小干扰RNA对前列腺癌表皮生长因子受体及其胞内效应蛋白表达的影响

Effects of RNA interference upon EGFR and its intracellular effective proteins in prostate cancer

目的应用RNA小干扰（SiRNA）技术影响表皮生长因子受体（EGFR）及其胞内效应蛋白水平的表达，观察其对激素非依赖前列腺癌细胞在体外以及在实验裸鼠模型体内的生长情况的影响。方法筛选出最有效的EGFRSiRNA，采用慢病毒为载体，转染激素非依赖前列腺癌细胞（HIPC）PC．3，MTT法检测细胞生长抑制情况，荧光实时定量PCR和Western印迹法检测细胞EGFR及其胞内效应蛋白Akt和MAPK的表达水平和磷酸化程度。同时，建市HIPC裸鼠模型，瘤体内注射EGFRSiRNA，观察肿瘤生长情况。结果慢病毒为载体的EGFRSiRNA对PC-3细胞转染率可稳定在75％，并显著抑制PC-3细胞牛长率，仅为40％～50％，其作用可能为沉默细胞EGFRmRNA及其蛋白的表达，抑制效率〉90％（P〈0．01）；而且，Akt和MAPK表达水平和磷酸化均明显降低，表达抑制率分别为76．49％和47．15％，P〈0．05。，与对照组比较，EGFRSiRNA体内抑瘤率为34．83％，P〈0．05。结论慢病毒介导的靶向EGFRSiRNAu，以在体内外显著抑制HIPC生长，其机制可能是沉默EGFR表达，进而抑制胞内效应蛋白的作用，后者叮作为未来靶向治疗的目标。

Objective To investigate the growth of prostate cancer in vitro or in vivo by inhibiting the expression of EGFR and its intracellular effective proteins with small RNA interference （SiRNA） . Methods The hormone independence prostate cancer （HIPC） cell line PC-3 was transfected by EGFR SiRNA synthesized and cloned into a recombinant lentivirus vector. The growth rate of transiected PC-3 cell was measured by MTT. The expression of EGFR and the expression and phosphorylation of its intracellular proteins, such as Akt and MAPK, were detected by fluorescent Real-Time PCR and Western blot respectively. Meanwhile nude mice were transplanted with PC-3 cell to establish the tumor model and the tumor growth was observed. Results The transfection efficiency was stable over 75% in PC-3 cell transfected with the recombinant lentivirus vector can-ying EGFR SiRNA and the survival rate of PC-3 cell was only 40% - 50%. Such depressant effects might be obtained by inhibiting the expression of EGFR mRNA and protein to only 10% as compared with their untreated levels （P 〈 0.01 ）; meanwhile, the expression level and phosphorylation of Akt and MAPK also obviously decreased to 76. 49% and 47.15% respectively （P 〈 0. 05）. Compared with the control group, the proliferation activity of tumors in nude mice was inhibited significantly by 34. 83% （ P 〈 0. 05）. Conclusion Lentivirus-mediated EGFR SiRNA can inhibit the growth of HIPC in vivo and in vitro by effectively suppressing the expression of EGFR and its intracellular proteins. The latter may be a potential candidate for future targeted therapy.