Abstract:

Objective To discuss the regulatory effect of microRNA-181a-5p（miR-181a-5p） targeting BTB and CNC homology 2 （BACH2） on the apoptosis and invasion of the acute lymphoblastic leukemia （ALL） cells，and to clarify its mechanism. Methods The BACH2 over-expression recombinant plasmid（overExpBACH2）， miR-181a-5p inhibitor，and inhibitor-NC were constructed in vitro，and the transfection effect after transfected with human ALL T lymphocytes CCRF-CEM was detected by real-time fluorescence quantitative PCR （RT-qPCR） and immunofluorescence staining. The CCRF-CEM cells were divided into control group， inhibitor-NC group， miR-181a-5p inhibitor group， BACH2 over-expression（overExpBACH2） group， miR-181a-5p inhibitor+empty vector（EV） group， and miR-181a-5p inhibitor+ overExpBACH2 group. The proliferation activities of cells in various groups were detected by CCK-8 assay； the percentages of cells at G₂ phase and apoptotic rates of cells in various groups were detected by flow cytometry； the expression levels of CyclinD3， B-cell lymphoma-2（Bcl-2），Bcl-2-associated X protein（Bax）， and cysteinyl aspartate specific proteinase-3（Caspase-3） proteins in cells in various groups were detected by Western blotting method； the numbers of invasion cells in various groups were detected by Transwell chamber test； the expression levels of matrix metalloproteinase-9（MMP-9），matrix metalloproteinase 14（MMP-14）， BACH2 mRNA， and miR-181a-5p in cells in various groups were detectd by RT-qPCR method. Results Both miR-181a-5p inhibitor and overExpBACH2 vector were successfully transfected into the CCRF-CEM cells. Compared with control group，the proliferation activities of the cells in overExpBACH2 group and miR-181a-5p inhibitor+EV group were decreased （P<0.05）， and the percentages of the cells at G₂ phase and the apoptotic rates were increased （P<0.05）； the expression levels of CyclinD3 protein in the cells were decreased （P<0.05）， the expression levels of Caspase-3 protein and the ratios of Bax/Bcl-2 were increased （P<0.05）， the numbers of invasion cells were decreased （P<0.05）， the expression levels of MMP-9， MMP-14 mRNA，and miR-181a-5p were decreased （P<0.05），and the BACH2 mRNA expression levels were increased （P<0.05）. Compared with miR-181a-5p inhibitor group and overExpBACH2 group， the change trends of the above indexes in miR-181a-5p inhibitor+overExpBACH2 group were more obvious. Conclusion miR-181a-5p can target BACH2 and regulate the apoptosis and invasion of the ALL cells， which provides the new ideas for the clinical target therapy of ALL.

Key words: Acute lymphoblastic leukemia, MicroRNA-181a-5p, BTB and CNC homology 2, Apoptosis, Cell invasion