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目的:构建针对人晚期糖基化终产物受体(receptor for advanced glycation end products,RAGE)基因的特异性短发夹RNA(small hairpin RNA,shRNA)表达载体,探讨其对前列腺癌细胞DU145增殖的抑制作用。方法:设计并合成4种针对RAGE基因的特异性短链寡核苷酸,构建含shRNA RAGE的表达载体,转染高表达RAGE的亚克隆细胞株sub DU145-2C1。荧光显微镜下观察细胞转染后的情况,实时荧光定量PCR(real-time fluorescence quantitative-PCR,RFQ-PCR)检测转染shRNA后对RAGE mRNA表达的影响,Western印迹法检测对RAGE蛋白表达的影响,CCK-8法检测对细胞增殖的影响,划痕实验观察对细胞迁移能力的影响。结果:构建获得的shRNA RAGE表达载体能够有效抑制RAGE基因的表达(P<0.05),其中以shRNA RAGE-1(R1)的抑制作用最强,其对RAGE mRNA表达的抑制率为84%,对蛋白表达的抑制率为27%。细胞增殖结果显示,转染shRNA RAGE后细胞增殖能力明显降低;而细胞迁移能力则无明显变化。结论:shRNARAGE能有效下调RAGE基因的表达水平,抑制细胞增殖。
OBJECTIVE: To construct a specific short hairpin RNA (shRNA) expression vector targeting human advanced glycation end products (RAGE) gene and investigate its effect on proliferation of prostate cancer cell line DU145 Inhibition. METHODS: Four specific short-chain oligonucleotides targeting RAGE gene were designed and synthesized. The expression vector containing shRNA RAGE was constructed and transfected into sub DU145-2C1 subline with high expression of RAGE. The expression of RAGE mRNA in transfected shRNA was detected by real-time fluorescence quantitative-PCR (RFQ-PCR). The expression of RAGE was detected by Western blotting , CCK-8 assay on cell proliferation, scratch test observed the impact on cell migration. Results: The constructed shRNA shRNA expression vector could effectively inhibit the expression of RAGE gene (P <0.05). The shRNA targeting RAGE-1 (R1) had the strongest inhibitory effect on RAGE mRNA expression. The inhibition rate of RAGE mRNA expression was 84% The inhibition rate of protein expression was 27%. The results of cell proliferation showed that the ability of transfecting shRNA RAGE was significantly decreased, while the ability of cell migration did not change significantly. Conclusion: shRNARAGE can effectively down-regulate the expression of RAGE gene and inhibit cell proliferation.