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OACSTPCD
Mechanism of microRNA-15a-5p in enhancing the sensitivity of glioma cells to temozolomide by modulating RUNX1
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 400 μmoL/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400 µmoL/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＜0.000 1).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＜0.000 1);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＜0.000 1).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 pro-liferation,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 400 μmoL/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＜0.000 1).Compared with Control group,the expression level of miR-15a-5p in TMZ-resistant H4 cells was significantly lower(t=18.89,P＜0.000 1);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＜0.000 1).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.MiR-15a-5p negatively regulated RUNX1 expression.Overexpression of RUNX1 reversed the inhibitory effects of miR-15a-5p upregulation and TMZ on glioma cell proliferation,in-vasion,and migration.Conclusion MiR-15a-5p negatively regulates RUNX1,thereby inhibiting the proliferation,invasion and migration of glioma cells and enhancing their sensitivity to TMZ.
居来提·阿扎提;马涛;王洋;张晶晶
新疆医科大学第二附属医院检验科,新疆乌鲁木齐,830028新疆医科大学第二附属医院神经外科,新疆乌鲁木齐,830028
临床医学
微小RNA-15a-5pRUNX1基因胶质瘤替莫唑胺
microRNA-15a-5pRUNX1 genegliomatemozolomide
《实用临床医药杂志》 2024 (019)
10-15,21 / 7
新疆维吾尔自治区自然科学基金资助项目(2022DO1C503)
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