CAJ | 학술논문

目的探讨采用化学合成小干扰RNA(siRNA)介导的RNA干扰(RNAi)技术是否能有效抑制乳腺癌细胞株MDA-MB231、MDA-MB-453S、ZR-75-30、ZR-75细胞表皮生长因子受体(EGFR)表达,以及抑制EGFR基因表达后乳腺癌细胞生物学特性的改变,以期为肿瘤治疗提供新的思路和实验依据.方法 (1)建立检测EGFR数量的方法,测定MDA-MB231、MDA-MB-453S、ZR-75-30、ZR-75细胞株EGFR的表达.(2)观察dsRNA-EGFR/Lipofectamine 2000对EGFR表达的抑制作用.(3)采用流式细胞仪测定细胞周期,采用ELISA法观察抑制EGFR表达后,MDA-MB231,MDA-MB-453S、ZR-75-30、ZR-75细胞生物学特性的改变.结果 MDA-MB231、MDA-MB-453S、ZR-75-30、ZR-75细胞株存在EGFR高表达.转染dsRNA-EGFR后可将MDA-MB231、MDA-MB-453S细胞对5-Fu的敏感性提高约4倍.细胞周期分析结果表明dsRNA-EGFR组G0-G1期细胞百分数较对照组增加了17.48%,进入S期的细胞百分数较对照组减少了19.20%.转染dsRNA-EGFR后可将ZR-75-30、ZR-75细胞对5-Fu的敏感性提高约7倍.结论 (1)乳腺癌细胞株存在EGFR高表达.(2)dsRNA-EGFR可序列特异性下调乳腺癌细胞EGFR基因水平,显著降低EGFR蛋白表达.(3)dsRNA-EGFR通过抑制EGFR表达可显著抑制细胞增生和细胞迁移,降低配体EGF的含量,将更多的细胞阻滞在G0-G1期,增加细胞对5-Fu的敏感性,有效逆转乳腺癌细胞的恶性表型,从而为乳腺癌基因治疗提供了新策略.
목적 탐토채용화학합성소간우RNA(siRNA)개도적RNA간우(RNAi)기술시부능유효억제유선암세포주MDA-MB231、MDA-MB-453S、ZR-75-30、ZR-75세포표피생장인자수체(EGFR)표체,이급억제EGFR기인표체후유선암세포생물학특성적개변,이기위종류치료제공신적사로화실험의거.방법 (1)건립검측EGFR수량적방법,측정MDA-MB231、MDA-MB-453S、ZR-75-30、ZR-75세포주EGFR적표체.(2)관찰dsRNA-EGFR/Lipofectamine 2000대EGFR표체적억제작용.(3)채용류식세포의측정세포주기,채용ELISA법관찰억제EGFR표체후,MDA-MB231,MDA-MB-453S、ZR-75-30、ZR-75세포생물학특성적개변.결과 MDA-MB231、MDA-MB-453S、ZR-75-30、ZR-75세포주존재EGFR고표체.전염dsRNA-EGFR후가장MDA-MB231、MDA-MB-453S세포대5-Fu적민감성제고약4배.세포주기분석결과표명dsRNA-EGFR조G0-G1기세포백분수교대조조증가료17.48%,진입S기적세포백분수교대조조감소료19.20%.전염dsRNA-EGFR후가장ZR-75-30、ZR-75세포대5-Fu적민감성제고약7배.결론 (1)유선암세포주존재EGFR고표체.(2)dsRNA-EGFR가서렬특이성하조유선암세포EGFR기인수평,현저강저EGFR단백표체.(3)dsRNA-EGFR통과억제EGFR표체가현저억제세포증생화세포천이,강저배체EGF적함량,장경다적세포조체재G0-G1기,증가세포대5-Fu적민감성,유효역전유선암세포적악성표형,종이위유선암기인치료제공료신책략.
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.