[In vitro transcription synthesis and effects of FLT3 targeted short hairpin RNA].
FMS-like tyrosine kinase 3 (FLT3) is a receptor of tyrosine kinase that is constitutively activated in most of acute myeloid leukemia patients and seems to give an adverse prognosis. In order to explore the silencing effect of FLT3 targeted short hairpin RNA (FLT3-shRNA) on acute leukaemia cell line THP-1, three FLT3-shRNAs (shRNA1, shRNA2, shRNA3) were designed and synthesized by transcription system in vitro and then transfected into THP-1 cells. FLT3 mRNA was analyzed by semi-quantitative RT-PCR, FLT3 protein was detected by Flow cytometry and immunofluorescence. The results indicated that FLT3 expression was downregulated by shRNA1 and shRNA3, and shRNA1 showed stronger inhibitory effect. At 48 hours following transfection, the inhibitory rate of 25 nmol/L shRNA1 was 72.95 +/- 2.07%, lasting 72 hours. The 5 nmol/L and more concentration of FLT3 shRNA1 could downregulate FLT3 mRNA level, which displayed a quantity-effect relation; the inhibitory rate of 15 nmol/L shRNA1 was 67.53 +/- 0.66%. FLT3 protein was located on THP-1 cell membrance, its expression was downregulated obviously by shRNA1, at 72 hours following transfection the inhibitory rate of shRNA1 was 79.67 +/- 0.66%. shRNA1 showed the best inhibitory effect on FLT3 protein, the optimal time of which was 72 hours with an inhibitory rate of 79.67%. It is concluded that FLT3-shRNA1 shows a desireable FLT3-targeted inhibitory effect, which can be used for further investigation of FLT3 mechanism or FLT3 targeting treatment.
Lu
Jie
J
Department of Pediatrics, The First Affiliated Hospital, Zhengzhou University, Zhengzhou, 450052, China.
Sheng
Guang-Yao
GY
Zou
Xiang
X
Fang
Ying-Qi
YQ
Zhao
Xiao-Min
XM
Xu
Xue-Ju
XJ
Bai
Song-Ting
ST
Xu
Bai-Rong
BR
Wang
Jian-Ren
JR
D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D051941:fms-Like Tyrosine Kinase 3 / Q000235:genetics*
D012333:RNA, Messenger; D034741:RNA, Small Interfering; C497970:FLT3 protein, human; D051941:fms-Like Tyrosine Kinase 3