Construction Of Eukaryotic Expression Vector MicroRNA -143 And Its Effect On The Expression Levels Of DNMT3A

Objective:To detect the expression of miR-143 in malignant hematopoietic cell lines and leukemia patients and investigate its clinical significance,then screene the malignant hematopoietic cell lines with low expression of miR-143. Furthermore,taked it as subject to research the relationship between miR-143 and target gene DNMT3A. Finally,a lentivirus expression vector,pMAGic 7.1-pre miR-143-GFP,Puro was constructed and expressed it in K562 cells in order to detecte its effect on the expression of DNMT3A, and on the situation of cell proliferation and apoptosis in K562 cells.Methods:The expression of miR-143 in 10 species of malignant hematopoietic cell lines and clinical samples was detected with quantitative real-time polymerase chain reaction method. Molecular cloning technique was used to construct a lentiviral expression vector miR-143,virus were collected after the vectors were co-transfected with the packaging plasmid and the envelope plasmid into 293T cells,then,infected it into K562 cells. Furthermore,a stable cell line expressing miR-143 was established and serial subcultivated. The CCK-8 assay and clone formation ratio methods were used to evaluate the effect on situation of cell proliferation in K562 cells after over-expression miR-143.The AO/EB and Hoechst 33258 fluorescent staining method were used to observe the situation of cell apoptosis. Apoptosis ratio was detected by flow cytometry with Annexin V,FITC/PI. After miR-143 infection into K562 cells by lentivirus vector, real-time PCR were applied to identify the mRNA expression lever of miR-143,DNMT3A,DNMT3B and DNMT1,and Western blotting were applied to identify the protein expression lever of procaspase-3,procaspase-9,DNMT3A,DNMT3B and DNMT1. Results:(1)The expression of miR-143 in acute leukemia patients was significantly decreased (P<0.05), while which have no significant correlation in a variety of factors, such as age, sex, bone marrow blasts, FAB classification, fusion gene abnormalities, chromosomal abnormalities. Besides, the expression of miR-143 in AL patients significantly increased after complete remission(P<0.05), which was inversely correlated with the mRNA expression of DNMT3A in clinical samples, a known target gene of miR-143(r=-0.665,P<0.05). (2)A lentiviral expression vector miR-143 was constructed successfully, real-time PCR and Western-blotting detection confirmed stable expression of miR-143 cell line was successfully created(.3)CCK-8 assay, clone formation ratio methods and cell cycle assay demonstrated that miR-143 could repress cell proliferation and G1-S transition maybe by regulating DNMT3A. (4)Compared with the normal control group, K562 cells over-expression of miR-143 were detected that apoptosis increased and nuclear stain were dense by the AO/EB and Hoechst 33258 fluorescence staining; and its apoptosis ratio increased by flow cytometry. MiR-143 may also promote the activation of procaspase-3 and procaspase-9, promoting apoptosis accordingly.Conclusion:(1)The low expression of miR-143 in leukemia has the function of tumor suppressor genes. ( 2) The study successfully constructed lentiviral expression vector pMAGic 7.1-pre miR-143-GFP,Puro. The experiment showed that it can expressed in leukemia cells efficiently and play a biological role through transforming to mature miR-143. Furthermore it showed this lentiviral expression vector could transfected into cells, which can be used for studying its function.(3)MiR-143 in vitro could repress cell proliferation and G1-S transition ,meanwhile,promote the K562 cells'apoptosis by inhibit the lever of its target gene-DNMT3A.