Decreased Proliferation of Myeloid Leukemia Cells through Down-Regulating LYL1 Expression with Small Interference RNA.
Authors: Meng Yuesheng , Liu Wei , Ma Xiaoxia , Meng Xiuqin , Ai Gongwen , Zhang Yanxiang 
Published: October 2019

Abstract
Ectopic expression of the basic helix-loop-helix transcription factor LYL1 has been implicated in T-cell acute lymphoblastic leukemia (T-ALL). It has also been found to be over-expressed in cells of acute myeloid leukemia (AML). Myeloid leukemia cells over-expressing LYL1 cDNA had accelerated growth rates, increased plating efficiency and a blockade of differentiation. To further investigate its role in the pathogenesis of leukemia, we used small interference RNA (siRNA) to silence the expression of LYL1 in human leukemia cell line K562, which expresses a moderate level of endogenous LYL1 protein. Three LYL1-specific RNA oligos, the Stealth Select RNAi HSS142834, HSS142835, and HSS142836, purchased from Invitrogen, were introduced into K562 cells by using Invitrogen transfection reagent Lipofectamine RNAiMAX. Two successive transfections at day 1 and day 2 were made according to manufacturer’s manual. Expression levels of LYL1 in LYL1 siRNA transfected cells and control cells (transfected with the Stealth RNAi Negative Controls) were determined with fluorescence real-time quantitative polymerase chain reaction assay. Our result showed that the application of any single RNAi oligo achieved observable inhibition of LYL1 expression levels (30–40%) while a combination of the three RNAi oligos remarkable inhibition (70.4%). The growth rates of K562 cells were not affected by any single RNAi oligo. However, a combination of three RNAi oligos did induce noticeable growth inhibition of cells. Plating efficiency assay showed that the clonogenic recovery rate of K562 cells treated with a combination of thee RNAi oligos was inhibited by 32.5% (P<0.05). The reduced growth rate and clonogenicity of cells was supposed to be secondary to the repressed expression of LYL1 because all other factors were controlled in our experiments. Further experiments are underway to define the changes of other genes in the LYL1-suppressed cells. We also tested the effect of specific siRNA on the expression of LYL1 and clonogenecity of leukemia cells in patient samples. Mononuclear cells separated from nine newly-diagnosed AML patients whose cells expressed comparatively higher levels of LYL1 were transfected with a combination of three LYL1 specific siRNA oligos for twice. We found that the siRNA oligos suppressed the expression of LYL1 in leukemia cells in most of the patients (7/9, decreased by 2 times or more) when compared with controls. Remarkably, the clonogenicity of AML cells in 3 patients was also inhibited by siRNA (P<0.05). In conclusion, the specific siRNA was effective to downregulate the expression of LYL1 in myeloid leukemia cells. It was also effective to affect cell proliferation in some cases. The data demonstrates that LYL1 plays a role for the malignant genotypes of leukemia cells and suggests that the RNA interference therapy targeting specific oncogenes might be clinically useful in the management of hematological malignancies.