Effects Of HL-60 Cell Proliferation And Apoptosis By Anti-c-myc Small Interference RNA

Backgrounds:C-myc gene is an oncogene regulating cell proliferation and differentiation. It has been found to be overexpressed in acute leukemia cell lines and cells in patients with acute leukemia and malignant lymphoma. It's one of the important genes influencing growth and progression of these malignancies.One common mechanism of elevated c-myc expression is related to deregulation of transcription due to mutations in the c-myc promoter region.The other mechanism of elevated c-myc expression is stabilization of c-myc mRNA as a result of mutations within its 3' or 5' untranslated regions. A lack of downregulation of total c-myc expression plays a key role in the development of tumors.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 in animals and plants,initiated by double-stranded RNA(dsRNA) that is homologous in sequence to the silenced gene.This phenomenon is conserved in a variety of organisms.RNAi is mediated by short interfering RNAs(siRNAs) that are produced from long dsRNAs of exogenous ro endougenous 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,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 fuctions of genes and was used widly in the fields of genemic function research and gene therapy. In this study,HL-60 cell line with high expression level of c-myc was chosen to be the object which was treated with the anti-c-myc siRNA .Effects of proliferation and apoptosis were detected.Objective:The aim of this study is to appraise the effects of anti-c-myc siRNA on apoptosis induction and proliferation inhibition and on c-myc protein and mRNA expression in human myeloid leukemia cell line HL-60 cells and is to find a new gene therapy method of leukemias.Methods:siRNA targeting the site 1762-1782 of c-myc mRNA was designed and synthesized by in vitro transcription using T7 RNA polymerase and short double-stranded DNA(dsDNA) templates.In vitro cultured HL-60 cells were transfected with lipofectamine 2000.Growth inhibition was detected by MTT assay and colony formation assay,and cell apoptosis by flow cytometry and DNA fragmentation.The expression of c-myc and hTERT was detected by RT-PCR and Western blot.Result: Anti-c-myc siRNA remarkably inhibited the cell proliferation,with an IC50 value of 150nM;(1) By MTT assay, c-myc siRNA has inhibition effects on the growth of HL-60 cells and significantly inhibited clone growth.(2) anti-c-myc siRNA induced decrease in c-myc and hTERT mRNA expression in HL-60 cells.(3) c -myc siRNA induced apoptosis in HL-60 cells that could be detected by flow cytometry and DNA fragment analysis.(4) c-myc siRNA can lower the expression levels of c-myc and hTERT proteins in HL-60 cells.Conclusion:. c-myc siRNA could significently inhibit HL-60 cells to proliferate and cause cells to apoptosis and may become one of the new tools of gene therapy to leukemia.