The Overexpression Of MiR-30a Promotes Apoptosis Of Leukemia K562 Cells

ObjectiveThe recombinant plasmid pEGFP-pre-miR-30 a was constructed and transfected in K562 cells to investigate the overexpression of miR-30 a on the pro-apoptotic effect and related mechanism of chronic myelocytic leukemia K562 cells.Laying the foundation for new ideas and strategies for CML treatment.Methods1.The precursor sequence pre-miR-30 a was chemically synthesized and the recombinant plasmid pEGFP-pre-miR-30 a was transfected in K562 cells.The expression level of miR-30 a was detected by real-time quantitative PCR(RT-qPCR)to determine whether the transfection was successful.2.The K562 cells transfected with recombinant plasmid pEGFP-pre-miR-30 a were selected as the experimental group,K562 cells transfected with empty vector as the negative control group,and the K562 cell group as the blank control group.Real-time quantitative PCR was used to detect the level of miR-30 a and bcr/abl,cell apoptosis was assessed by apoptosis detection kit annexinV-FITC/PI and flow cytometry.Western blot was used to test the level of BCR/ABL fusion protein,apoptosis related protein BCL-2 and BAX,PTEN,AKT,p-AKT.3.Western blot was used to test the expression of PTEN/AKT signaling pathway related protein.Results1.The results of restriction enzyme digestion and sequencing showed that the recombinant plasmid pEGFP-pre-miR-30 a was successfully constructed.2.Compared with the pEGFP-C1-K562 negative control group and K562 blank control group,the obvious up-regulation of miR-30a in k562 cells transfected with recombinant plasmid pEGFP-pre-miR-30 a was observed.3.Compared with the pEGFP-C1-K562 negative control group and K562 blank control group,the expression of bcr/abl mRNA and BCR/ABL fusion protein were both significantly decreased.Apoptotic rate had a obvious improvement(both P<0.05),and the expression of anti-apoptotic protein BCL-2 was down-regulated while pro-apoptotic protein BAX was up-regulated.The difference was statistically significant(p<0.05).4.The level of PTEN was significantly up-regulated ompared with negative and blank control groups,no variation was found in total AKT,but the expression of p-AKT was down-regulated.and the differences were statistically significant(p<0.05).Conclusion1.The results of restriction enzyme digestion and sequencing showed that the recombinant plasmid pEGFP-pre-miR-30 a was successfully constructed.2.The overexpression of miR-30 a was abled to inhibit the expression of bcr/abl mRNA and BCR/ABL fusion protein,and increase apoptotic rate by up-regulating pro-apoptotic protein BAX and down-regulating anti-apoptotic protein BCL-2 which indicated that overexpression of miR-30 a could inhibit the expression of oncogene at the molecular level and promote the apoptosis of K562 cells in CML.3.Overexpression of miR-30 a may induce apoptosis of K562 cells in CML by inhibiting the activity of BCR/ABL-PTEN/AKT signaling pathway.