miR-146b-5p对胶质瘤TJ905细胞生长的抑制作用及其机制探讨

Inhibitory Action of miR-146b-5p on Growth and Mechanism of TJ905 Glioma Cells

目的:在TJ905胶质母细胞瘤细胞系中观察miR-146b-5p对MMP16的调控作用及其对细胞侵袭、迁移、增殖和凋亡的影响。方法:分别用无义序列表达质粒(对照组)和miR-146b-5p表达质粒(P-miR-146b-5p组)转染TJ905细胞,用qRT-PCR和Western blot检测两组细胞miR-146b-5p的表达水平及MMP16 mRNA和蛋白的表达水平,用体外迁移侵袭实验及流式细胞术检测转染细胞迁移、侵袭、细胞周期分布和凋亡水平,并分析这些变化的相互关系。结果:与对照组相比,p-miR-146b-5p组MMP16 mRNA和蛋白表达量明显降低,二者间呈正相关且均与miR-146b-5p表达量呈负相关。p-miR-146b-5p组侵袭和迁移细胞数均明显低于对照组,并均与同组MMP16蛋白表达量呈正相关;而凋亡水平明显高于对照组,并与同组miR-146b-5p表达量呈正相关,但两组细胞周期时相分布无显著性差异。结论:miR-146b-5p是胶质瘤的抑瘤miRNA;补充外源性miR-146b-5p可促进胶质瘤细胞凋亡,并通过抑制靶基因MMP16表达阻止其侵袭迁移;提示miR-146b-5p在恶性胶质瘤基因治疗方面具有重要的潜在应用价值。

To clarify whether miR-146b-5p can modulate the expression of MMP-16 and to observe its effects on migration, invasion, proliferation, and apoptosis of the glioblastoma cell line. Methods： TJ905 cells were transfected with the expression plasmid of the nonsense scrambled sequence （ Group 1 ） or that of miR-146b-5p （ Group 2 ）. The expression levels of miR-146b-5p, MMP 16 mRNA, and MMP 16 protein were measured by quantitative real-time polymerase chain reaction （ qRT-PCR ） and Western blot assay. Migratory and invasive abilities and cell cycle distribution and apoptosis in the two groups were assessed by in vitro migration assays and by invasion and flow cytometry. Results： Compared with Group 1, the expression levels ofMMP16 mRNA and MMP16 protein significantly decreased in Group 2. MMP16 mRNA and MMP16 protein expression levels positively correlated with each other, whereas they negatively correlated with the expression level of miR-146b-5p. The number of migrated and invaded cells was significantly lower in Group 2 than in Group 1, which was positively correlated with the expression level of MMP 16. The apoptotic level was significantly higher in Group 2 than in Group 1 and was positively correlated with the miR-146b-5p expression level in Group 2. However, the two groups shared a similar pattern on cell cycle distribution. Conclusion： MiR-146b-5p is a tumor-suppressive mRNA against glioma. Exogenous miR-146b-5p can effectively promote apoptosis of the glioma cells and can inhibit invasive and migratory abilities by down-regulating the expression level of MMP16. Our results suggest a potential value of miR-146b-5p in the gene therapy of malignant gliomas.