| ObjectiveScreen the shRNA sequence targeting CDX2 gene that can silence the CDX2 gene expression specially and effectively, then investigate the effects of RNA interference targeting CDX2 gene and chemotherapeutics on the proliferation and apoptosis of human leukemia cells, and then study the possible mechanisms in order to research the new ideas and methods for the treatment of leukemia.Methods1. Design the four shRNA sequences which target the different coding regions of CDX2 gene: CDX2-shRNA : CDX2-shRNA-1443 ,CDX2-shRNA-2008, CDX2-shRNA-921 and the negative control sequence CDX2-shRNA-NC, and the tansfect the human carcinoma of colon cells SW620 using X-tremeGENE HP DNA Transfection Reagent respectively. Realtime PCR and Western Blot were used to detect the CDX2 mRNA and protein expression in order to screen the shRNA sequence that can silence the CDX2 gene expression most effectively; Then the effects of shRNA targeting CDX2 gene on the proliferation and apoptosis of SW620 cells were researched by MTT and flow cytometry analysis to do the follow-up experiments.2. The human chronic myeloid leukemia cells were transfected with the shRNA sequence that can silence the CDX2 expression most effectively, and the CDX2 mRNA and protein expression were detected by Realtime PCR and Western Blot;MTT and flow cytometry analysis were used to detect the effects of silence of CDX2 gene on the proliferation and apoptosis of K562 cells.3. The shRNA sequence and classical chemotherapeutics were used on leukemia cells and Realtime PCR and Western Blot were used to detect the CDX2 mRNA andprotein expression. Then the changes of cell proliferation and apoptosis were detected with MTT and flow cytometry analysis. At the same time, the BCR-ABL fusion gene and relative apoptosis proteins were studied as well to investigate the possible mechanisms.Results1. Of the four designed shRNA sequence, the CDX2-shRNA-921 can silence the CDX2 gene expression most effectively. Compared with the normal group, the expression of CDX2 mRNA and protein decreased 66.2% and 68.2% respectively, and the differences have statistical significance; Meanwhile, the expression of CDX2 of negative control group has no distinct change; The MTT and flow cytometry analysis showed that the CDX2-shRNA-921 can significantly inhibit the cell proliferation and promote its apoptosis. It indicate that the CDX2 shRNA sequence can silence the CDX2 expression effectively which can be used in the follow-up experiments.2. The K562 cells were transfected with CDX2-shRNA-921 and the expression of CDX2 gene were reduced distinctly. The expression of CDX2 mRNA and protein were decreased by 60.5% and 47.7% respectively while the CDX2-shRNA-NC group has no significant change; And the relative apoptosis proteins were detected as follow: The expression of Survivin reduced apparently while the Caspase-9 and Bax expression increased obviously; The MTT and flow cytometry analysis showed that the cell proliferation inhibition rate and apoptosis rate of CDX2-shRNA-921 group increased apparently compared with the normal cell group and CDX2-shRNA-NCgroup.3. The CDX2-shRNA-921 and VCR were acting on K562 cells collectively and the MTT and flow cytometry analysis showed that the cell proliferation inhibition rate and apoptosis rate of CDX2-shRNA-921+VCR group were increased obviously compared with the VCR group and CDX2-shRNA-921 group.The difference has great statistic significanceConclusions1. The shRNA sequence can silence the CDX2 gene expression, and It as well can inhibit the proliferation of SW620 and promote the cell apoptosis.2. Using the CDX2-shRNA-921 to transfect the human leukemia cells K562,it can silence the CDX2 gene expression in K562 cells effectively. it can also suppress the proliferation of K562 cells and as well promote its apoptosis.3.The CDX2-shRNA combined with Vincristine can inhibit the proliferation of k562 cells obviously, and the apoptosis rate was increased apparently as well.In other words, the silence of CDX2 expression in K562 cells can increase the sensibility to VCR of K562 cells. |
