Abstract:

Background and purpose: Apollon gene is highly expressed in leukemia and other tumors. The study aimed to discuss whether RNAi technology can reverse multidrug resistance of chronic myeloid leukemia cell line K562 through constructing a eukaryotic vector of short hairpin RNA (shRNA) targeting at Apollon gene. Methods: The eukaryotic vector pGPHI-GFP-Neo-Apollon with shRNA targeting at Apollon gene was constructed and then transfected into K562 cells by Lipofectamine^TM2000, and G418 pressure selection. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence were used to detect the expression of Apollon mRNA and protein after Apollon was transfected stably in K562 cells. The changes of sensitivity of K562 cells to leurocristine (VCR) and etoposide (VP16) after transfection with shRNA-Apollon were detected by MTT method, and the apoptosis rate was detected by flow cytometry. Results: pGPHI-GFP-Neo-Apollon carrier was constructed successfully and expressed stably in K562 cells, and after G418 screening, it silenced Apollon mRNA and protein expression effectively. According to the result of MTT, the sensitivity of K562 cells to VCR and VP16 increased significantly in the group of gene interference, with half of its inhibition concentration (half-inhibitory, IC₅₀) value significantly lower than the control group (P<0.05); Flow cytometry showed that the cell apoptosis rate was increased significantly (P<0.05), but there was no statistically significant difference in the apoptosis rate between shRNA negative control group and normal control group (P>0.05). Conclusion: pGPHI-GFP-Neo-Apollon carrier can enhance the abilities of VCR and VP16 to induce the apoptosis of K562 cells, namely an increase of sensitivity to these chemotherapeutics in K562 cells, it is hinted that RNA interference targeting Apollon gene may reverse the multidrug resistance of leukemia cells in some degree.

Key words: Leukemia, RNA interference, Apollon genes, Multiple drug resistance, Apoptosis, K562 cells