摘要：

目的 探讨胶质瘤miRNA-449b表达异常减少对细胞增殖活性和侵袭能力的影响。方法 采用锁定寡核苷酸探针原位杂交法检测60例不同级别胶质瘤患者和10例非肿瘤对照脑组织miRNA-449b表达变化，Stem-loop实时定量逆转录聚合酶链反应（qRT-PCR）检测胶质母细胞瘤细胞系U251、LN229和永生化星形胶质细胞UC2 miRNA-449b相对表达量，EdU染色、MTS法和流式细胞术检测外源性miRNA-449b对胶质瘤细胞增殖活性和细胞周期的影响，Transwell侵袭实验和荧光染色检测miRNA-449b对胶质瘤细胞侵袭能力的影响。结果 各级别胶质瘤miRNA-449b阳性标记指数均低于对照组（均P<0.001），且随胶质瘤恶性程度的增加，miRNA-449b阳性标记指数降低（均P<0.001）。U251细胞、LN229细胞miRNA-449b相对表达量低于UC2细胞（P=0.001，0.002）。U251-miRNA-449b组miRNA-449b相对表达量高于U251-Scr组（P<0.001）、EdU阳性细胞率（P=0.029）和细胞增殖活性（P=0.004，0.001，0.002）低于U251-Scr组，LN229-miRNA-449b组miRNA-449b相对表达量高于LN229-Scr组（P<0.001）、EdU阳性细胞率（P=0.032）和细胞增殖活性（P=0.003，0.001，0.004）低于LN229-Scr组。U251-miRNA-449b组和LN229-miRNA-449b组G₀期/G₁期细胞百分比分别高于U251-Scr组（P<0.001）和LN229-Scr组（P=0.018），侵袭细胞数目分别低于U251-Scr组（P=0.002）和LN229-Scr组（P=0.005）。U251-Scr组和LN229-Scr组F-actin局部聚集于细胞突起部位以及胞膜内侧，U251-miRNA-449b组和LN229-miRNA-449b组F-actin散在分布于肿瘤细胞内。结论 miRNA-449b是胶质瘤重要的抑瘤miRNA，其表达异常减少可能是导致胶质瘤发生发展的重要因素，可以作为评价胶质瘤恶性程度的重要参考指标；补充外源性miRNA-449b可以有效抑制胶质瘤细胞的增殖活性和侵袭能力，在恶性胶质瘤的治疗中具有潜在应用价值。

关键词: 神经胶质瘤, 微RNAs, 细胞增殖, 细胞侵袭(非MeSH词)

Abstract:

Objective To investigate the expression of miRNA-449b in gliomas and its effect on cell proliferation and invasion. Methods The expression level of miRNA-449b was detected by locked-oligonucleotide-probe in situ hybridization (ISH) in 60 human glioma specimens of different WHO grades and 10 non-tumoral control brain tissues. miRNA-449b was quantified by Stem-loop quantitation real-time polymerase chain reaction (qRT-PCR) with U6 as the internal control in U251, LN229 and UC2 cells. EdU staining, MTS method, flow cytometry assay (FCM), Phalloidin staining and Transwell assays were used to evaluate the effect of miRNA-449b on the proliferation and invasion of U251 and LN229 cells. Results The expression of miRNA-449b was decreased in gliomas compared with that in the non-tumoral control brain tissues (P<0.001, for all), and was significantly declined with the elevation of glioma grades (P<0.001, for all). miRNA-449b level was significantly lower in U251 and LN229 cells than that in UC2 cells (P=0.001, 0.002). The relative expression of miRNA-449b in U251-miRNA-449b group was higher (P<0.001), while EdU-positive rates (P=0.029) and cell proliferation (P=0.004, 0.001, 0.002) was lower than U251-Scr group; the relative expression of miRNA-449b in LN229-miRNA-449b group was higher (P<0.001), while EdU-positive rates (P=0.032) and cell proliferation (P=0.003, 0.001, 0.004) was lower than LN229-Scr group. The percentages of each phase G₀/G₁ in U251-miRNA-449b group and LN229-miRNA-449b group were higher than those in U251-Scr group (P<0.001) and LN229-Scr group (P=0.018) respectively, cell proliferation were lower than U251-Scr group (P=0.002) and LN229-Scr group (P=0.005). F-actin was centralized mostly on cell cortex or lamellae in the groups of U251-Scr and LN229-Scr, whereas diffusely distributed in the groups of U251-miRNA-449b and LN229-miRNA-449b. Conclusions miRNA-449b is an important tumor-suppressive miRNA and its downregulation may be an important cause leading to tumorigenesis of glioblastoma, which could be used as a valuable biomarker to assess the malignant degree of gliomas. Furthermore, the exogenous miRNA-449b could effectively inhibit the proliferation and invasion abilities of glioma cells, confirming it has potential value in malignant gliomas therapy.

Key words: Glioma, MicroRNAs, Cell proliferation, Cell invasion (not in MeSH)