RNA干扰技术抑制非小细胞肺癌表皮生长因子受体的表达

目的　观察RNA干扰技术是否能有效抑制非小细胞肺癌 (NSCLC)细胞株SPC A 1细胞表皮生长因子受体 (EGFR)的表达。方法　体外化学合成EGFR序列特异性双链RNA(dsRNA) ,与Lipofectamine 2 0 0 0结合后转染细胞 (分 4个组 ,A组 :加入无血清DMEM 5 0 0 μl;B组 :加入Lipofectamine2 0 0 0稀释液 2 5 0 μl及无血清DMEM 2 5 0 μl;C组 :加入非特异性dsRNA/Lipofectamine复合物 5 0 0 μl;D组 :加入dsRNA EGFR/Lipofectamine复合物 5 0 0 μl) ,用Westernblot和流式细胞仪检测EGFR表达。流式细胞仪测细胞周期 ,结合集落形成、化疗敏感性分析观察SPC A 1细胞生物学特性的改变。结果　与A组比较 ,D组EGFR数量减少了 71 31% (P <0 0 0 1) ,B组和C组分别降低了 9 0 %和 16 2 % (P >0 0 5 )。与A组比较 ,D组细胞生长抑制了 78 3% ,集落形成抑制了 6 6 8%。D组G0 G1期细胞百分数较A组增加了 17 4 8% ,D组进入S期的细胞百分数较A组减少了 19 2 0 %。据Origin 6 0计算的IC50 ,dsRNA EGFR可将细胞对顺铂的敏感性提高约 7倍。结论　dsRNA EGFR可有效抑制SPC A 1细胞EGFR表达 ,将更多的细胞阻滞在G0 G1期 ,抑制细胞增生 ,提高细胞对顺铂的敏感性 ,RNA干扰技术为NSCLC基因治疗提供了新策略。

Effects of RNA interference on epidermal growth factor receptor expression in SPC-A-1 cells

Objective To investigate whether RNA interference (RNAi) induced by small interference RNA (siRNA) could suppress epidermal growth factor receptor (EGFR) expression in non-small-cell lung carcinoma (NSCLC) cells. Methods SPC-A-1 cells were transfected using chemically synthesized double stranded RNA (dsRNA) formulated with Lipofectamine 2000. The EGFR numbers were determined by both Western blot and flow cytometry. The antiproliferative effects of dsRNA-EGFR were assessed using cell counts and colony assay. Cell cycle analysis was carried out via flow cytometry. The chemosensitivity of transfected cells to cisplatin was determined by MTT. Results Sequence specific siRNAs targeting EGFR down-regulated EGFR expression significantly. Compared with the control group,dsRNA-EGFR reduced the cell numbers by 78.3% and decreased the colonies by 66.8%. Cell cycle analysis showed that dsRNA-EGFR induced accumulation of cells in G 0-G 1 phase by 17.48% with a significant decrease in the percentage of cells in S-phase by 19.20% relative to the control. Based on the value of IC 50 obtained by Origin 6.0 software,we concluded that dsRNA-EGFR increased the sensitivity of SPC-A-1 to cisplatin by seven-fold. Conclusions Sequence specific siRNAs targeting EGFR was capable of suppressing EGFR expression,and therefore,significantly inhibiting cellular proliferation and inducing cell cycle arrest. The finding from chemosensitivity assay further revealed that dsRNA-EGFR was associated with an addictive or synergistic effect on tumor growth inhibition when combined with cisplatin. The successful application of dsRNA-EGFR for inhibition of proliferation in EGFR overexpressing cells extends the list of available therapeutic modalities in the treatment of human cancer.