miR-216b-5p Promotes Ferroptosis in Glioblastoma Cells by Targeting NCOA3
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摘要:
目的 主要探讨miR-216b-5p通过靶向NCOA3对胶质瘤细胞铁死亡的影响，以期为胶质母细胞瘤的临床治疗提供新的靶点。 方法 U251细胞分为DMSO处理组，Erastin处理组，Erastin + mimic NC组，Erastin + miR-216b-5p mimic组，Erastin + pcDNA 3.1组，Erastin + pcDNA-NCOA3组，pcDNA+miR-216b-5p mimic+ pcDNA-NCOA3组。通过实时荧光定量PCR检测和Western blot 检测miR-216b-5p和NCOA3的表达以及铁死亡相关蛋白PTGS2， NOX1， ACSL4，GPX4， FTH1的蛋白表达量，CCK-8和流式细胞术检测细胞增殖和凋亡，通过试剂盒检测MDA，亚铁（Fe2+），谷氨酰胺，谷氨酸，α-酮戊二酸的含量。 结果 （1） miR-216b-5p在人胶质母细胞瘤细胞株LN229和U251中均高表达，Erastin刺激下的U25细胞活力回升且细胞凋亡率下降；（2）转染miR-216b-5p mimics后，Erastin刺激下的U251中的MDA，亚铁（Fe2+），谷氨酰胺，谷氨酸，α-酮戊二酸均有明显下降，铁死亡相关蛋白PTGS2， NOX1， ACSL4的蛋白表达量在Erastin的刺激下明显上升，而GPX4， FTH1的蛋白量明显下降；（3）获得9个在胶质母细胞瘤中与铁死亡相关，且与miR-261b-5p有直接靶向关系的基因，即FOXO4，NCOA3，PARP8，PARP11，LIG3，MAPK9，TLR4，RB1，PTEN，miR-216b-5p与 NCOA3直接结合向并负向调控NCOA3；4. 转染NCOA3使细胞活力下降，MDA，亚铁（Fe2+），谷氨酰胺，谷氨酸，α-酮戊二酸含量上升，铁死亡相关蛋白PTGS2，NOX1，ACSL4的蛋白表达量上升，而GPX4，FTH1的蛋白量下降；但miR-216b-5p mimic 的转染可逆转以上结果。 结论 miR-216b-5p直接靶向NCOA3，且通过NCOA3调控胶质母细胞瘤细胞的铁死亡，以此促进肿瘤的凋亡。对胶质母细胞瘤的新的治疗靶点以及后续研究提出了新的可能性，以期为胶质母细胞瘤的治疗提供新思路。 -
关键词:
- 胶质母细胞瘤 /
- miR-216b-5p /
- NCOA3 /
- 铁死亡
Abstract:Objective To investigate the effect of miR-216b-5p on ferroptosis in glioblastoma cells by targeting NCOA3, with a view to providing new targets for the clinical treatment of glioblastoma. Methods U251 cells were divided into DMSO-treated group, Erastin-treated group, Erastin + mimic NC group, Erastin + miR-216b-5p mimic group, Erastin + pcDNA 3.1 group, Erastin + pcDNA-NCOA3 group, pcDNA + miR-216b-5p mimic+ pcDNA-NCOA3 group. The expression of miR-216b-5p and NCOA3 and the expression of ferroptosis -related proteins PTGS2, NOX1, ACSL4, GPX4, FTH1 were detected by real-time fluorescence quantitative PCR assay and Western blot, and cell proliferation and apoptosis were detected by CCK-8 and flow cytometry, and MDA, Fe2+, glutamine, glutamic acid and α-ketoglutarate by kit. Results miR-216b-5p was highly expressed in human glioblastoma cell lines LN229 and U251, and the viability of Erastin-stimulated U25 cells increased and the apoptosis rate decreased. After transfection with miR-216b-5p mimics, MDA, Fe2+, glutamine, glutamic acid and α-ketoglutarate were all decreased significantly, and the expression of ferroptosis-related proteins PTGS2, NOX1, and ACSL4 increased significantly upon Erastin stimulation, while the protein amount of GPX4 and FTH1 decreased significant. 9 genes associated with ferroptosis in glioblastoma and directly targeted to miR-261b-5p were obtained, namely FOXO4, NCOA3, PARP8, PARP11, LIG3, MAPK9, TLR4, RB1, PTEN, miR-216b-5p, which directly bind to and negatively regulate NCOA3. Transfection with NCOA3 decreased cell viability, MDA, Fe2+, glutamine, glutamic acid, α-ketoglutarate levels, the protein expression of PTGS2, NOX1 and ACSL4 increased and that of GPX4 and FTH1 decreased; however, transfection of miR-216b-5p mimic reversed these results. Conclusion This study found that miR-216b-5p directly targets NCOA3 and regulates ferroptosis in glioblastoma cells through NCOA3, thereby promoting apoptosis. This study suggests new therapeutic targets for glioblastoma as well as new possibilities for subsequent research, in order to provide new ideas for the treatment of glioblastoma. -
Key words:
- Glioblastoma /
- miR-216b-5p /
- NCOA3 /
- Ferroptosis
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图 1 miR-216b-5p参与Erastin介导的细胞凋亡
A：miR-216b-5p在人脑胶质细胞HEB、人胶质母细胞瘤细胞株LN229和U251中的表达；B：qRT-PCR检测miR-216b转染效率；C：CCK-8检测细胞活力；D（a）：Annexin V-FITC/PI-流式检测凋亡流式图；D（b）：数据统计柱状图。**P < 0.01；***P < 0.001；****P < 0.0001。
Figure 1. miR-216b-5p was involved in Erastin-mediated apoptosis
图 2 miR-216-5p 的上调缓解胶质母细胞瘤细胞在Erastin介导的脂质过氧化和铁离子累积
A：MDA水平检测；B：亚铁离子（Fe2+）水平检测；C：谷氨酰胺浓度检测；D：谷氨酸浓度检测；E：a-酮戊二酸浓度检测；F～K：western blot检测PTGS2， NOX1， FTH1， GPX4， ACSL4的蛋白表达。*P < 0.05；**P < 0.01；****P < 0.0001。
Figure 2. Upregulation of miR-216-5p alleviates Erastin-mediated lipid peroxidation and iron accumulation in glioblastoma cells
图 3 miR-216b-5p直接靶向并负向调控NCOA3
A：Venn图获取铁死亡相关基因，胶质母细胞瘤相关基因，miR-216b-5p的靶向基因中的共有基因；B：GO/KEGG富集分析；C：NCOA3与miR-216b-5p之间的结合位点预测；D：双荧光素酶对miR-216b-5p和NCOA3的靶向关系进行验证；E：qPCR检测NCOA3的mRNA表达；F，G：western blot 检测NCOA3的蛋白表达及对应灰度值。**P < 0.01；***P < 0.001；****P < 0.0001。
Figure 3. miR-216b-5p directly targets and negatively regulates NCOA3
图 4 miR-216b-5p 通过NCOA3调控胶质母细胞瘤细胞的铁死亡
A：CCK-8检测细胞活力；B：MDA水平检测；C：亚铁离子（Fe2+）水平检测；D：谷氨酰胺浓度检测；E：谷氨酸浓度检测；F：a-酮戊二酸浓度检测；G～M）：western blot检测PTGS2，NOX1， FTH1，GPX4，ACSL4的蛋白表达。*P < 0.05；**P < 0.01；***P < 0.001；****P < 0.000 1。
Figure 4. miR-216b-5p regulates iron death in glioblastoma cells via NCOA3
表 1 荧光定量PCR扩增引物列表
Table 1. Nucleotide sequences of the primers used for real-time quantitative PCR
引物名称 引物序列 NCOA3 forward 5’-AAGTGAAGAGGGATCTGGA-3’ NCOA3 reverse 5’-CAGATGACTACCATTTGAGG-3’ miR-216b-5p forward 5ʹ-TGAGAACTGAATTCCATGGGTT-3ʹ miR-216b-5p reverse 5ʹ-CCCAUGGAAUUCAGUUCUCAUU -3ʹ. U6 forward 5’-GCTTCGGCAGCACATATACTAA-3’ U6 reverse 5’-CGAATTTGCGTGTCATCCTT-3’ GAPDH forward 5’-CGAATTTGCGTGTCATCCTT-3’ GAPDH reverse 5’-CGAATTTGCGTGTCATCCTT-3’ 
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表 2 Western blot中所用一抗
Table 2. Primary antibodies used in western blot
一抗 公司 货号 稀释比 分子量 种属 PTGS2 Abcam ab179800 1∶1000 69 Rabbit NOX1 Abcam ab131088 1∶1000 65 Rabbit ACSL4 Abcam ab155282 1∶10000 79 Rabbit GPX4 Abcam ab125066 1∶1000 22 Rabbit FTH1 Abcam ab75972 1∶500 21 Rabbit NCOA3 Abcam ab133611 1∶1000 160 Rabbit GAPDH Abmart P30008M 1∶1000 37 Rabbit
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