An in vitro study on the suppressive effect of glioma cell growth induced by plasmid-based small interference RNA (siRNA) targeting human epidermal growth factor receptor
An in vitro study on the suppressive effect of glioma cell growth induced by plasmid-based small interference RNA (siRNA) targeting human epidermal growth factor receptor
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DOI：
10.1007/s11060-004-8322-z
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发表时间：
2005-09-01
影响因子：
3.9
通讯作者：
Wang, GX
中科院分区：
文献类型：
--
作者：
Kang, CS;Pu, PY;Wang, GX
目的：研究靶向人表皮生长因子受体（EGFR）的siRNA质粒对TJ905胶质母细胞瘤细胞增殖和侵袭的抑制作用。方法：通过Lipofectamine介导，将两个靶向人EGFR胞外域（516-536）和催化域（2400-2420）的siRNA表达构建体转染TJ905细胞。采用免疫荧光法和Western blotting检测EGFR的表达。流式细胞术检测细胞周期，MTT法检测细胞增殖活性。采用Western blotting和明胶酶谱法检测MMP9的表达和酶活性。采用Transwell法评价细胞侵袭能力。结果：免疫荧光法和Western blotting结果显示，转染siRNA构建物组EGFR表达分别下调90和92。流式细胞术分析显示，转染sirna的细胞的S相分数（SPF）均低于亲本细胞和空载体转染的细胞。MTT实验显示，与亲代细胞和空载体转染的细胞相比，转染sirna的胶质瘤细胞在植入后第1天的存活率显著下降（P < 0.05）。同时，在Transwell的研究中，转染了sirna的TJ905细胞中，MMP9的表达和酶活性显著降低，细胞侵袭潜能也被大大抑制。结论：靶向EGFR的siRNA表达构建物可特异性抑制EGFR表达，抑制细胞生长，诱导细胞周期阻滞，抑制侵袭。基于质粒的siRNA靶向人EGFR方法有望成为恶性胶质瘤基因治疗的新策略。
Objectives:To study the inhibitory effects of plasmid-based siRNA targeting human epidermal growth factor receptor (EGFR) on tumor proliferation and invasion of TJ905 glioblastoma cells. Methods: Two siRNA expression constructs targeting human EGFR extracellular domain (516-536) and catalytic domain (2400-2420) were transfected into TJ905 cell as mediated by Lipofectamine. Immunofluorescence assay and Western blotting were used to detect EGFR expression. Cell cycle was analyzed by flow cytometry, cell proliferative activity was measured by MTT assay. The expression and enzymatic activity of MMP9 were measured by Western blotting and gelatin zymography. Cell invasive capability was evaluated by Transwell method. Results: The expression of EGFR was knocked-down by 90 and 92, respectively in siRNA constructs transfected groups as indicated by immunofluorescence assay and Western blotting. The flow cytometric analysis showed that the S phase fraction (SPF) was lowered in both siRNAs transfected cells than that in parental cells and the cells transfected with empty vector. Compared to parental cells and the cells transfected with empty vector, the survival rates of glioma cells transfected with the siRNAs dramatically dropped down from the first day after implantation (P < 0.05) as indicated by MTT assay. Meanwhile, the expression and enzymatic activity of MMP9 decreased significantly in siRNAs transfected in TJ905 cells, and cell invasive potential was also greatly inhibited in the Transwell study. Conclusion: The siRNA expression constructs targeting EGFR could specifically suppress EGFR expression, inhibit cell growth, induce cell cycle arrest and suppress invasion. The plasmid-based siRNA targeting human EGFR approach should be a new strategy for gene therapy of malignant gliomas.