ST8SIA3在胶质瘤细胞替莫唑胺耐药性的作用

Role of ST8SIA3 in temozolomide resistance of glioma cells

目的探讨ST8α-乙酰神经氨酸酶α-2,8-唾液酸转移酶3(ST8SIA3)对胶质瘤产生耐替莫唑胺(Temozolomide,TMZ)特性的影响。方法将胶质母细胞瘤细胞株U87-MG进行慢病毒转染,分为对照组、下调组(ST8SIA3表达下调)、过表达组(ST8SIA3过表达),使用qRT-PCR检测3组ST8SIA3相对表达,通过Western-blot检测3组A2B5表达与O6-甲基鸟嘌呤-DNA甲基转移酶(O6-methylguanine-DNA methyltransferese,MGMT)表达。采用不同TMZ浓度(0μmol/L、5μmol/L、10μmol/L、20μmol/L、50μmol/L、100μmol/L、200μmol/L)与3组细胞培养48h后,使用CCK-8检测并绘制TMZ浓度-细胞生存率曲线。通过单细胞成球实验观察下调组和过表达组ST8SIA3对胶质瘤干细胞(glioma stem cells,GSC)自我更新能力及干细胞标志物(CD133、SOX2)的影响。采用TCGA及CGGA数据库的人脑胶质瘤临床数据,分析ST8SIA3表达与患者预后关系。裸鼠种瘤后记录裸鼠出现恶病质的时间并进行生存分析统计,免疫组织化学方法检测裸鼠原位移植瘤标本MGMT和A2B5表达。结果过表达组A2B5和MGMT相对表达增加。CCK-8实验结果显示:随着ST8SIA3表达增加,TMZ的LC_(50)(半致死浓度)逐渐升高。单细胞成球实验显示:随着ST8SIA3表达增高,成球细胞数量及体积逐渐增加。裸鼠原位移植瘤模型结果显示:过表达ST8SIA3明显缩短裸鼠生存期;随着ST8SIA3表达增加,Ki-67、A2B5、MGMT表达随之增加。结论ST8SIA3通过合成干细胞标志物A2B5从而促进U87-MG细胞成球能力,进而表现出GSC耐TMZ特性。

Objective To investigate the influence of ST8α-N-acetylneuraminidaseα-2,8-sialyltransferase 3(ST8SIA3)on the development of temozolomide(TMZ)resistance in glioma cells.Methods The glioblastoma U87-MG cell lines were subjected to lentiviral transfection and divided into three groups:control group,downregulation group(reduced ST8SIA3 expression)and overexpression group(increased ST8SIA3 expression).The relative expressions of ST8SIA3 in these groups were detected using qRT-PCR,while A2B5 and O6-methylguanine-DNA methyltransferase(MGMT)expressions were analyzed via Western-blot.After culturing the three groups with varying concentrations of TMZ(0μmol/L,5μmol/L,10μmol/L,20μmol/L,50μmol/L,100μmol/L,200μmol/L)for 48 hours,the TMZ concentration-cell survival curves were plotted using CCK-8 assays.The effects of ST8SIA3 in downregulation group and overexpression group on the self-renewal capacity of glioma stem cells(GSC)and stem cell markers(CD133,SOX2)were observed through single-cell sphere formation experiments.The relationship between ST8SIA3 expression and patient prognosis was analyzed using clinical data from The Cancer Genome Atlas(TCGA)and Chinese Glioma Genome Atlas(CGGA)databases.Survival analysis and the recording of the onset of cachexia were conducted in nude mouse tumor models,and immunohistochemical methods were used to detect MGMT and A2B5 expressions in orthotopic transplanted tumor samples from nude mice.Results The relative expressions of A2B5 and MGMT increased in the overexpression group.CCK-8 assay results showed that the LC_(50)(lethal concentration 50%)of TMZ gradually increased with increase of ST8SIA3 expression.The single-cell sphere formation experiments demonstrated that the number and size of spheres gradually increased with increase of ST8SIA3 expression.Results from the nude mouse orthotopic tumor models indicated that overexpression of ST8SIA3 significantly shortened the survival period of nude mice,and expressions of Ki-67,A2B5 and MGMT increased with increase of ST8SIA3 expression.Conclusions ST8SIA3 promotes the sphere-forming ability of U87-MG cells by synthesizing the stem cell marker A2B5,thereby conferring GSC resistance to TMZ.