The Effect Of New Gene CGI-100 On Differentiation And Apoptosis Of K562 Cells Induced By Matrine

It has become a tendency in Chinese traditional herbal drugs researches to look for new anticancer drug and to do new explorations and explanations for their pharmacological actions by using modern molecular biological techniques. Matrine is one of the active ingredients extracted from the traditional Chinese medical herb sophora flavesscens Ait. Our previous researches have proved that matrine with certain concentrations may induce erythroid differentiation and apoptosis of K562 human leukemia cells and evidently influence the cell cycle and telomerase activity. Differential gene expression results in these cellular phenotypic changes. Through gene expression profile, we have found several related differentially expressed genes, including those with known functions and those with unknown functions.CGI-100 is one of those genes with unknown functions. This study aims at exploring the relationship between gene CGI-100 and differentiation and apoptosis of K562 induced by matrine. Furthermore, we hope to pioneer a new approach for the researches on tumor generation and reversion, to provide experimental basis for seeking for a new target of cancer therapy, to explore anti-tumor function of matrine and to develope new drugs against tumor.Methods:1. The expression level distinction of CGI-100 was evaluated by RT-PCR in K562 cells before and after being treated with matrine.2. The coding domain sequence (CDS) of human CGI-100 gene was amplified from human leukemia K562 cells with RT-PCR and was cloned into pMD18-T Simple vector. It was sub-cloned into pIRES2-EGFP eukaryotic expressive plasmid.3. The pIRES2-EGFP/ CGI-100 recombinant plasmid was transfected into K562 cells and the positive clones were selected by G418 pressure. The monoclonal K562 cells were obtained by limited dilution.4. The changes of CGI-100 gene expression level, cell cycle of the modified K562 cells were detected by RT-PCR, FCM. Their morphological changes were observed under the optical and electron microscopes.Results:1. The expression of CGI-100 mRNA decreased in a dose- manner in the K562 cells treated with 0.1~1.0mg/ml matrine. It reduced remarkably in 3 hours and kept a steady low expression level till 48h when the cells were treated with 0.1mg/ml matrine.2. The combined pIRES2-EGFP / CGI-100 eukaryotic expression vector was constructed successfully with correct direction and sequence. 3. The combined pIRES2-EGFP / CGI-100 were transfected into K562 cells. The monoclone modified K562 cells named as K562-CGI-100 was selected by G418 pressure and limited dilution.In K562-CGI-100 cells, the fluorescent light was observed under the inverted fluorescence microscope; over expression of CGI-100 mRNA was detected by RT-PCR.4. Heterochromatin decreased, euchromatin and the proportion of S phase increased in K562-CGI-100 cells, growth curves indicated enhanced cell proliferation.Conclusions:1. The expression level of CGI-100 decreases when K562 cells are induced to differentiation and apoptosis by matrine. It suggests that matrine can reduce the expression level of CGI-100, and indicates a possible involvement of CGI-100 gene in the early response of the K562 cells to matrine.2. Recombinant eukaryotic expressive plasmid containing CGI-100 gene ---pIRES2-EGFP/ CGI-100 was reconstructed successfully, and the sequence was confirmed correct.3. Over-expression of CGI-100 elevates the proliferation and the immaturity level of K562- CGI-100 cells.