells induced by hypoxic microenvironment in a concentration-dependent manner.It also down-regulated the mRNA expression levels of stem cell markers Nanog,OCT4 and SOX2,and reduced the number and volume of forming cell pellets.Moreover,SME promoted the SUMOylation of HIF-1αprotein,down-regulated the protein expression levels of SENP-1 and HIF-1α,up-regulated the protein expression level of SUMO1,and inhibited the translocation of HIF-1αprotein to the nucleus.However,overexpression of SENP-1 reversed the inhibi-tory effect of SME on the stemness of TPC-1 cells under hypoxic microenvironment.It also up-regulated the protein expres-sion level of HIF-1α,and decreased the SUMOylation level of HIF-1αprotein.CONCLUSION:Treatment with SME in-hibited the stemness of TPC-1 cells by promoting the SUMOylation of HIF-1αprotein under hypoxia,thus reducing the sta-bility and transcriptiona
作者
綦彬
范浩
王博
袁燕
毛伟明
QI Bin;FAN Hao;WANG Bo;YUAN Yan;MAO Weiming(Yangtze University Health Science Center,Huanggang 434023,China;Huanggang Central Hospital,Huanggang 438000,China;Yangtze University,Jingzhou 434023,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2023年第11期2003-2010,共8页
Chinese Journal of Pathophysiology
基金
湖北省卫生健康委科研资助项目(No.WJ2023M175)。
