Effects Of U937 Cell Proliferation And Apoptosis By Anti-RPL36A Small Interference RNA

【Objectives】To compare the expression level of RPL36A (ribosome protein 36a) in the primary acute myeloid leukemia (AML) cells, human myeloid leukemia cell line U937 and normal mononuclear cells (MNC), and to investigate the influence on cell proliferation and cell apoptosis of U937 cells with the inhibition of RPL36A.【Methods】In the present study, the RPL36A mRNA expression in the primary AML cells, U937 and MNC was determinated by RT-PCR; Small interfering RNA (siRNA) that are designed to target to RPL36A was first synthesized chemically, and then transfected into U937 cells using LipofectamineTM 2000 system. The influence of RPL36A siRNA on cell proliferation was analyzed by MTT assay, while the influence on cell apoptosis and cell cycle of U937 cells was examined by acridine orange/ethidium bromide (AO/EB) double staining, TUNEL and Annexin V/FITC. RPL36A mRNA and protein expression levels by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively.【Results】RPL36A expression in either the primary acute myeloid leukemia (RPL36A/actin 1.0546±0.22892)or U937 cells(RPL36A/actin 1.1642±0.1112) was significantly higher than normal mononuclearcell (RPL36A/actin 0.3227±0.1528); Cell proliferation of U937 cells were remarkably inhibited by RPL36A siRNA, with an IC50 value of 150nM. The average OD value of U937 cells transfected with RPL36AsiRNA were 0.266±0.027(24h),0.315±0.0102(48h),0.391±0.0099(72h),0.418±0.0142(96h), by MTT assay respectively, was significantly lower compared to that of untreated controls, indicated the inhibition effect of RPL36A SiRNA on cell proliferation. Remarkable cell apoptosis was recorded in U937 cell treated with RPL36A SiRNA using AO-EB and TUNEL analysis. Moreover, it is showed that RPL36A SiRNA induced cell apoptosis of U937 cells in a time-dependent manner.Annexin V/FITC assay showed that the apoptosis rate of U937 cells treated with anti-RPL36A siRNA were 25.31±3.01%(24h),35.78±2.30%(48h),53.25±2.71%(72h) respectively . With the cell cycle analysis it is revealed S- and G2- phase cell cycle arrests when U937 cells transfected with RPL36AsiRNA: in 24h, 48h and 72h, percentage of G2- and S- phase were 34.6±1.8, 26.8±2.8 and 14.5±3.7 respectively.(3) RPL36A mRNA and protein expression level of U937 cells treated with siRNA were significantly declined in a time-dependent manner.【Conclusion】RPL36A mRNA expression in the primary acute myeloid leukemia and U937 cells was much higher than that of normal controls. RPL36A siRNA could significantly inhibit U937 cells proliferation and induce cell apoptosis which might due to cell cycle arrest. Thus, RPL36A gene may play a valuable role in the pathogemesis and development of acute myelogenous leukemia.