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miR-149-5p通过MSH5/Wnt信号通路调控胶质瘤细胞恶性生物学行为

冯毅 王小峰 白西民 姚胜 党俊涛 赵云洁 蔡冰

冯毅, 王小峰, 白西民, 姚胜, 党俊涛, 赵云洁, 蔡冰. miR-149-5p通过MSH5/Wnt信号通路调控胶质瘤细胞恶性生物学行为[J]. 昆明医科大学学报, 2023, 44(8): 59-70. doi: 10.12259/j.issn.2095-610X.S20230823
引用本文: 冯毅, 王小峰, 白西民, 姚胜, 党俊涛, 赵云洁, 蔡冰. miR-149-5p通过MSH5/Wnt信号通路调控胶质瘤细胞恶性生物学行为[J]. 昆明医科大学学报, 2023, 44(8): 59-70. doi: 10.12259/j.issn.2095-610X.S20230823
Yi FENG, Xiaofeng WANG, Ximin BAI, Sheng YAO, Juntao DANG, Yunjie ZHAO, Bing CAI. miR-149-5p Regulates Malignant Biological Behavior of Glioma Cells Through MSH5/WNT Signaling Pathway[J]. Journal of Kunming Medical University, 2023, 44(8): 59-70. doi: 10.12259/j.issn.2095-610X.S20230823
Citation: Yi FENG, Xiaofeng WANG, Ximin BAI, Sheng YAO, Juntao DANG, Yunjie ZHAO, Bing CAI. miR-149-5p Regulates Malignant Biological Behavior of Glioma Cells Through MSH5/WNT Signaling Pathway[J]. Journal of Kunming Medical University, 2023, 44(8): 59-70. doi: 10.12259/j.issn.2095-610X.S20230823

miR-149-5p通过MSH5/Wnt信号通路调控胶质瘤细胞恶性生物学行为

doi: 10.12259/j.issn.2095-610X.S20230823
基金项目: 陕西省渭南市重点科技计划项目(2019-ZDYF-SFCX-13);陕西省渭南市首批自然科学和工程技术类青年人才支持项目
详细信息
    作者简介:

    冯毅(1978~),男,陕西西安人,副主任医师,主要从事颅脑肿瘤及脊柱脊髓相关疾病的诊治工作

    通讯作者:

    蔡冰,E-mail:caibing0101@163.com

  • 中图分类号: R739.41

miR-149-5p Regulates Malignant Biological Behavior of Glioma Cells Through MSH5/WNT Signaling Pathway

  • 摘要:   目的  探索miR-149-5p调控MSH5/Wnt信号通路影响胶质瘤细胞恶性生物学行为的具体作用机制。  方法  RT-qPCR检测miR-149-5p在胶质瘤组织和细胞系A172、U251、HS683、H4中的表达。CCK-8检测细胞活力,EDU染色检测细胞增殖,Transwell小室检测细胞的迁移和侵袭,流式细胞术检测细胞周期和细胞凋亡。双荧光素酶报告基因实验验证miR-149-5p和MSH5的靶向关系。Western blot检测MSH5、GAPDH、GSK3β、β-catenin、AXIN2的蛋白表达。  结果  miR-149-5p在胶质瘤组织(P < 0.0001)和细胞系中表达降低(P < 0.0001),过表达miR-149-5p可显著抑制A172细胞的增殖(P < 0.01)、迁移(P < 0.05)和侵袭(P < 0.01)、细胞周期(P < 0.01),并诱导凋亡(P < 0.01),敲降miR-149-5p则具有相反的作用。双荧光素酶报告基因实验证明miR-14-5p靶向MSH5。敲降miR-149-5p可逆转敲降MSH5对A172细胞恶性生物学行为的作用。过表达MSH5通过抑制GSK3β、激活β-catenin/AXIN2通路,促进A172细胞的增殖、迁移和侵袭和细胞周期,并抑制细胞凋亡。  结论  miR-149-5p通过靶向上调MSH5,抑制AXIN2表达激活β-catenin/AXIN2通路,从而抑制间质瘤细胞的恶性生物学行为。
  • 图  1  miR-149-5p在胶质瘤组织和细胞中的表达

    A:RT-qPCR检测胶质瘤组织中miR-149-5p的表达,与Para-cancer组相比,****P < 0.0001;B:RT-qPCR检测miR-149-5p在人正常星形胶质细胞阿和人胶质瘤细胞中的表达,与HA1800组相比,**P < 0.01,****P < 0.0001。

    Figure  1.  miR-149-5p was downregulated in glioma tissues and cells

    图  2  miR-149-5p对A172细胞恶性生物学行为的影响

    A:RT-qPCR检测miR-149-5p的表达;B:CCK-8检测细胞活力;C和F:EDU检测细胞增殖;D和G:Transwell检测细胞迁移能力;E和H:Transwell检测细胞侵袭能力;与NC mimic组相比,*P < 0.05,**P < 0.01,****P < 0.000 1;与NC inhibitor组相比,#P < 0.05。

    Figure  2.  Effect of miR-149-5p on malignant biological behavior of A172 cells

    图  3  miR-149-5p对A172细胞恶性生物学行为的影响

    A和B:流式细胞术检测细胞周期;C和D:流式细胞术检测细胞凋亡率;与NC mimic组相比,**P < 0.01;与NC inhibitor组相比,#P < 0.05。

    Figure  3.  Effect of miR-149-5p on malignant biological behavior of A172 cells

    图  4  miR-149-5p靶向MSH5

    A:starbase数据库预测miR-149-5p和MSH5的结合序列;B:双荧光素酶报告基因实验验证miR-149-5p和MSH5的靶向关系,与NC mimic组相比,*P < 0.05;C:GEPIA数据库预测MSH5在胶质瘤组织中的表达,与Para-cancer组相比,**P < 0.01;D:Western blot检测MSH5的蛋白表达,与NC组相比,**P < 0.01。

    Figure  4.  miR-149-5p targeted MSH5

    图  5  miR-149-5p靶向MSH5调控A172细胞的恶性生物学行为

    A和B:Western blot检测MSH5蛋白表达;C:CCK-8检测细胞活力;D和E:EDU实验检测细胞增殖;F和G:Tranwell实验检测细胞迁移能力;H和I:Tranwell实验检测细胞侵袭能力;与sh-NC组相比,*P < 0.05,**P < 0.01,***P < 0.001;与sh-MSH5组相比,#P < 0.05。

    Figure  5.  miR-149-5p targeted MSH5 to regulate the malignant biological behavior of A172 cells

    图  6  miR-149-5p靶向MSH5调控A172细胞的恶性生物学行为

    A和B:流式细胞术检测细胞周期;C和D:流式细胞术检测细胞凋亡率;与sh-NC组相比,**P < 0.01,***P < 0.001;与sh-MSH5组相比,#P < 0.05。

    Figure  6.  miR-149-5p targeted MSH5 to regulate the malignant biological behavior of A172 cells

    图  7  MSH5调控Wnt信号通路对A172细胞的作用

    A:Western blot检测MSH5,B:GSK3β,C:β-catenin,D:和AXIN2,E:的蛋白表达;F:CCK-8检测细胞活力;G和H:EDU检测细胞增殖;I和J:Transwell检测细胞迁移能力;K和L:Transwell检测细胞迁移能力;与pcDNA-NC组相比,*P < 0.05,**P < 0.01,***P < 0.001;与pcDNA-MSH5组相比,#P < 0.05,##P < 0.01,###P < 0.001。

    Figure  7.  Effect of MSH5 regulating Wnt signaling pathway on A172 cells

    图  8  MSH5调控Wnt信号通路对A172细胞的作用

    A和B:流式细胞术检测细胞周期;C和D:流式细胞术检测细胞凋亡率;与pcDNA-NC组相比,*P < 0.05;与pcDNA-MSH5组相比,#P < 0.05。

    Figure  8.  Effect of MSH5 regulating Wnt signaling pathway on A172 cells

    表  1  引物序列

    Table  1.   Primer sequences

    目的基因引物序列 (F:正向序列,R:反向序列,5′-3′)
    miR-149-5p F: CTCTGGCTCCGTGTCTTCAC
    R: CTGCCCCAGCACAGCC
    U6 F: TGCGGGTGCTCGCTTCGGCAGC
    R: CCAGTGCAGGGTCCGAGGT
    下载: 导出CSV
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  • 收稿日期:  2023-06-13
  • 网络出版日期:  2023-09-04
  • 刊出日期:  2023-08-30

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