微小RNA-15a-5p通过RUNX1增强胶质瘤细胞对替莫唑胺的敏感性机制研究  

Mechanism of microRNA-15a-5p in enhancing the sensitivity of glioma cells to temozolomide by modulating RUNX1

作　　者：居来提·阿扎提 马涛[1] 王洋[1] 张晶晶[2] Julaiti·AZHATI;MA Tao;WANG Yang;ZHANG Jingjing(Department of Laboratory Medicine,the Second Affiliated Hospital of Xinjiang Medical University,Urumqi,Xinjiang,830028;Department of Neurosurgery,the Second Affiliated Hospital of Xinjiang Medical University,Urumqi,Xinjiang,830028)

机构地区：[1]新疆医科大学第二附属医院检验科,新疆乌鲁木齐830028 [2]新疆医科大学第二附属医院神经外科,新疆乌鲁木齐830028

出　　处：《实用临床医药杂志》2024年第19期10-15,21,共7页Journal of Clinical Medicine in Practice

基　　金：新疆维吾尔自治区自然科学基金资助项目(2022D01C503)。

摘　　要：目的探讨微小RNA-15a-5p(miR-15a-5p)影响胶质瘤细胞增殖、侵袭、迁移及胶质瘤细胞对替莫唑胺(TMZ)敏感性的机制。方法选取胶质瘤细胞H4、SHG-44、正常星形胶质细胞HA1800及TMZ耐药H4细胞,将miR-NC、miR-15a-5p mimics质粒分别转染至H4细胞,记为miR-NC组、miR-15a-5p组;将Vector、OE-RUNX1质粒分别转染至miR-15a-5p组细胞,并分别用400μmoL/L TMZ处理,分别记为miR-15a-5p+Vector组、miR-15a-5p+RUNX1组、TMZ+Vector组及TMZ+RUNX1组。miR-NC组、miR-15a-5p组细胞用400μmoL/L TMZ处理,记为TMZ+miR-NC组、TMZ+miR-15a-5p组。未进行任何处理的细胞设为Control组。采用四甲基偶氮唑蓝(MTT)检测细胞增殖能力。采用Transwell实验检测细胞侵袭、迁移能力。采用荧光定量聚合酶链反应(RT-PCR)检测细胞RUNX1 mRNA及miR-15a-5p表达水平。采用Western blot检测细胞RUNX1蛋白表达水平。采用TargetScan在线网站预测miR-15a-5p与RUNX1的结合位点。采用双荧光素酶报告基因实验验证miR-15a-5p与RUNX1的靶向关系。结果H4、SHG-44细胞的RUNX1 mRNA及其蛋白表达水平高于HA1800细胞,H4、SHG-44细胞miR-15a-5p表达水平低于HA1800细胞,差异有统计学意义(P<0.0001)。与Control组比较,TMZ耐药H4细胞miR-15a-5p表达水平降低,差异有统计学意义(t=18.89,P<0.0001);与Control组比较,TMZ耐药H4细胞RUNX1 mRNA及其蛋白表达水平升高,差异有统计学意义(t=34.11、18.07,P<0.0001)。上调miR-15a-5p、TMZ均可抑制细胞的增殖、侵袭及迁移,而上调miR-15a-5p联合TMZ可显著抑制细胞的增殖、侵袭及迁移。miR-15a-5p可负性调控RUNX1表达。RUNX1过表达可逆转上调miR-15a-5p、TMZ对胶质瘤细胞增殖、侵袭及迁移的抑制作用。结论miR-15a-5p负性调控RUNX1抑制胶质瘤细胞的增殖、侵袭、迁移,以及提高肿瘤细胞对TMZ的敏感性。Objective To investigate the effects of microRNA-15a-5p(miR-15a-5p)on the proliferation,invasion and migration of and its mechanism of sensitivity of glioma cells to temozolomide(TMZ).Methods Glioma cell lines H4 and SHG-44,normal astrocytes HA1800 and TMZ-resistant H4 cells were selected.H4 cells were transfected with miR-NC or miR-15a-5p mimics and recorded as the miR-NC group and the miR-15a-5p group,respectively.The cells of miR-15a-5p group was further transfected with Vector or OE-RUNX1 plasmids and treated with 400μmoL/L TMZ,and recorded as miR-15a-5p+Vector group,miR-15a-5p+RUNX1 group,TMZ+Vector group and TMZ+RUNX1 group.Additionally,the miR-NC and miR-15a-5p groups were treated with 400μmoL/L TMZ,and recorded as TMZ+miR-NC group and TMZ+miR-15a-5p group,respectively.Untreated cells served as the Control group.Cell proliferation was assessed using the Methyl Thiazolyl Tetrazolium(MTT)assay.Invasion and migration were evaluated by Transwell assays.RUNX1 mRNA and miR-15a-5p expression levels were determined by quantitative real-time polymerase chain reaction(RT-PCR).RUNX1 protein expression was analyzed by Western blot.The binding sites between miR-15a-5p and RUNX1 were predicted using the TargetScan online database.The targeting relationship between miR-15a-5p and RUNX1 was validated by dual-luciferase reporter assays.Results RUNX1 mRNA and its protein expression levels were significantly higher in H4 and SHG-44 cells compared to HA1800 cells,while miR-15a-5p expression was significantly lower in H4 and SHG-44 cells(P<0.0001).Compared with Control group,the expression level of miR-15a-5p in TMZ-resistant H4 cells was significantly lower(t=18.89,P<0.0001);compared with Control group,the expression level of RUNX1 mRNA and protein in TMZ-resistant H4 cells was significantly higher(t=34.11,18.07,P<0.0001).Upregulation of miR-15a-5p and TMZ treatment both inhibited cell proliferation,invasion and migration,and the combination of miR-15a-5p upregulation and TMZ significantly enhanced these inhibitory effects.

关 键 词：微小RNA-15a-5p RUNX1基因 胶质瘤 替莫唑胺 

分 类 号：R739.41[医药卫生—肿瘤] R329.2[医药卫生—临床医学] R446