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An in vitro study on the suppressive effect of glioma cell growth induced by plasmid-based small interference RNA
Chun-Sheng Kang1, Pei-Yu Pu, Yan-He Li
1Department of Neurosurgery Laboratory of Neuro-Oncology, Tianjin Medical University General Hospital, Tianjin Neurological Institute, Tianjin 300052 , PR China.
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.
Insights
Plasmid-based small interfering RNA (siRNA) targeting the human epidermal growth factor receptor (EGFR) effectively suppressed glioblastoma cell proliferation and invasion. This approach shows promise as a novel gene therapy strategy for malignant gliomas.
Area of Science:
- Oncology
- Molecular Biology
- Gene Therapy
Background:
- Malignant gliomas, including glioblastoma, are aggressive brain tumors with poor prognosis.
- The human epidermal growth factor receptor (EGFR) is frequently overexpressed in gliomas and plays a critical role in tumor growth and invasion.
Purpose of the Study:
- To investigate the efficacy of plasmid-based small interfering RNA (siRNA) targeting EGFR in inhibiting the proliferation and invasion of TJ905 glioblastoma cells.
Main Methods:
- Two siRNA expression constructs targeting different domains of EGFR were transfected into TJ905 cells.
- EGFR expression was assessed using immunofluorescence and Western blotting.
- Cell cycle, proliferation, MMP9 expression and activity, and invasive potential were evaluated using flow cytometry, MTT assay, Western blotting, gelatin zymography, and Transwell assays, respectively.
Main Results:
- siRNA transfection significantly reduced EGFR expression by 90-92%.
- EGFR siRNA inhibited cell proliferation, induced cell cycle arrest at the S phase, and decreased the survival rate of glioma cells.
- The expression and enzymatic activity of matrix metalloproteinase-9 (MMP9) were significantly reduced, leading to inhibited cell invasion.
Conclusions:
- Plasmid-based siRNA targeting EGFR effectively suppresses glioblastoma cell growth and invasion.
- This strategy offers a potential new avenue for the gene therapy of malignant gliomas.
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