| K562 cell is a highly malignant human erythroleukemia cell line that can be induced in vitro to apoptosis by a variety of factors. Apoptosis (meaning a dropping off) is a phenomenon induced by the intrinsic activation of cellular suicide program, which is universal in metazoan organisms. Strictly speaking, apoptosis is defined by morphological features. It is characterized by cellular shrinkage, violent blebbing of the plasma membrane and chromatin condensation. Furthermore, apoptosis is related to the activation, expression and regulation of many genes.The proto-oncogene c-myc is at the center of a transcription factor network that regulates cellular proliferation, replication, growth, differentiation and apoptosis. c-myc functions through the transcriptional activation or repression of its target genes. Whether c-myc expression induces apoptosis or proliferation is dependent onthe growth factor status of the cell, c-myc drives proliferation through activation of growth regulators. In cells without mitogenic stimulation, c-myc expression leads to apoptosis. In K562 cells, c-myc overexpression leads to cellular unrestrained proliferation and the blockage differentiation.RNAi (RNA interference) refers to the post-transcriptional gene silencing. Endogenous or exogenous dsRNA triggers expressional restraining of genes of homologous sequence, which leads to the decrease expression of specific protein. RNAi is an evolutionarily conserved multi-step process that involves generation of active small interfering RNA (siRNA) in vivo through the action of an RNase III endonuclease, Dicer, in an ATP dependent manner. The resulting 21- to 23-nt siRNA mediates degradation of the complementary homologous RNA. RNAi technology has developed very rapidly as a revolutionary tool for experimental biology in a variety of organisms.In our study, using RNAi technology, siRNAs targeting the different sites of c-myc mRNA molecule were designed to interfere the expression of this gene. By testing with RT-PCR, MTT and FACS, the level of c-myc mRNA was obviously decreased and more induced K562 cells apoptosis. But there were differently interfering efficacy in different target sites.Total RNA was isolated from K562 cells before and after RNA interference and mRNA were purified. Both mRNA from the treated K562 cells and the untreated control K562 cells were reverse transcribed into cDNA. Restriction Display (RD) technique was applied combined with labeling by extension in corporate Cy-dNTP. Cy3/Cy5 (two different fluorescence dyes) separately labeled the control and thetreated samples. The labeled cDNA were hybridized to the K562 microarray at 42 °C for 12-18h. The gene expression microarray were scanned by using a dual-laser scanner (GenePix 4100A) and the data were statistically analyzed with the Quantarray software package. Among the 616 target genes, 455 down-regulated genes (Cy5/Cy3<0.5) were identified after RNA interference. Most down-regulated genes were correlated with cell cycle regulation, metabolism and transformation. 12 up-regulated genes (Cy5/Cy3>2.0) were identified, including cellular apoptosis, cell cycle regulation genes. These results suggest that cellular transformation and apoptosis induced by c-myc may occur through overlapping and non-overlapping pathway.In summary, RNAi was used to knock out the c-myc expression in K562 cells, which were systematically studied with the high-throughput, efficient and sensitive method of cDNA microarray to analyze the differentially expressed genes after RNAi interference. This study could provide theoretical basis for further elucidation of the molecular mechanism of tumor cells apoptosis and can provide reference in genetic level to explore novel scheme leading to effective therapeutics against erythroleukemia.
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