| 张坤虎,陈勃勃,王保江,等.微小 RNA-520a-5p通过靶向调控鼠双微体基因影响胶质瘤细胞的增殖、凋亡、迁移和侵袭[J].安徽医药,2022,26(12):2502-2507. |
| 微小 RNA-520a-5p通过靶向调控鼠双微体基因影响胶质瘤细胞的增殖、凋亡、迁移和侵袭 |
| MiR-520a-5p affects the proliferation, apoptosis, migration and invasion of glioma cells by targeting MDM2 gene |
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| DOI：10.3969/j.issn.1009-6469.2022.12.037 |
| 中文关键词: 神经胶质瘤 DNA，环状 鼠双微体基因(MDM2) 微小 RNA-520a-5p 细胞增殖 凋亡 迁移 侵袭 |
| 英文关键词: Glioma DNA,circular Murine double mimute 2(MDM2) MiR-520a-5p Cell proliferation Apoptosis Migra. tion Invasion |
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| 中文摘要: |
| 目的探讨微小 RNA-520a-5p(miR-520a-5p)在胶质瘤发生发展中的作用和机制。方法选取 2016年 5月至 2018年 11月在宝鸡高新人民医院治疗的胶质瘤病人肿瘤组织作 56例为研究对象。实时荧光定量 PCR(qRT-PCR)检测 56例胶质瘤组织和 56例正常脑组织中 miR-520a-5p表达水平。体外培养胶质瘤 U251细胞，转染 miR模拟对照序列( miR-NC)、 miR-520a-5p9模拟物( miR-520a-5p mimics)至 U251细胞，分别采用四甲基噻唑蓝染色法( MTT)、流式细胞仪、 Transwell和蛋白质印迹法检测过表达 miR-520a-5p对 U251细胞增殖、凋亡、迁移和侵袭及细胞中鼠双微体基因( MDM2)、细胞周期蛋白 D1(cyclin D1)、 P21、B淋巴细胞瘤 -2相关蛋白( Bax)、 B淋巴细胞瘤 -2(Bcl-2)、基质金属蛋白酶 2(MMP-2)和 E-钙黏蛋白( E-cadherin)蛋白表达的影响。双萤光素酶报告基因实验验证 miR-520a-5p与 MDM2靶向关系，蛋白质印迹法检测转染 miR-520a-5p mimics、miR-520a-5p抑制剂( anti-miR-520a-5p)对 U251细胞中 MDM2蛋白表达的影响。共转染 miR-520a-5p mimics和 MDM2过表达载体( pcD. NA3.1-MDM2)至 U251细胞，上述相同方法观察过表达 MDM2能否逆转过表达 miR-520a-5p对 U251细胞增殖、迁移和侵袭及相关蛋白表达的影响。结果与正常脑组织相比，胶质瘤组织中 miR-520a-5p表达降低( P<0.05)。与转染 miR-NC的 U251细胞比较，转染 miR-520a-5p mimics的 U251细胞 24 h、48 h和 72 h的 OD值［(0.36±0.04)比( 0.50±0.05)、(0.49±0.05)比( 0.95±0.09)、(0.71±0.07)比( 1.36±0.13)］、迁移细胞数［(67±6.26)比( 144±13.38)］、侵袭细胞数［(59±5.47)比( 135±13.12)］均降低( P<0.05)，凋亡率［( 21.17±2.06)%比( 7.82±0.77)%］升高( P<0.05)细胞中 cyclin D1、Bcl-2和 MMP-2的蛋白表达水平均降低( P<0.05)而 P21、Bax和 E-cadherin的蛋白表达水平均升高( P<0.05miR-520a-5p可与 MDM2的 3’UTR靶向结合，mimics的 U251细胞中 MDM2蛋白水平显著低于转染 miR-NC的细胞，而转染 anti-miR-520a-5p的 U251细胞中 MDM2的蛋白水平显著高于转染 anti-miR-NC的细胞。与共转染 miR-520a-5p mimics与 pcDNA3.1的 U251细胞比较，共转染 miR-520a-5p mim. ics与 pcDNA3.1-MDM2的 U251细胞 24 h、48 h和 72 h的 OD值、迁移细胞数、侵袭细胞数均升高( P<0.05)凋亡率降低( P< 0.05)。，同时转染miR-520a-5p，细胞中 cyclin D1、Bcl-2和 MMP-2的蛋白表达水平均升高( P<0.05)，而 P21、Bax和 E-cadherin的蛋白表达水平均降低( P<0.05)。结论 miR-520a-5p在胶质瘤组织中表达降低，过表达 miR-520a-5p可能通过靶向负调控 MDM2抑制胶质瘤 U251细胞增殖、迁移和侵袭，并诱导其凋亡。 |
| 英文摘要: |
| Objective To investigate the role and mechanism of miR-520a-5p in the occurrence and development of glioma.Meth. ods Fifty-six tumor tissues of glioma patients treated in Baoji High-tech People's Hospital from May 2016 to November 2018 were se. lected as the research objects. qRT-PCR was used to detect the expression of miR-520a-5p in 56 glioma tissues and 56 normal brain tis. sues. Glioma U251 cells were cultured in vitro, and miR-NC or miR-520a-5p mimics was transfected into U251 cells. Then Methyl Thi.azolyl Tetrazolium (MTT) staining, flow cytometry, Transwell and Western Blotting were used to detect the effect of over-expressing miR-520a-5p on the proliferation, apoptosis, migration and invasion of U251 cells, and the protein expression levels of mouse doublemicrosomal gene (MDM2), cyclin D1, P21, Bax, Bcl-2, MMP-2 and E-cadherin in cells. The dual luciferase reporter gene assay verified the targeting relationship between miR-520a-5p and MDM2, and Western blotting was used to detect the effect of transfection of miR-520a-5p mimics or anti-miR-520a-5p on the expression of MDM2 protein in U251 cells. miR-520a-5p mimics and pcDNA3.1-MDM2 were co-transfected into U251 cells, and then the same methods as above were used to observe whether the overexpression of MDM2could reverse the effect of the overexpression of miR-520a-5p on the proliferation, migration, invasion and related protein expression of U251 cells.Results Compared with normal brain tissue, the expression level of miR-520a-5p in glioma tissues decreased (P<0.05). Compared with the U251 cells transfected with miR-NC, the OD values [(0.36±0.04) vs. (0.50±0.05), (0.49±0.05) vs. (0.95±0.09), (0.71± 0.07) vs. (1.36±0.13)] of U251 cells transfected with miR-520a-5p mimics at 24 h, 48 h and 72 h, the number of migrating cells [(67± 6.26) vs. (144±13.38)], and the number of invading cells [(59±5.47) vs. (135±13.12)] decreased (P<0.05), the apoptosis rate [(21.17± 2.06) % vs. (7.82±0.77) %] increased (P<0.05), the protein expression levels of cyclin D1, Bcl-2 and MMP-2 decreased (P<0.05), while the protein expression levels of P21, Bax and E-cadherin increased (P<0.05). miR-520a-5p could target the 3'UTR of MDM2, and the level of MDM2 protein in U251 cells transfected with miR-520a-5p mimics was significantly lower than that in cells transfected with miR-NC, while the protein level of MDM2 in U251 cells transfected with anti-miR-520a-5p was significantly higher than that in cells transfected with anti-miR-NC. Compared with the U251 cells co-transfected with miR-520a-5p mimics and pcDNA3.1, the OD values of U251 cells co-transfected with miR-520a-5p mimics and pcDNA3.1-MDM2 at 24 h, 48 h and 72 h, the number of migrating cells and the number of invading cells increased (P<0.05), the apoptosis rate decreased (P<0.05), the protein expression levels of cyclin D1, Bcl-2 and MMP-2 increased (P<0.05), while the protein expression levels of P21, Bax and E-cadherin decreased (P<0.05).Conclusions The expression of miR-520a-5p reduced in glioma tissues. Overexpression of miR-520a-5p may inhibit the proliferation, migration and invasion of glioma U251 cells and induce its apoptosis by negatively regulating MDM2. |
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