敲低花生四烯酸5脂加氧酶(Alox5)基因促进K562/ADM细胞凋亡

Knockdown of Alox5 gene promotes apoptosis of K562/ADM cells

目的研究短发夹RNA(shRNA)敲低花生四烯酸5脂加氧酶(Alox5)基因对慢性粒细胞白血病耐药细胞K562/ADM细胞凋亡的影响。方法设计并合成3对针对人Alox5基因的靶向shRNA片段,插入到p Genesil-1干扰载体中,经过酶切和测序验证,成功构建p Genesil-1-shRNA-Alox5重组质粒,转染K562/ADM细胞,实时定量PCR和Western blot法检测Alox5 mRNA及蛋白水平,筛选最好干扰组。选择较高干扰效率的p Genesil-1-shRNA-Alox5重组质粒组作为实验组,采用实时定量PCR检测K562/ADM细胞bcr/abl mRNA水平、Western blot法检测BCR/ABL融合蛋白水平、流式细胞术检测细胞凋亡率。结果酶切及测序结果显示重组质粒构建成功。与阴性对照组(p Genesil-1-K562/ADM)和空白K562/ADM细胞组相比,转染p Genesil-1-shRNA-Alox5重组质粒组的K562/ADM细胞Alox5 mRNA和蛋白水平均明显降低。敲低K562/ADM细胞Alox5后,bcr/abl mRNA、BCR/ABL融合蛋白水平均显著降低、细胞凋亡率显著增加。结论敲低Alox5基因后,K562/ADM细胞bcr/abl mRNA和BCR/ABL融合蛋白表达水平下降,细胞凋亡率增加。

Objective To investigate the effect of short hairpin RNA（shRNA） knockdown of arachidonate 5-lipoxygenase（Alox5） gene on the apoptosis of resistant chronic myeloid leukemia K562/ADM cells. Methods Three pairs of shRNA fragment targeting human Alox5 gene were synthesized and inserted into p Genesil-1 interference vector. Enzyme digestion and sequencing were performed to identify the recombinant plasmid p Genesil-1-shRNA-Alox5. The plasmid was then transfected into K562/ADM cells. Real-time quantitative PCR and Western blotting were used to detect the Alox5 mRNA and protein levels to get the best interference group. The p Genesil-1-shRNA-Alox5 plasmid group with higher interference efficiency was selected as the experimental subjects. Real-time quantitative PCR was adopted to detect the expression level of bcr/abl mRNA and Western blotting to detect the BCR/ABL fusion protein,and the apoptotic rate was assessed by flow cytometry. Results The enzyme digestion and sequencing confirmed the successful construction of recombinant plasmid.Compared with the negative p Genesil-1-K562/ADM control group and blank K562/ADM cell group,the Alox5 mRNA and protein levels of K562/ADM cells transfected with p Genesil-1-shRNA-Alox5 recombinant plasmid significantly decreased,and after the knockdown of Alox5,the levels of bcr/abl mRNA and BCR/ABL fusion protein significantly decreased and the apoptosis rate increased obviously. Conclusion The knockdown of Alox5 gene can induce the decreased levels of bcl/abl mRNA and BCR/ABL fusion protein in the K562/ADM cells and increased apoptosis rate.