Objective

To investigate the function of Ras-guanine nucleotide release factor 1 (Ras-GRF1) in glioma through mediating H-Ras/ERK signaling pathway.

Methods

Ras-GRF1, H-Ras, K-Ras and N-Ras expressions in glioma and normal brain tissues were detected via Immunohistochemistry. Glioma cells (U87 cells, U251 cells and primary human glioma cells) were transfected with Ras-GRF1 siRNA, H-Ras siRNA and/or Ras-GRF1 lentivirus activation particles. Then, the following aspects were evaluated: cell proliferation by MTT assay, clonogenic ability by the plate clone formation experiment, cell migration and invasion by Wound-healing and Transwell assays, and cell apoptosis by Annexin-V-FITC/PI staining. The protein expressions were measured by Western blotting. Subcutaneous and orthotopic mouse models of glioma were conducted to determine the role of Ras-GRF1 in glioma tumorigenesis.

Results

Ras-GRF1, H-Ras, K-Ras and N-Ras expressions were upregulated in the glioma tissues, which were correlated with the WHO grade of glioma. Besides, Ras-GRF1 expression was positively related to H-Ras expression. Ras-GRF1 siRNA could reduce the expression of H-Ras and p-ERK/ERK in glioma cell. H-Ras siRNA inhibited the proliferation, clone formation, migration and invasion, and enhance the apoptosis of glioma cells, which, however, were reversed by Ras-GRF1 lentivirus activation particles. In vivo experiments also revealed that Ras-GRF1 shRNA reduced the volume and weight of the tumors in the nude mice, with down-regulations of H-Ras and p-ERK/ERK.

Conclusion

Ras-GRF1 was upregulated in glioma tissues and correlated with its malignancy and prognosis. Silencing Ras-GRF1, through mediating H-Ras/ERK pathway, may suppress the growth and metastasis of glioma.

中文翻译：

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Ras-鸟嘌呤核苷酸释放因子1介导的H-Ras/ERK信号通路对胶质瘤的影响

 客观的

探讨Ras-鸟嘌呤核苷酸释放因子1（Ras-GRF1）通过介导H-Ras/ERK信号通路在胶质瘤中的功能。

 方法

采用免疫组化法检测胶质瘤和正常脑组织中Ras-GRF1、H-Ras、K-Ras和N-Ras的表达。神经胶质瘤细胞（U87细胞、U251细胞和原代人神经胶质瘤细胞）用Ras-GRF1 siRNA、H-Ras siRNA和/或Ras-GRF1慢病毒激活颗粒转染。然后，通过MTT法评估细胞增殖能力，通过板克隆形成实验评估细胞克隆形成能力，通过伤口愈合和Transwell实验评估细胞迁移和侵袭能力，通过Annexin-V-FITC/PI染色评估细胞凋亡。通过蛋白质印迹法测量蛋白质表达。研究人员建立了神经胶质瘤的皮下和原位小鼠模型，以确定 Ras-GRF1 在神经胶质瘤发生中的作用。

 结果

Ras-GRF1、H-Ras、K-Ras和N-Ras在胶质瘤组织中表达上调，且与胶质瘤的WHO分级相关。此外，Ras-GRF1的表达与H-Ras的表达呈正相关。 Ras-GRF1 siRNA可降低胶质瘤细胞中H-Ras和p-ERK/ERK的表达。 H-Ras siRNA 抑制胶质瘤细胞的增殖、克隆形成、迁移和侵袭，并增强其凋亡，但 Ras-GRF1 慢病毒激活颗粒可逆转这种情况。体内实验还表明，Ras-GRF1 shRNA 减少了裸鼠肿瘤的体积和重量，同时下调了 H-Ras 和 p-ERK/ERK。

 结论

Ras-GRF1 在神经胶质瘤组织中表达上调，并与其恶性程度和预后相关。通过介导H-Ras/ERK通路沉默Ras-GRF1可能抑制胶质瘤的生长和转移。