Midkine Antisense Oligodeoxynucleotides Enhances The Sensitivity Of5-FU Chemotherapy Of Choriocarcinoma Cell

Objective:Through transfectting Midkine(MK) antisense oligodeoxynucleotide (ASODN) into choriocarcinoma cell line JEG-3cells and decreasing the expression of MK gene, to explore the effect of MK-ASODN on5-fluorouracil sensitivity of chemotherapy in JEG-3cells and study the related mechanisms of this effect.Method:1. MK-ASODN and the corresponding control with antisense oligodeoxynucleotide (MK-NSODN) were transfected into JEG-3cells using Oligofectamine, and then the transfection effect was examined by RT-PCR.2. The JEG-3cells of MK-ASODN group and MK-NSODN control group were treated by different concentrations of5-FU, and then the cell growth inhibition was tested by MTS test.3. Using Anexin V-FITC flowcytometry measuring the apoptosis cells, which devided into MK-ASODN group, MK-ASODN+5-FU group and the corresponding control groups.4.With Western blot detecting the expreesion of p-PI3K, p-Akt and its downstream of Bcl-2protein in MK-ASODN+5-FU group and the corresponding control group JEG-3cells. Result:1. Compared with the MK-NSODN control group and blank control group, the expression of MKmRNA was significantly decreased in the group cells transfected by MK-ASODN.2. In the same concentration of5-FU, MK-ASODN experimental group cell’s growth inhibition rate was obviously higher than the control group; Downregulation of expression of MK with MK-ASOND can enhance5-Fu sensitivity of chemotherapy in JEG-3cells, and IC50values was declined from54.83±4.86mg/L to35.69±3.04mg/L (P<0.05).3. The MK-ASODN group cell’s total apoptosis rate(0.54±0.10%) and early apoptosis rate(0.27±0.09%) were rising compared to the control group which the total apoptosis rate was0.09±0.03%and the early apoptosis rate0.04±0.01%(P<0.05); MK-ASODN increased the cell total apoptosis rate in JEG-3cell treated by5-FU from28.99±2.04%to49.14±4.12%(P<0.05), and more significant was the early apoptosis rate increasing from16.63±2.45%to38.28±4.41%(P<0.05).4. MK-ASODN combined with5-FU can reduce the activity of PI3K/Akt signal (p-PI3K,p-Akt) and expression of downstream protein Bcl-2. Conclusion:1. MK-ASODN could inhibit MKmRNA expression in JEG-3cells.2. MK-ASODN could decrease IC50of5-FU in JEG-3cells, and MK-ASODN could promote the choriocarcinoma JEG-3cell apoptosis after adding the5-FU, which explained MK-ASODN enhancing5-FU sensitivity of chemotherapy in JEG-3cells.3. The synergistic effect of5-FU chemotherapy in choriocarcinoma JEG-3cells may be related to the inhibition of PI3K/Akt signaling pathway by MK-ASODN.