Construction Of Antisense Bmi-1 And Its Inhibition Effect On K562 Cells In Vitro

Objectives Leukemia is a malignant tumor which threatens the life of patients seriously. The current way of treating leukemia is to eliminate the proliferative leukemic cells, however, only very few leukemic stem cells were in their proliferative state and therefore, resist therapy. In order to prevent the leukemia in its source, identifying the gene determining the self-renewal of leukemic stem cells and finding a way to inhibit its expression would be new approach to stop leukemic cell proliferation. Bmi-1 plays a central role in normal and leukemia hematopoietic stem cells self-renewal. The proliferative potential of leukemic stem and progenitor cells lacking Bmi-1 is compromised and leukemic stem cells would have a progressive failure. We constructed a plasmid , antisense Bmi-1 plasmid then transferred it into K562 cells, and to see if it can inhibit the proliferation of K562 cells and upgrade activity of p16. Methods 1.Using the total RNA extracted from a human erythroleukemia cell line (K562) as the template, the Bmi-1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) with a pair of specific primers containing the restriction sites of XhoI and ClaI. The amplified fragment of Bmi-1 gene digested with XhoI and ClaI was subcloned into cloning vector pMD18-T and then into expression vector pLNCX2. 2.An antisense plasmid was constructed by reverse designing of PCR primers, and the PCR products were subsequently cloned to plasmid pLNCX2. 3.G418 was added into the medium after the plamid was successfully introduced into K562 cells by using lipofectactin- mediated DNA transfection. 4.The effects of on the proliferation of K562 cells were analyzed by means of trypanblue staining,MTT and colony forming. 5.Cell cycle was analyzed by cytometric. 6.The p16 activity of K562 cells was studied by immune chemistry methods. 7. Apoptosis was analyzed by DNA ladder. Results 1. The growth rate of antisense Bmi-1 transfected K562 cells was significantly slower than those of the controls. In contrast to control cells the colony forming ability of antisense Bmi-1 gene transfected cells decreased significantly (p<0.01). However, the survival rate of antisense K562 cells have no significantly difference when compared with empty plasmid K562 cells and parental K562 cells. 2. Cell cycle analysis using flow cytometry showed that percentage of antisense K562 cells in G0/G1 increased significantly when compared with empty plasmid K562 cells and parental K562 cells. 3. The p16 activity of antisense Bmi-1 transfected cells was significantly upgrade than those of controls. 4. The result of DNA ladder is negative and the experimental cells have no significantly apoptosis. Conclusion Antisense Bmi-1 can inhibit the growth of K562 cells and upgrade activity of p16 in vitro.