[Knockdown of Alox5 gene promotes apoptosis of K562/ADM cells].

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 pGenesil-1 interference vector. Enzyme digestion and sequencing were performed to identify the recombinant plasmid pGenesil-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 pGenesil-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 pGenesil-1-K562/ADM control group and blank K562/ADM cell group, the Alox5 mRNA and protein levels of K562/ADM cells transfected with pGenesil-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.