下调 Stathmin 基因对 U373 和 U87-MG 脑胶质瘤细胞生物学行为的影响。

The effect of downregulation of Stathmin gene on biological behaviors of U373 and U87-MG glioblastoma cells.

机构信息

Department of Oncology, Peace Hospital of Changzhi Medical College, Changzhi, 046000, Shanxi, China.

出版信息

Biol Res. 2018 Jun 8;51(1):16. doi: 10.1186/s40659-018-0160-0.

DOI:10.1186/s40659-018-0160-0

PMID:29880026

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5992777/

Abstract

BACKGROUND

Stathmin as a critical protein involved in microtubule polymerization, is necessary for survival of cancer cells. However, extremely little is known about Stathmin in glioblastoma. So, this study was designed to elucidate the function of Stathmin gene in the tumorigenesis and progression of glioblastoma cells.

METHOD

The lentiviral interference vector pLV3-si-Stathmin targeting Stathmin gene and the control vector pLV3-NC were established for the co-transfection of 293T cells together with the helper plasmids. Viral titer was determined via limiting dilution assay. Then pLV3-si-Stathmin and pLV3-NC were stably co-transfected into U373 and U87-MG glioblastoma cells. Expression levels of Stathmin protein in each group were determined by using Western Blot, and the proliferation and migration ability of the cells with downregulated Stathmin were evaluated through CCK8 assay and transwell invasion assay, respectively. Cell cycles and cell apoptosis were detected with flow cytometry. Finally, the effect of Stathmin in tumor formation was determined in nude mice.

RESULT

DNA sequencing and viral titer assay indicated that the lentiviral interference vector was successfully established with a viral titer of 4 × 10 TU/ml. According to the results from Western Blotting, Stathmin protein expression level decreased significantly in the U373 and U87-MG cells after transfected with pLV3-si-Stathmin, respectively, compared with those transfected with pLV3-NC. In glioblastoma cells, the cell proliferation and migration were greatly inhibited after the downregulation of Stathmin protein. Flow cytometry showed that much more cells were arrested in G2/M phasein Stathmin downregulated group, compared with the non-transfection group and NC group. But Stathmin downregulation did not induce significant cell apoptosis. Tumor formation assay in nude mice showed that tumor formation was delayed after Stathmin downregulation, with a reduction in both tumor formation rate and tumor growth velocity.

CONCLUSION

Stathmin downregulation affected the biological behaviors of U373 and U87-MG glioblastoma cells, inhibiting the proliferation and migration of tumor cells. Stathmin gene may serve as a potential target in gene therapy for glioblastoma.

摘要

背景

作为一种参与微管聚合的关键蛋白，Stathmin 对于癌细胞的存活是必需的。然而，目前对于神经胶质瘤中 Stathmin 的了解甚少。因此，本研究旨在阐明 Stathmin 基因在神经胶质瘤细胞的发生和进展中的作用。

方法

构建靶向 Stathmin 基因的慢病毒干扰载体 pLV3-si-Stathmin 和对照载体 pLV3-NC，与辅助质粒共转染 293T 细胞，通过有限稀释法测定病毒滴度。然后将 pLV3-si-Stathmin 和 pLV3-NC 稳定共转染至 U373 和 U87-MG 神经胶质瘤细胞。采用 Western Blot 检测各组 Stathmin 蛋白的表达水平，通过 CCK8 检测和 Transwell 侵袭实验分别评估下调 Stathmin 后细胞的增殖和迁移能力，采用流式细胞术检测细胞周期和细胞凋亡。最后，在裸鼠体内检测 Stathmin 对肿瘤形成的影响。

结果

DNA 测序和病毒滴度测定表明，慢病毒干扰载体构建成功，病毒滴度为 4×10 TU/ml。Western Blotting 结果显示，与 pLV3-NC 转染组相比，pLV3-si-Stathmin 转染的 U373 和 U87-MG 细胞中 Stathmin 蛋白表达水平显著降低。在神经胶质瘤细胞中，下调 Stathmin 蛋白表达后，细胞增殖和迁移能力受到显著抑制。流式细胞术显示，Stathmin 下调组中更多的细胞被阻滞在 G2/M 期，而非转染组和 NC 组。但 Stathmin 下调并未诱导明显的细胞凋亡。裸鼠肿瘤形成实验表明，下调 Stathmin 后肿瘤形成延迟，肿瘤形成率和肿瘤生长速度均降低。