Interference With Alox5 Gene Promotes Apoptosis And Reverses Drug Resistance In K562/ADM Cells

ObjectiveThe pGenesil-1-shRNA-Alox5 recombinant plasmid was constructed to study the effect of interference Alox5 gene on proapoptotic and reversal of multidrug resistance in adriamycin-resistant chronic myeloid leukemia(CML)K562/ADM cells.To lay a foundation for the clinical multidrug resistance of CML.Methods1.Three pairs of specific shRNA oligonucleotides of Alox5 gene were designed,the pGenesil-1-shRNA-Alox5 vectors were constructed and transfected into K562/ADM cells.The expression of Alox5 gene and its protein were detected by real-time PCR and Western blot.The best interference group was screened out.2.The optimal interference group of the Alox5 gene was selected as the experimental group,the Sh-NC empty vector group as the negative control group,and the K562/ADM cell group as the blank control group.RT-PCR and real-time PCR were used to detect the expression of bcr/ablfusion gene,Western blot was used to detect the expression of BCR/ABL fusion protein,pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2in K562/ADM cells.Flow cytometry(FCM)was used to detect the apoptosis rate of K562/ADM cells.3.Western blot was used to detect the expression of MDR1,P-gp,BCRP and the expression of PI3K/AKT signaling pathway related proteins in K562/ADM cells.Results1.The recombinant plasmid structure of shAlox5-153,shAlox5-461 and shAlox5-1478 groups were successfully confirmed by enzyme digestion and sequencing.2.Compared with Sh-NC vector group and K562/ADM control group,the expression of Alox5 gene and its protein in K562/ADM cells transfected with the recombinant plasmids were significantly decreased,and the shAlox5-461 transfection group was obviously down-regulated.3.The shAlox5-461 group with the highest interference efficiency was selected as the experimental group.The expression levels of bcr/abl fusion gene and BCR/ABL fusion protein were significantly decreased compared with the Sh-NC empty group and K562/ADM blank group.The apoptosis rate increased,the expression level of pro-apoptotic protein Bax increased,but the expression level of anti-apoptotic protein Bcl-2 decreased.The difference was statistically significant(p<0.05).4.Compared with Sh-NC empty group and K562/ADM blank group,the expression levels of MDR1,P-gp and BCRP were significantly decreased in K562/ADM cells in shAlox5-461 group,the expression levels of PI3K/AKT signal pathway related proteins were down-regulated,and the differences were statistically significant(p<0.05).Conclusion1.ShAlox5-461 recombinant plasmid group can significantly reduce the expression of bcr/abl fusion gene and BCR/ABL fusion protein in K562/ADM cells;increase the expression of pro-apoptosis protein Bax but decrease the expression of anti-apoptosis protein Bcl-2.The apoptosis rate of K562/ADM cells increased.2.Interfering with Alox5 gene can inhibit the expression of multidrug resistance-associated proteins MDR1,P-gp and BCRP in K562/ADM cells by regulating PI3K/AKT signaling pathway and reverse it's drug resistance.