[Regulatory effect of STAT5 decoy oligonucleotides on trans-activation of bcl-x gene promoter in K562 cells].
OBJECTIVE
Constitutive activation of signal transducers and activators of transcription 5 (STAT5) plays an important role in malignant transformation of chronic myeloid leukemia (CML) cells. This study was to explore regulatory effect of STAT5 decoy oligonucleotides (ODNs) on trans-activation of its downstream target bcl-x gene in K562 cells.


METHODS
STAT5 decoy ODNs, mismatched ODNs (M-ODNs), and FAM-decoy ODNs were designed and synthesized. FAM-decoy ODNs were used as control, and transfected into K562 cells by cationic liposomes, analyzed by flow cytometry (FCM) and fluorescent inversive microscopy. The bcl-x promoter fragment acquired by polymerase chain reaction (PCR) was inserted into pGL3-basic to construct luciferase report plasmid pGL3b-bclxp, which was co-transfected with decoy ODNs or M-ODNs into K562 cells. The activity of luciferase was detected. After transfection of decoy ODNs, and M-ODNs, expression of bcl-xL mRNA in K562 cells was detected by reverse transcription-PCR (RT-PCR),cell apoptosis was detected by FCM.


RESULTS
FAM-decoy ODNs were incorporated into K562 cells in a dose-dependent manner. The incorporation efficiency reached 99.1% at the concentration of 4 micromol/L 24 h after transfection, and green fluorescence could be observed in cells under fluorescent inversive microscope. The luciferase activity was significantly lower in STAT5 decoy ODNs group than in control group [(181.48+/-204.46) RLU/microg protein vs. (675.26+/-62.91) RLU/mug protein, P < 0.05], but that of M-ODNs group [(632.07+/-98.95) RLU/microg protein] has no significant difference with that of control group (P > 0.05). mRNA level of bcl-xL was decreased by STAT5 decoy ODNs, but not by M-ODNs. Moreover, Sub G1 peak was detected in STAT5 decoy ODNs group by FCM.


CONCLUSIONS
Transfection of STAT5 decoy ODNs can down-regulate the trans-activation of bcl-x in K562 cells.
Zeng
Jian-Ming
JM
Department of Clinical Hematology, Chongqing University of Medical Sciences, Chongqing, 400016, P. R. China.
Feng
Wen-Li
WL
Wang
Xiao-Zhong
XZ
Shi
Mei
M
Tu
Zhi-Guang
ZG
Huang
Zong-Gan
ZG
D017209:Apoptosis / Q000187:drug effects; D015536:Down-Regulation; D006801:Humans; D020014:K562 Cells; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D050799:STAT5 Transcription Factor / Q000235:genetics*; D015533:Transcriptional Activation / Q000187:drug effects*; D014162:Transfection; D025521:Tumor Suppressor Proteins; D051020:bcl-X Protein / Q000096:biosynthesis* / Q000235:genetics
C494811:BCL2L1 protein, human; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D050799:STAT5 Transcription Factor; C494092:STAT5A protein, human; D025521:Tumor Suppressor Proteins; D051020:bcl-X Protein