Construction of antisense Bmi-1 expression plasmid and its inhibitory effect on K562 cells proliferation.
BACKGROUND
Bmi-1 gene determines the proliferative capacity of normal and leukemia stem cells. Expression of Bmi-1 has been found in all types of myeloid leukemia cells in both humans and mice. This study aimed at assessing the effect of antisense Bmi-1 expression on K562 cells proliferation and p16 protein (p16) expression.


METHODS
A transcriptional repressor, Bmi-1 cDNA was cloned by reverse transcriptase polymerase chain reaction (RT-PCR) of its mRNA from K562 cells. A plasmid expressing antisense Bmi-1 mRNA was then constructed by reverse design of PCR primers and cloned to the plasmid pLNCX2; G418 was added to the medium after the plasmid was successfully introduced in K562 cells by lipofectin-mediated DNA transfection. The effects of the antisense expression on the proliferation of K562 cells were analyzed by using microculture tetrazolium and colony forming. Cell cycle was analyzed by using flow cytometry. The p16 expression of K562 cells was observed by immunofluorescence histochemical stain.


RESULTS
K562 cells transfected with antisense Bmi-1 plasmid grew significantly slower than that of controls (the parental K562 and cells transfected with empty plasmid). The colony forming ability of antisense Bmi-1 plasmid transfected cells decreased significantly (P < 0.01) compared with controls. The p16 expression of cells transfected with antisense Bmi-1 was upgraded more apparently than that of controls.


CONCLUSIONS
The antisense Bmi-1 gene can inhibit the growth of K562 cell and upgrade expression of p16 in K562 cells.
Meng
Xiu-xiang
XX
Department of Laboratory Hematology, School of Laboratory Medicine, Dalian Medical University, Dalian116027, China. xiuxiang_meng@sina.com
Liu
Wei-hong
WH
Liu
Dan-dan
DD
Zhao
Xin-yu
XY
Su
Ben-li
BL
D002453:Cell Cycle; D049109:Cell Proliferation; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000032:analysis; D006801:Humans; D020014:K562 Cells; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D010957:Plasmids; D063150:Polycomb Repressive Complex 1; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D016372:RNA, Antisense / Q000502:physiology*; D012097:Repressor Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics
C069077:BMI1 protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016372:RNA, Antisense; D012097:Repressor Proteins; D063150:Polycomb Repressive Complex 1