Effects Of Acute Lymphoblastic Leukemia Cell Line Jurkat Cell Proliferation And Apoptosis By Anti C-myc Small Interference RNA

Backgrounds: c-myc gene is an oncogene regulating proliferation and differentiation of cell. It has been found to be overexpressed in acute leukemia cell lines and cells of acute leukemia and malignant lymphoma patients. It's one of the important genes influencing growth and progression of these malignancies. RNA interference(RNAi) is the sequence-specific gene silencing induced by double-stranded RNA(dsRNA), is the process of sequence-specific post-transcriptional gene silencing(PTGS) in animals and plants, initiated by double-stranded RNA that is homologous in sequence to the silenced gene. This phenomenon is conserved in a variety of organisms. RNAi is mediated by small interfering RNAs(siRNAs) that are produced from long dsRNAs of exogenous or endogenous origin by an endonuclease-â…¢type, called Dicer. The resulting siRNAs are about 21-23 nucleotides(nt) long and are then incorporated into a nuclease complex, the RNA-inducing silencing complex (RISC), which then targets and cleaves mRNA containing a sequence identical to that of the siRNA. Recently RNAi has became the new tool used to study the functions of genes and was used widly in the fields of genemic function research and gene therapy. In this study, Jurkat cell line with rather high expression level of c-myc which plays an important role in its proliferation and apoptosis was chosen to be the object which was treated with the anti c-myc siRNA.Objective: The aim of this study is to investigate the effects of anti c-myc siRNA on apoptosis and proliferation and on c-myc protein and mRNA expression in human lymphoblastic leukemia cell line Jurkat cells and is to provide a target for treatment and new gene therapy method of leukemia.Methods: siRNA targeting the site 1545-1565 of c-myc mRNA was designed and chemically synthesized. In vitro cultured Jurkat cells were transfected with transfection agent. Growth inhibition was detected by MTS assay and colony formation assay, and cell apoptosis by flow cytometry and detection of TdT mediated dUTP nick end labeling(TUNEL). The expression of c-myc and hTERT was detected by RT-PCR and Western blot.Result: (1) c-myc siRNA remarkably inhibited the cell proliferation, and the inhibitory concentration 50%(IC50) after 48 hour of treatment was about 75nM. By MTS assay, c-myc siRNA had inhibition effects on the growth of Jurkat cells and inhibited clone growth significantly. (2) c-myc siRNA induced apoptosis in Jurkat cells that could be detected by flow cytometry and detection of TdT mediated dUTP nick end labeling(TUNEL), These also showed that c-myc siRNA induced apoptosis in Jurkat cells in time-dependent manner. (3) c-myc siRNA can decrease the expression levels of c-myc and hTERT mRNA expression in Jurkat cells. (4) c-myc siRNA can decrease the expression levels of c-myc and hTERT proteins in Jurkat cells.Conclusion: c-myc siRNA chemically synthesized could inhibit significently Jurkat cells proliferation and induce apoptosis. c-myc siRNA decreased c-myc and hTERT mRNA and proteins expression in Jurkat cells, it may become one of the new tools of gene therapy to acute leukemia.