RNA interference‐mediated silencing of focal adhesion kinase inhibits growth of human malignant glioma xenograft in nude mice
RNA interference‐mediated silencing of focal adhesion kinase inhibits growth of human malignant glioma xenograft in nude mice
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DOI：
10.1042/cbi20100243
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发表时间：
2011-08
影响因子：
3.9
通讯作者：
Guan Wang;Yongping Ma;Yang Yang-Yang;N. Zhang;Wei Wang;Sheng-yong Liu;Li-Juan Chen;Yu Jiang;Xia Zhao;Yuquan Wei;H. Deng
中科院分区：
文献类型：
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作者：
Guan Wang;Yongping Ma;Yang Yang-Yang;N. Zhang;Wei Wang;Sheng-yong Liu;Li-Juan Chen;Yu Jiang;Xia Zhao;Yuquan Wei;H. Deng
粘着斑激酶（focal adhesion kinase，FAK）在人脑恶性胶质瘤中过度表达，在介导细胞增殖、存活和迁移中起关键作用。FAK的表达随肿瘤分级和分期的增高而增高。基于这些观察结果，我们推测FAK表达的减弱可能对恶性胶质瘤的生长具有抑制作用。在本研究中，使用阳离子脂质体将含有U6启动子驱动的针对人FAK的shRNA（小发夹RNA）的质粒转染人胶质瘤细胞系U251。通过使用流式细胞术和PI（碘化丙啶）染色测定来分析体外U251细胞中FAK敲低的影响。基于令人鼓舞的FAK沉默体外结果，编码FAK靶向shRNA的质粒被DOTAP（二油酰三甲基丙烷铵）：Chol（胆固醇）阳离子脂质体包裹，并通过尾静脉注射，以评估其抑制肿瘤生长的治疗效率在人胶质瘤异种移植模型中。分别采用PCNA（增殖细胞核抗原）、CD34免疫染色和TUNEL（末端脱氧核苷酸转移酶介导的dUTP缺口末端标记）检测肿瘤血管生成、凋亡和增殖的变化。结果表明，DOTAP：Chol阳离子脂质体可将治疗性质粒全身递送至肿瘤异种移植物，从而抑制肿瘤生长。用编码FAK靶向shRNA的质粒治疗使平均肿瘤体积减少约10%。与对照组比较，有显著性差异（P<0.05），并伴有血管生成抑制（P<0.05）、肿瘤细胞增殖抑制（P<0.05）和凋亡诱导（P<0.05）。综上所述，我们的结果表明，shRNA介导的FAK沉默可能是一种潜在的治疗人类恶性胶质瘤的方法。
FAK (focal adhesion kinase), which plays a pivotal role in mediating cell proliferation, survival and migration, is frequently overexpressed in human malignant glioma. The expression of FAK increases with the advance of tumour grade and stage. Based on these observations, we hypothesized that attenuation of FAK expression may have inhibitory effects on the growth of malignant glioma. In the present study, human glioma cell line U251 was transfected with plasmids containing U6 promoter‐driven shRNAs (small‐hairpin RNAs) against human FAK using cationic liposome. The effects of FAK knockdown in U251 cells in vitro were analysed by using flow cytometry and PI (propidium iodide)‐staining assays. Based on the encouraging in vitro results with FAK silencing, plasmids encoding FAK‐targeted shRNA were encapsulated by DOTAP (dioleoyltrimethylammonium propane): Chol (cholesterol) cationic liposome and injected via tail vein to evaluate its therapeutic efficiency on suppressing tumour growth in a human glioma xenograft model. PCNA (proliferating‐cell nuclear antigen), CD34 immunostaining and TUNEL (terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labelling) assay were used to assess the changes in tumour angiogenesis, apoptosis and proliferation respectively. The results indicated that DOTAP:Chol cationic liposome could deliver therapeutic plasmids systemically to tumour xenografts, resulting in suppression of tumour growth. Treatment with plasmid encoding FAK‐targeted shRNA reduced mean tumour volume by approx. 70% compared with control groups (P<0.05), accompanied with angiogenesis inhibition (P<0.05), tumour cell proliferation suppression (P<0.05) and apoptosis induction (P<0.05). Taken together, our results demonstrated that shRNA‐mediated silencing of FAK might be a potential therapeutic approach against human malignant glioma.