作者单位：315000 宁波，宁波明州医院神经外科(江汉清)；510630 广州，中山大学附属第三医院(罗仑，梁朝峰)

营养缺乏自噬因子1对人脑胶质瘤U251细胞增殖侵袭能力的影响

江汉清罗仑梁朝峰

【摘要】目的探讨营养缺乏自噬因子1对人脑胶质瘤U251细胞增殖侵袭能力的影响。 方法 复苏培养人脑胶质瘤U251细胞，建立转染小干扰RNA(siRNA)的NAF1组(si-NAF1组)、阴性对照组(si-NC组)、未转染siRNA对照组(control组)，采用CCK-8实验检测细胞增殖能力、平板克隆形成实验检测细胞克隆形成能力、细胞划痕实验检测各组细胞迁移能力、Transwell法观察各组细胞侵袭能力变化。结果与control组比较，NAF1-siRNA干预U251细胞后，si-NAF1组NAF1的mRNA和蛋白表达水平均明显降低(P均<0.05)，其增殖细胞核抗原(PCNA)、基质金属蛋白酶-9(MMP-9)蛋白表达水平降低，细胞增殖能力下降，克隆形成能力明显减弱，迁移能力明显降低，穿过Transwell膜的细胞数量明显减少(P均<0.05)。si-NC组与control组各项结果比较差异无统计学意义(P>0.05)。结论转染siRNA后NAF1表达下降，进而下调PCNA水平，抑制人脑胶质瘤U251细胞的增殖能力；同时MMP-9蛋白表达水平下降，抑制U251细胞的迁移和侵袭能力。

【关键词】脑胶质瘤；RNA干扰；细胞增殖；肿瘤侵袭

DOI：10.3969/g.issn.0253-9802.2015.02.004

通讯作者，梁朝峰，E-mail：lcfjeff@163.com

收稿日期：(2014-10-09)

Influence of nutrient-deprivation autophagy factor-1 on the proliferation and invasion of human glioma cell line U251JiangHanqing,LuoLun，LiangChaofeng.DepartmentofNeurosurgery，NingboMingzhouHospital,Ningbo315000,China

Correspondingauthor,LiangChaofeng,E-mail:lcfjeff@163.com

Abstract【】ObjectiveTo investigate the effect of nutrient-deprivation autophagy factor-1 (NAF1) upon the proliferation and invasion of human glioma cell line U251. MethodsResuscitation of human glioma cell line U251 was conducted. The cultured U251 cells were divided into the si-NAF1, si-NC and control groups. CCK-8 assay was performed to assess the proliferation of U251 cells. Plate cloning formation assay was utilized to evaluate the formation of cell cloning. Wound healing assay was conducted to investigate the migration of U251 cells. Changes in the migration of U251 cells were determined by Transwell invasion assay. ResultsThe expression of NAF1 in U251 cells transfected with NAF1-siRNA at both protein and RNA levels was significantly down-regulated compared with those in si-NC and control groups (both P<0.05). The protein expression of proliferating cell nuclear antigen (PCNA) and matrix metalloprotein-9 (MMP-9) was significantly down-regulated. The proliferation, formation of cell cloning and migration of U251 cells in the si-NAF1 group were obviously suppressed, and the count of U215 cells trespassing the Transwell membrane was significantly decreased compared with those in the control group (all P<0.05). There was no statistically significant difference between Si-NC group and the control group in above indexes(P>0.05). ConclusionsAfter transfection with siRNA, the expression of NAF1 was decreased, thereby down-regulating the expression of PCNA and inhibiting the proliferation of U251 cells. Down-regulated expression level of MMP-9 protein suppressed the proliferation and migration of U251 cells.

【Key words】Glioma; RNA interference; Cell proliferation; Neoplasm invasion

脑胶质瘤是最常见的中枢神经系统恶性肿瘤[1]。脑胶质瘤的手术、放射治疗、化学治疗等治疗方法在不断改进，但其疗效仍然未得到明显提高，患者预后较差，中位生存期仅13个月左右[2-3]。阐明脑胶质瘤生存过程中导致增殖和侵袭的机制，有利于发现新的治疗策略[4]。因此，寻找和研究在脑胶质瘤的生物学过程中的关键分子具有重要的科学意义和临床应用价值。营养缺乏自噬因子1(NAF1)基因定位于人4号染色体长臂，是一种新发现的、与细胞能量代谢和增殖凋亡相关的蛋白[5]。近年研究表明，其表达与乳腺癌等多种恶性肿瘤的增殖、侵袭密切相关[6]。……