摘要
目的:探讨低氧诱导的长链非编码核内小RNA宿主基因14(long non-coding small nucleolar RNA host gene 14,lncRNA SNHG14)在胶质瘤替莫唑胺(temozolomide,TMZ)耐药中的作用和潜在机制。方法:根据不同处理将实验分为常氧组、低氧组、对照组(NC组)和TMZ组,利用real-time PCR和蛋白质印迹法分别检测胶质瘤细胞SNB19和U251中lncRNA SNHG14和O6-甲基鸟嘌呤DNA甲基转移酶(O6-methylguanine DNA methyltransferase,MGMT)的表达水平,分析lncRNA SNHG14表达水平与低氧和TMZ处理的关系。利用siRNA干扰胶质瘤细胞中lncRNA SNHG14表达,将转染后的胶质瘤细胞分为si-对照组(si-NC组)和si-SNHG14组,利用real-time PCR检测干扰效率,并采用蛋白质印迹法检测TMZ敏感性调控关键因子MGMT的表达变化,采用流式细胞术检测细胞凋亡;此外,增设常氧组和低氧组,应用MTT法检测不同TMZ浓度梯度下各组胶质瘤的细胞活性,分析lncRNA SNHG14对胶质瘤TMZ敏感性的影响。利用在线工具针对性地预测与lncRNA SNHG14和MGMT结合的miRNAs。应用realtime PCR观察不同环境下si-NC组、si-SNHG14组、常氧组和低氧组miR-143的丰度变化。利用miR-143拟似剂(mimics)和抑制剂(inhibitor)改变胶质瘤细胞中miR-143水平,将实验设置为NC inhibitor组、miR-143 inhibitor组、NC mimics组和miR-143 mimics组,采用real-time PCR检测干扰效率,应用蛋白质印迹法检测MGMT的表达水平,分析miR-143对MGMT水平的影响。对NC inhibitor组、miR-143 inhibitor组、NC mimics组和miR-143 mimics组进行不同干预,采用双荧光素酶报告实验观察lncRNA SNHG14和MGMT荧光素酶的活性变化,验证lncRNA SNHG14、miR-143和MGMT之间的靶向调控关系。最后,设置NC组和lncRNA SNHG14过表达组,通过RNA结合蛋白免疫沉淀实验检测各组中miR-143和MGMT的丰度变化,分析lncRNA SNHG14、miR-143和MGMT间的竞争结合关系。结果:与常氧组相比,低氧组可以促进胶质瘤细胞中lncRNA SNHG14表达;与NC组相
Objective:This study aims to investigate the role of hypoxia-induced long non-coding small nucleolar RNA host gene 14(lncRNA SNHG14)in glioma temozolomide(TMZ)resistance and underlying mechanisms.Methods:According to different treatments,the experiment was divided into a normoxia group and a hypoxia group,a control group and a TMZ group.The lncRNA SNHG14 and O6-methylguanine DNA methyltransferase(MGMT)levels in glioma SNB19 and U251cell line were detected by real-time PCR and Western blotting,respectively,and the association of lncRNA SNHG14 level with hypoxia and TMZ treatment was analyzed.siRNA was used to knockdown the lncRNA SNHG14 expression in glioma cells,and the transfected glioma cells were divided into a negative control group(si-NC group)and a siSNHG14 group.The interference efficiency was examined by real-time PCR,the key factor MGMT of lncRNA SNHG14 sensitivity regulation was detected by Western blotting,and the cell apoptosis was detected by cytometry.In addition,MTT method was used to detect the cell viability of gliomas in the different groups under the different TMZ concentrations,and the effect of lncRNA SNHG14 on TMZ sensitivity of gliomas was analyzed.Online tools were used to predict miRNAs that could specifically bind to lncRNAs SNHG14 and MGMT.A si-NC group,a si-SNHG14 group,a normoxia group and a hypoxia group were set up,and the changes of miR-143 abundance in different environments were observed by real-time PCR.miR-143 mimics and inhibitor were used to change the level of miR-143 in glioma cells.A NC inhibitor group,a miR-143 inhibitor group,a NC mimics group and a miR-143 mimics group were set up,the interference efficiency was detected by real-time PCR,the expression level of MGMT was detected by Western blotting,and the effect of miR-143 on the level of MGMT were analyzed.The NC inhibitor group,the miR-143 inhibitor group,the NC mimics group and the miR-143mimics group were treated with different interventions,and the dual luciferase reporter assay was used to observe the changes of
作者
赵海婷
孟莉
廖新斌
刘燚
莫鑫
龚梦麒
廖艺玮
ZHAO Haiting;MENG Li;LIAO Xinbin;LIU Yi;MO Xin;GONG Mengqi;LIAO Yiwei(Department of Neurology,Xiangya Hospital,Central South University,Changsha 410008;Department of Radiology,Xiangya Hospital,Central South University,Changsha 410008;Department of Neurosurgery,Xiangya Hospital,Central South University,Changsha 410008,China)
出处
《中南大学学报（医学版）》
CAS
CSCD
北大核心
2022年第7期829-838,共10页
Journal of Central South University ：Medical Science
基金
国家自然科学基金(81702490)。
