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.
OBJECTIVE
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.


CONCLUSIONS
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.
Kang
Chun-Sheng
CS
Department of Neurosurgery Laboratory of Neuro-Oncology, Tianjin Medical University General Hospital, Tianjin Neurological Institute, Tianjin 300052 , PR China.
Pu
Pei-Yu
PY
Li
Yan-He
YH
Zhang
Zhi-Yong
ZY
Qiu
Ming-Zhe
MZ
Huang
Qiang
Q
Wang
Guang-Xiu
GX
D015153:Blotting, Western; D001932:Brain Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015316:Genetic Therapy / Q000379:methods*; D005910:Glioma / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D009361:Neoplasm Invasiveness; D010957:Plasmids; D034741:RNA, Small Interfering; D014162:Transfection
D034741:RNA, Small Interfering; D066246:ErbB Receptors; D020780:Matrix Metalloproteinase 9