Effect Of Survivin Gene Silence On Cell Differentiation And Drug Resistance Of K562 Cells With RNA Interference

Objective:To explore the effects of survivin small interference RNA (siRNA) on the cell differentiation and sensitivity of K562 cell line to Adriamycin (ADM), which will offer an important theory evidence for chemotherapy of leukemia.Method: 1.The expression of survivin in K562 cells after treated with recombinant plasmid pshRNA-survivin. Recombinant plasmid were delivered in the form of complexes with lipofectamine2000. The K562 cells were divided into there groups: RNA interference (RNAi) group, lipofectamine group and control group. The expression of survivin was examined by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry (SP). 2. The cell differentiation of K562 cells effected by pshRNA-survivin. The K562 cells were also divided into there groups: RNAi group, lipofectamine group and control group. 48h after transfection, the morphologic changes were observed by light microscope. Then CD13 and CD33,the two surface markers, were determined by flow cytometry and the expression of c-myc,c-fos were analysised by RT-PCR and immunohistochemistry (SP) in the there groups. 3. Detect the inhibition ratio of K562 cells treated with Adriamycin (ADM). Analysis the inhibition ratio of K562 cells exposed to ADM in different concentration by MTT assay. The K562 cells were divided into two groups: RNAi group and control group. 4. To investigate whether down-regulation of survivin expression has the potential to sensitize K562 cells to chemotherapy, the mean fluorescence intensity of ADM in K562 cells was determined by flow cytometry. K562 cells were divided into three groups: RNAi group, lipofectamine group and control group. Then we detected the protein level of P-glycoprotein (P-gp), TOPO-2, GST-Ï€using immunohistochemistry methods to find out the possible mechanism of drug resistance of K562 cells.Result: 1. The protein of survivin is overexpress in K562 cells. After transfection with recombinant plasmid pshRNA-survivin, the expression of survivin in K562 cells was decreased significantly in mRNA and protein level (P<0.05). 2. 48h later thansfection, the morphologic characteristics of K562 cells changed and the expression of c-myc in K562 cells was decreased significantly in mRNA and protein level in RNAi group (P<0.05). The mean fluorescence intensity of CD33 in K562 cells were increased in RNAi group comparing with lipofectamine group and control group (P<0.05). 3. The sensibility of K562 cells to ADM was increased in RNAi group comparing with lipofectamine group and control group (P<0.05). 4. The protein level of GST-Ï€expression was drawdown in RNAi group comparing with the 2 control groups (P<0.05).Conclusion: 1. RNA interference with survivin can down regulate the protein expression of survivin. 2. Survivin gene is related to the differentiation of K562 cells. 3. RNA interference with survivin can increase the sensitivity of K562 cells to ADM, maybe through the mechanism of decreasing the expression of GST-Ï€.