miR-149-5p Regulates Malignant Biological Behavior of Glioma Cells Through MSH5/WNT Signaling Pathway
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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。 Abstract:Objective To explore the mechanism of miR-149-5p regulating the malignant biological behavior of glioma cells through MSH5/Wnt signaling pathway. Methods RT-qPCR was used to detected the expression of miR-149-5p in glioma tissues and cell lines A172, U251, HS683 and H4. CCK-8 was used to detected cell viability, and EDU staining was performed to measure cell proliferation, Transwell assay was used to detected cell migration and invasion, and flow cytometry examined cell cycle and apoptosis. The dual luciferase reporter gene experiment verified the targeting relationship between miR-149-5p and MSH5. The protein expressions of MSH5, GAPDH, GSK3β, β-catenin and AXIN2 were detected by Western blot. Results The expression of miR-149-5p was decreased in glioma tissues （P < 0.0001） and cell lines （P < 0.0001）. Overexpression of miR-149-5p significantly inhibited the proliferation （P < 0.01）, migration （P < 0.05）, invasion （P < 0.01）, and cell cycle （P < 0.01） of A172 cells, and induced cell apoptosis （P < 0.01）. However, knocking down miR-149-5p had the opposite effect. Dual luciferase assay verified that miR-149-5p targeted MSH5. Knockdown of miR-149-5p reversed the effect of knocking down MSH5 on the malignant biological behavior of A172 cells. Overexpression of MSH5 promoted the proliferation, migration and invasion of A172 cells, cell cycle, and inhibited apoptosis by inhibiting GSK3β and activating β-catenin/AXIN2 pathway. Conclusions miR-149-5p inhibited the AXIN2 expression and activated the β-catenin/AXIN2 pathway by targeting up-regulation of MSH5, thereby inhibited the malignant biological behavior of stromal tumor cells. -
Key words:
- Glioma /
- miR-149-5p /
- MutS Homolog 5 /
- Malignant biological behavior /
- Proliferation /
- Metastasis
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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 
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