Study Of The Effects Of SiRNA Targeting LSD1Gene On Histone Modulation In Molt-4Cell Line

Objective: RNA interference (RNAi) is applied to silence the gene expression ofhistone lysine-specific demethylase1(LSD1) in human T-cell acute lymphoblasticleukemia MOLT-4cells. After LSD1gene is silenced by the corresponding smallinterfering RNA, its effects on the proliferation and apoptosis of MOLT-4cells, histonemethylation and acetylation modification and DNA methylation are examined.Methods:(1) Four small interfering RNA segments targeted at LSD1gene weredesigned and transfected into MOLT-4cells by LipofectamineTM2000. The optimalsegment of siRNA was then screened out by RT-PCR.(2) MTS was applied to measurecell proliferation in MOLT-4cells. Cell apoptosis was analyzed by flow cytometry.(3)The state of methylation of histone H3K4and H3K9and histone H3acetylation as wellas the expression of apoptosis-related proteins, namely cyclin-dependent kinaseinhibitor P15, methyltransferase DNMT1, Bcl-2and procaspase-3were measured bywestern blot.Results:(1) The optimal siRNA: sense5’-CCACGAGUCAAACCUUUAUTT-3’;Anti-sense5’-AUAAAGGUUUGACUCGUGGTT-3’.(2) LSD1siRNA inhibitedMOLT-4cell proliferation. LSD1siRNA in0,30,60,120nM trnasfection for24hour,the cell proliferation rate was （99.35±1.38）%,（83.02±1.69）%，（65.72±2.16）%，（41.15±2.23）%；p＜0.05;60nM LSD1siRNA transfection for0,24,48,72hours,the cell proliferation rate was（99.86±1.35）%，（65.72±2.16）%，（48.26±1.92）%，（37.86±1.66）%，p＜0.05，which showed time and concentration dependent.(3) LSD1siRNA decreased expression of apoptosis-related proteins such as Bcl-2,procaspase-3and induced cell apoptosis. LSD1siRNA with60,120,240μMol for24hours， apoptosis cell was (12.16±1.74)%,(32.74±2.47)%,(54.64±2.58)%respectively，as compared to control(3.35±1.26)%p＜0.05.(4) LSD1siRNAinhibited the expression of histone LSD1and LSD1mRNA. It improved the histonemethylation of H3K4me1, me2and histone acetylation of H3. variation oftrimethylation of H3K4and methylation of H3K9has not seen.(5) LSD1siRNAdown-regulated DNA methyltransferase (DNMT1) and P15was de novo. Conclusions:(1) With RNAi technology, LSD1siRNA could effectively interfereLSD1gene expression in MOLT-4cells.((2) LSD1siRNA inhibite cell proliferationand induce cell apoptosis.(3) silencing LSD1gene promote histone methylation ofH3K4me1, me2and the acetylation of histone H3in MOLT-4cells. However, it doesn’taffect the trimethylation of histone methylation H3K4and the expression of H3K9methylation.(4) LSD1siRNA inhibits DNMT1expression, thus causing thereexpression of P15gene. LSD1gene might be one of the target of anti-leukemia.