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目的观察RNAi对人前列腺癌PC3细胞EphB4基因表达的抑制效应,寻求高效率的干扰片段。方法设计并化学合成特异性针对EphB4基因的siRNA(siRNA-1、siRNA-2、siRNA-3)为实验组,另设一无意义siRNA为对照组,脂质体转染PC3细胞。荧光显微镜观察siRNA的转染效率;分别应用RT-PCR以及Western Blot、免疫荧光组织化学染色方法检测EphB4 mRNA和蛋白的表达,以及应用MTT法检测siRNA对PC3细胞生长的影响,比较实验组与对照组的差异及评估siRNA阻抑效应。结果实验组siRNA对PC3细胞EphB4 mRNA和蛋白的表达较对照组均有不同程度的下降,统计学分析其差异具有统计学意义(P<0.05)。siRNA对PC3细胞的生长具有抑制作用。结论RNAi可以有效地抑制人前列腺癌PC3细胞EphB4的表达,找到了具有较高抑制效率的siRNA,为进一步探究EphB4的生物学功能、体内实验和临床抗肿瘤药物研究打下基础。
Objective To observe the inhibitory effect of RNAi on the expression of EphB4 gene in human prostate cancer PC3 cells and to find a highly efficient interference fragment. Methods siRNAs (siRNA-1, siRNA-2, siRNA-3) specific to EphB4 gene were designed and synthesized chemically, and a nonsignificant siRNA was designed as the control group. Lipofectamine was transfected into PC3 cells. The transfection efficiency of siRNA was observed by fluorescence microscopy. The expression of EphB4 mRNA and protein was detected by RT-PCR and Western Blot. The expression of EphB4 mRNA and protein was detected by immunofluorescence histochemistry staining. The effect of siRNA on PC3 cell growth was detected by MTT assay. Group differences and evaluate siRNA suppression effects. Results The expression of EphB4 mRNA and protein in PC3 cells of experimental group decreased to some extent compared with the control group, and the difference was statistically significant (P <0.05). siRNA has an inhibitory effect on the growth of PC3 cells. Conclusion RNAi can effectively inhibit the expression of EphB4 in human prostate cancer PC3 cells and find a siRNA with high inhibitory efficiency, which lays the foundation for further exploring the biological functions of EphB4, in vivo experiments and clinical anti-tumor drugs.