[Effects of FMS-like tyrosine kinase 3 targeted RNA interference on proliferation and apoptosis of acute monocytic leukemia cell line THP-1].
OBJECTIVE
FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is constitutively activated in (70-90)% pediatric patients with acute myeloid leukemia (AML) and appears to confer an adverse prognosis. Although several FLT3-selective small molecule inhibitors and antibodies were developed with varied degrees of success, to address the specificity and resistance, new approaches for specifically targeted FLT3 are needed and RNA interference is a promising choice. The aim of the present study was to investigate the efficacy of suppression of FLT3 induced by small hairpin interfering RNA (shRNA) on myeloproliferation and apoptosis in an acute monocytic leukemia (AMOL) cell line THP-1.


METHODS
FLT3-targeted small hairpin interfering RNA (FLT3-shRNA) was designed and synthesized by transcription system in vitro was transfected into THP-1 cells. Firstly FLT3 mRNA level was detected by semi-quantitative RT-PCR and FLT3 protein level was detected by flow cytometry (FCM) to verify the efficacy on FLT3-shRNA interference at 48 h after transfection. Cell growth viability was measured at 24 h, 48 h and 72 h after treatment with CCK-8. The distribution of cell cycle was assayed by FCM, and apoptosis was analyzed by DNA Ladder and Annexin V-FITC Staining at 48 h.


RESULTS
FLT3 targeted shRNAs was synthesized successfully and the concentration of 15 nmol/L for 48 h could obtain desirable downregulation of FLT3 expression, the inhibitory percentages of FLT3 mRNA and protein were (72.95 +/- 2.07)% and (65.39 +/- 5.57)%, respectively. The suppression of FLT3 induced by FLT3-shRNA resulted in marked inhibition of cell growth and the inhibitory percentages were (36.66 +/- 3.67)% at 48 h, (35.56 +/- 0.73)% at 72 h. FLT3-shRNA induced the inhibition of cell cycle from G(0)/G(1) phase to S phase, the percentage of sub-G(0)/G(1) phase (65.71 +/- 4.47)% was higher than those in the PBS-control group (52.23 +/- 2.98)%, NC-shRNA control group (51.81 +/- 1.44)%, P < 0.01; the percentage of S phase (25.11 +/- 2.70)% was lower than those in the PBS-control group (34.41 +/- 4.07)% and NC-shRNA control group (32.50 +/- 1.46)%, P < 0.05. Furthermore treatment with FLT3-shRNA for 48 h resulted in clear apoptosis ladder, the percentage of early apoptosis detected by Annexin V-FITC was (18.59 +/- 2.07)% which was significantly higher than that in the PBS-control group (4.00 +/- 0.50)% and the NC-shRNA control group (6.06 +/- 0.70)%, P < 0.001.


CONCLUSIONS
The suppression of FLT3 induced by the shRNA can effectively inhibit cell proliferation, and apoptosis induction on THP-1 cells, which indicates that this approach may bear the therapeutic potential on childhood AMOL.
Lu
Jie
J
Department of Pediatrics, First Affiliated Hospital, Zhengzhou University, Henan Key Laboratory of Molecular Medicine, Zhengzhou 450052, China.
Sheng
Guang-yao
GY
Zou
Xiang
X
Xu
Xue-ju
XJ
Zhao
Xiao-ming
XM
Bai
Song-ting
ST
Xu
Pei-rong
PR
D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects*; D002648:Child; D006801:Humans; D007948:Leukemia, Monocytic, Acute / Q000201:enzymology / Q000473:pathology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D034622:RNA Interference / Q000502:physiology; D034741:RNA, Small Interfering / Q000494:pharmacology*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D051941:fms-Like Tyrosine Kinase 3 / Q000378:metabolism*
D034741:RNA, Small Interfering; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; C084944:TYRO3 protein, human; D051941:fms-Like Tyrosine Kinase 3