RNA interference for inhibiting expression of epidermal growth factor receptor in breast cancer cells

Abstract

Objective To investigate whether RNA interference (RNAi) produced by small inter- ference RNA (siRNA) could induce gene silencing in breast cancer cells and to assess the degree of epidermal growth factor (EGF) receptor gene silencing and its effect on functional outcome. Methods Overexpression of EGF receptor gene was determined in breast cancer cells and a variety of other types of cancer cells. Then breast cancer cell lines MDA-MB231, MDA-MB-453S, ZR-75-30, and ZR-75 were transfected with target se- quence-specific dsRNA as well as various controls. Immune fluorescent labeling, flowcytometry and Western blot were used to monitor the reduction in production of the EGF receptor protein. Quantitative reverse- transcriptase PCR was used to detect the silencing of EGF receptor gene levels. Cell count, colony assay, scratch assay, MTT assay, cell cycle analysis, and ELISA were used to assess the functional effects of RNAi. Results EGF receptor gene was overexpressed in breast cancer cells. In cell line MDA-MB231 and MDA-MB-453S, we displayed sequence specific silencing of the EGF receptor with 71.31% of downregulation of EGF receptor protein production and 37.04% of silencing of EGF receptor gene. The reduction in EGF receptor caused growth inhibition of the cells, reducing the total cell numbers by 85.0% and colony number by 63.3%. This also retarded the migration of MDA-MB231 and MDA-MB-453S by more than 80% at 24h and 48h, enhanced the chemosensitivity to cisplatin by four-fold. Cell cycle analysis showed that dsRNA-EGFR induced accumulation of cells in Go-Gi phase by 12.67% with a decrease in the percentage of cells in S-phase by 6.56% relative to control. The results from ELISA showed EGF level was reduced by 27.74% and 11.07% in medium and cell extraction respectively. In cell line ZR-75-30 and ZR-75, we displayed sequence specific silencing of the EGF receptor with 71.78% of downregulation of EGF receptor protein production and 50.00% of silencing of EGF receptor gene. The reduction in EGF receptor caused growth inhibition of the cells, reducing the total cell numbers by 78.3% and colony number by 66.8%. This also retarded the migration of ZR-75-30 and ZR-75 by more than 80% at 24h and 48h, enhanced the chemosensitivity to cisplatin by seven-fold. Cell cycle analysis showed that dsRNA-EGFR induced accumulation of cells in G0-G1 phase by 17.48% with a significant decrease in the percentage of cells in S-phase by 19.20% relative to control. ELISA showed EGF level was reduced by 45.65% and 22.45% in medium and cell extraction, respectively. Conclusions These sequence specific siRNAs showed a blockbuster effect in downregulation of EGF receptor gene expression, inhibition of the cellular proliferation and motility, arrest of the cell cycle, and enhancement of the cellular chemosensitization. The successful application of dsRN A-EGFR to reverse the neoplastic phenotype of EGF receptor overexpressing cells extends the list of available therapeutic modalities in the treatment of human cancer. Key words: Epidermal growth factor receptor;  RNA interference;  Small interference RNA;  Double stranded RNA;  Breast cancer