[Effect of specific siRNA targeting against bcr-abl chimeric gene on chronic myelogenous leukemia cells].
OBJECTIVE
To investigate the effect of specific small interfering RNA (siRNA) targeting against bcr-abl chimeric gene on the biological traits of chronic myelogenous leukemia (CML) cells.


METHODS
CML cells of the line K561 transcribing a type of b3: a2 mRNA of bcr-abl chimeric gene were cultured. A 21nt siRNA targeting against the chimeric location of the b3: a2 mRNA of bcr-abl chimeric gene was designed, synthesized, and transfected into the K562 cells as RNA interference group. Another K562 cells were transfected with fluorescein enzyme gene specific siRNA as indifferent controls, or with lipid alone as blank vector controls. Some K562 cells without treatment were used as normal controls. 48 hours after the transfection Western blotting was used to detect the expression of P210bcr-abl fusion protein. 3H-TdR incorporation was used to detect the proliferation activity of K562. Annexin V-fluorescencein isothiocyanate (FITC)/phosphatidylinositol (PI) staining was used to detect the apoptosis of K2562 cells. Flow cytometry was used to observe the cell cycle of K562 cells. Benzidine staining was used to detect the differentiation of K562 cells towards erythrocytic series. Western blotting was used further to detect the expression of apoptosis-related protein Bcl-xL/Bax.


RESULTS
(1) In contrast with the control groups, the expression level of bcr-abl chimeric gene was much lower in the RNAi group. (2) (3)H-TdR incorporation test showed time-dependent inhibition of proliferation of K562 cells, reflected in decrease of counts per minute (CPM) value in RNAi group 24 h, 48 h, 72 h, and 96 h after siRNA transfection by 33.06%, 52.25%, 57.64%, and 70.87% respectively (F=17.7, P < 0.01). (3) About 43.2% of K562 cells in the RNAi group were apoptotic 48 h after siRNA transfection (F=13.6, P < 0.01). (4) In contrast with the control groups, the expression of apoptosis-associated protein Bcl-xL was greatly down-regulated; however, the expression of Bax protein showed little change. (5) The percentage of benzidine-positive cells in the RNAi group was 23.5% +/- 3.2%, significantly higher than those in the indifferent control group, blank vector group, and normal control group (2.4% +/- 0.3%, 4.5% +/- 0.5%, and 3.6% +/- 0.2% respectively, all P < 0.01), which meant that part of the K562 cells differentiated towards erythrocytic series. (6) The percentage of G1 phase of K561 cells in the RNA1 group was significantly higher than those of the other groups (F = 6.2, P < 0.05), showing a capture in G1-phase of cell cycle.


CONCLUSIONS
The specific siRNA distinctly inhibits the expression of bcr-abl chimeric gene and influences essential biological traits of K562 cells, which will ultimately result in differentiation or apoptosis of K562 cells.
Wang
Sha
S
Department of Hematology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Chai
Yu-bo
YB
Liu
Fei
F
Zhang
Xiao-yong
XY
Jia
Wei
W
Xie
Xin
X
Yu
Wen-qiang
WQ
Shang
Zhen-chuan
ZC
Jin
Bo-quan
BQ
Sun
Bing-zhong
BZ
D004305:Dose-Response Relationship, Drug; D016044:Fusion Proteins, bcr-abl / Q000235:genetics / Q000502:physiology*; D016313:Genes, abl; D006801:Humans; D020014:K562 Cells / Q000378:metabolism; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000032:analysis; D034741:RNA, Small Interfering / Q000494:pharmacology*; D014162:Transfection
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016044:Fusion Proteins, bcr-abl