[Study on the targeting effects of M1-GS RNA on K562 cells].
OBJECTIVE
To determine the effects of M1-GS RNA (M1 RNA) on bcr-abl mRNA and oncoprotein after M1 RNA with guide sequence (M1-GS RNA) targeting the oncogene was transfected into K562 cells.


METHODS
pAVGS4 (an eukaryocyte expression vector containing M1-GS RNA sequence) and pNAV-1 (as the control) were transfected into K562 cells by X-tremeGENE Q2. Total RNA was extracted at 24, 48, 72 and 96 hours after transfection. Then RT-PCR was done to compare the products at different time point. After collecting pAVGS4-transfected cells and the control cells at 48 and 96 hours after transfection, total protein was extracted and quantified. Change of P210 was determined by Western blot. Colony formation was analyzed at 96 hours after transfection.


RESULTS
RT-PCR based on transfected cells at different time point showed that the amount of bcr-abl mRNA began to decrease at 24 hours and reduced to 9.2% and 2.5% respectively at 48 and 72 hours after transfection. Western blot showed that the expression of P210 in the pAVGS4 group reduced to 10.4% of the control at 48 hours and 6.7% of the control at 96 hours after transfection. The inhibition rate of colony formation was 81.3% after K562 cells were transfected by pAVGS4.


CONCLUSIONS
pAVGS4 can efficiently destroy bcr-abl mRNA in K562 cells. The transcript level of bcr-abl mRNA was reduced with the time after transfection. The expression of P210 was decreased significantly at 48 and 96 hours after transfection. K562 cell colony formation was prominently inhibited.
Chen
Bo-bin
BB
Department of Hematology, Huashan Hospital, Fudan University, Shanghai 200040, China.
Lin
Guo-wei
GW
Lu
Hua-zhong
HZ
Fan
Hua-hua
HH
Gao
Li
L
D029968:Escherichia coli Proteins / Q000235:genetics; D016044:Fusion Proteins, bcr-abl / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D020014:K562 Cells; D012329:RNA, Bacterial / Q000235:genetics; D016337:RNA, Catalytic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D043262:Ribonuclease P / Q000235:genetics; D013997:Time Factors; D014162:Transfection / Q000379:methods
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D012333:RNA, Messenger; D016044:Fusion Proteins, bcr-abl; D043262:Ribonuclease P; C470865:ribonuclease P, E coli