CAJ | 학술논문

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EGFR SiRNA,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,建立HIPC裸鼠模型,瘤体内注射EGFR SiRNA,观察肿瘤生长情况.结果慢病毒为载体的EGFR SiRNA对PC-3细胞转染率可稳定在75%,并显著抑制PC-3细胞生长率,仅为40%～50%,其作用可能为沉默细胞EGFR mRNA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,EGFR SiRNA体内抑瘤率为34.83%,P<0.05.结论慢病毒介导的靶向EGFR SiRNA可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,后者可作为未来靶向治疗的目标.
목적 응용RNA소간우(SiRNA)기술영향표피생장인자수체(EGFR)급기포내효응단백수평적표체,관찰기대격소비의뢰전렬선암세포재체외이급재실험라서모형체내적생장정황적영향.방법 사선출최유효적EGFR SiRNA,채용만병독위재체,전염격소비의뢰전렬선암세포(HIPC)PC-3,MTT법검측세포생장억제정황,형광실시정량PCR화Western인적법검측세포EGFR급기포내효응단백Akt화MAPK적표체수평화린산화정도.동시,건립HIPC라서모형,류체내주사EGFR SiRNA,관찰종류생장정황.결과만병독위재체적EGFR SiRNA대PC-3세포전염솔가은정재75%,병현저억제PC-3세포생장솔,부위40%～50%,기작용가능위침묵세포EGFR mRNA급기단백적표체,억제효솔>90%(P<0.01);이차,Akt화MAPK표체수평화린산화균명현강저,표체억제솔분별위76.49%화47.15%,P<0.05.여대조조비교,EGFR SiRNA체내억류솔위34.83%,P<0.05.결론 만병독개도적파향EGFR SiRNA가이재체내외현저억제HIPC생장,기궤제가능시침묵EGFR표체,진이억제포내효응단백적작용,후자가작위미래파향치료적목표.
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 transfected PC-3 cell was measured by MTr. 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 carrying 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.