The Effects Of Decreasing CGI-100 Gene Expressions On Proliferation And Differentiation Of K562 Cells

Objective: This study aims at explaining the effects on proliferation and differentiation of K562 cells, and the molecular mechanism of induced differertiation of K562 cells after silencing CGI-100 gene expression by RNA interference. Furthermore,we hope to provide experimental basis for seeking for a new target of cancer gene therapy.Methods:Three oligonucleotides targeting to CGI-100 gene and a pair of negative control which has the same composition but different sequence, were devised and chemically synthesized. They were annealed to form double-strand DNA and inserted into plasmid pGenesil-1 which digested by BamHI and HindIII. The recombinant vectors were then transfected respectively into K562 cells with lipofectamineTM 2 000 ,The CGI-100 mRNA level in the transfected K562 cells was analyzed by RT-PCR and dot blot hybridization. The recombinated K562 cells with the highest inhibition of CGI-100 expression were chosen for following experiments, named K562/shRNA-CGI-100 cells.The changes of cell proliferation,cell cycle,erythroid differentiation and ultrastructures of K562/shRNA-CGI-100 cells were detected respectively with MTT,flow cytometry,benzidine staining and electron microscope. K562/shRNA-CGI-100 cells and control K562 cells( including K562/shRNA-pGenesil cells and untransfected K562 cells) were treated respectively with 0.2mg/ml matrine and 30μmol/L hemin.The proliferation were observed by MTT. The expressions of GPA(CD235a),Gfi-1B mRNA and GPA,CD14,CD15 proteins were detected respectively by RT-PCR and flow cytometry.Results: The three recombinant plasmids of pGenesil-1-CGI-100 and negative control plasmid—pGenesil-1-Negative were successfully constructed. The three recombinant plasmids of pGenesil-1-CGI-100,pGenesil-1-Negative and blank plasmids of pGenesil-1 were transfected respectively into K562 cells. The modified K562 cells named K562/shRNA-CGI-100,K562/shRNA-Negative,K562/shRNA-pGenesil were selected by G418 pressure ,the fluorescent light might been observed under the inverted fluorescence microscope; The expression of CGI-100mRNA was detected by RT-PCR and dot blot hybridization. One of the modified K562 cells ,which was named K562/shRNA-CGI-100 cells in the following experiments, exhibited the highest inhibition efficiency in CGI-100 mRNA level, up to 54%; however, the pGenesil-1- Negative vector and pGenesil-1 plasmid did not decrease CGI-100 gene expression.Compared with control K562 cells, K562/shRNA-CGI-100 cells showed CGI-100 gene mRNA expression decrease by 54%,enchromation decreased while heterochromation increased, the absorbance value decreased by 23% with MTT(P<0.05), percentage of G0/G1 phase cells increased from 21% to 35%, While that of S phase decreased from 56% to 38%(P<0.05); PI (proliferation index of cells) decreased from 78% to 63%(P<0.05). The percentage of benzidine-positive cells increased from 3% to 7% (P<0.05). The inhibitory rate of proliferation of K562/shRNA-CGI-100 cells increased from 41% to 62%( P<0.05) after exporing to 0.2mg/ml matrine, while the percentage of benzidine-positive cells increased from 16% to 23%( P<0.05), the mRNA expression of GPA and Gfi-1B were obviously increased( P<0.05),the mean fluorescence intensity(MFI) of GPA was increased by 31%( P<0.05).Expressions of CD14 which was the marker of monocytes andmacrophages were not found. Although the granulocytic marker-CD15expressed, it has no statistical significance( P>0.05).Compared with control cells, the MFI of GPA increased by 20% in K562/shRNA-CGI-100 cells treated with 30μmol/L hemin ( P<0.05). The MFI of GPA in K562/shRNA-CGI-100 cells treated with 30μmol/L hemin was 1.7 times as high as that with 0.2 mg/ml marine(P<0.05).Conclusion:The RNA interference vectors targeting to CGI-100 gene were successfully constructed and one recombinated interfering vector with highest inhibition efficiency was screened.Low expression of CGI-100 can inhibit the proliferation of K562 cells and reduce the immaturation level. Inhibition of the expression of CGI-100 can induce erythroid differentiation of K562 cells as well as elevate the sensitivity to matrine and hemin. Hemin which was generally accepted as erythroid differentiation inducer have more powerful erythroid differentiation ability than Matrine, while,With CGI-100 knockdown, K562 cells showed higher sensitivity to Matrine than to Hemin.