Study On The Mechanism Of Limonin A1542 Acting On Leukemia Through Cholesterol Biosynthesis Pathway

Objective :This project aims to explore the molecular mechanism of the anti-leukemia activity of limonin compound A1542,in order to provide a better therapeutic strategy for the treatment of leukemia.Methods:(1)The effects of compound A1542 on HEL gene in leukemia cells were analyzed by RNASeq and Real Time PCR;(2)MTT colorimetric method was used to detect the growth curve of cholesterol on leukemia cell line HEL;(3)SREBP1(sh SREBP1)cloned in a lentivirus was used to silence the SREBP1 gene in HEL cells.Real time-PCR technology was used to detect the expression of genes related to cholesterol biosynthesis and to measure the effect of SREBP1 knockout gene on the proliferation of leukemia cell line HEL;(4)RNAseq,Western Blottin,Real time-PCR technologies were used to detect the effect of compound A1542 on the expression level of EGR1 on HEL cells;(5)RNAseq and Real time-PCR technologies were used to detect the expression level of related genes AP1(FOS + JUN)after treatment with drugs in order to determine the mechanism of A1542 anti-leukemia activity;(6)F-Mu LV virus was used to generate a mouse model of erythroleukemia,in order to examine the effects of Cholesterol,HDL,and LDL on leukemia progression.Results:(1)RNASeq and Real time-PCR results showed that A1542 compound induced cell death was related to cholesterol biosynthesis gene(p<0.05);(2)Using MTT assay,addition of cholesterol to leukemia HEL cells was then significantly inhibited proliferation(p<0.05);(3)Knocking out the SREBP1 transcription factor inhibited only expression of several genes involved in cholesterol biosynthesis while it also promoted the proliferation of leukemia cell line HEL(p<0.05);(4)In RNAseq analysis combined with Western Blotting and Q-RT-PCR,A1542 treatment of HEL cells up-regulated the EGR1 expression known to be involved in cholesterol biosynthesis(p<0.05);(5)RNAseq data also revealed that A1542 increases the expression of other regulators of cholesterol biosynthesis genes including AP1(FOS+ JUN).Indeed,drug mediated inhibition of AP1 significantly inhibited expression of all cholesterol biosynthesis genes;(6)The experimental results of leukemia mice showed that: Cholesterol,HDL and LDL significantly delayed the leukemia process.Conclusions:Limonin compound A1542 induced the expression of the cholesterol biosynthesis genes through upregulation of SREBP1,EGR1 and AP1,and increases the cholesterol content in leukemia cells,thereby exerting anti-leukemia effect and providing new ideas and theoretical basis for the treatment of leukemia.