Intervention Study Of Metformin On Proliferation Of Human Endometrial Carcinoma Cells

Objective:To observe the effect of Metformin on expression of REDD1(regulated in development and DNA damage responses-1) in human endometrial carcinoma cell lines Ishikawa cell in vitro, and the role of possible REDD1 / mTOR signaling pathway(independent of AMPK pathway),to investigate the potential effect of REDD1 on proliferation of human endometrial carcinoma cells.Methods:Human endometrial carcinoma Ishikawa cells were recoveried, and cultured in a humidifier 5 % CO2 incubator at 370 C. The RPMI 1640 medium contained 10% fetal bovine serum(FBS). Inoculating the cells into 25 cm2 culture bottle with 5 x 105 cells each bottle when the cells confluenced. The medium was changed every 2 days.When 90% confluenced,the Ishikawa cells was inoculated into serum-free RPMI1640 medium for 12 hours, and then carried out the following drug intervention respectively: 1 mmol/l、5 mmol/l、10 mmol/l meiformin intervened for 48 hours, the same volum of serum-free RPMI 1640 culture acts as the control group.After intervention, Ishikawa cells proliferation rate was measured by MTT assay, then total RNA and protein was extracted. The expression of gene mTOR and REDD1 was detected by RT-PCR techniques. Expression levels of protein mTOR and REDD1 were examined by Western-blot.Results:( 1) Metformin inhibited endometrial carcinoma Ishikawa cells proliferation.With the increase of concentration of metformin, inhibition of cell proliferation rate has a tendency to increase. At the concentration of 10 mmol/l, metformin has the strongest inhibitory effects of cell proliferation.The difference was statistically significant(P < 0.05).( 2) Metformin can upregulate the expression of REDD1 and inhibite the expression of mTOR.The expression of 1mmol/l、5mmol/l、10 mmol/l REDD1 mRNA is 1.09 times 、 1.15 times 、 1.29 times than that of controlgroup, respectively, the difference was statistically significant(P <0.05). The expression of 1mmol/l、5mmol/l、10 mmol/l mTOR mRNA is 0.90 times 、0.38 times 、 0.24 times than that of control group, respectively, the difference was statistically significant(P <0.05).( 3) Metformin can increase the expression of REDD1 protein which is in the REDD1 / mTOR signaling pathway, and inhibit its downstream mTOR protein expression. The expression of 1mmol/l、5mmol/l、10mmol/l REDD1 protein is 4.29 times 、4.45 times、8.66 times than that of control group, respectively; The expression of 1mmol/l、5mmol/l、10 mmol/l mTOR protein is 0.70 times 、0.51 times、0.20 times than that of control group, respectively.The most significant effect was in the concentration of 10 mmol/l of both protein. The difference was statistically significant(P <0.05).Conclusion:Metformin obviously upregulated the expression of REDD1, the REDD1 /mTOR signaling pathway may be one of the key signaling pathways involved in the Ishikawa cells proliferation. Metformin may promote the expression of REDD1 /mTOR signaling pathway associated protein REDD1 and inhibit the associated protein mTOR to exert its inhibition effect of the Ishikawa cells. Suggesting that metformin may play an important role in the process of treatment of human endometrial carcinoma, and REDD1 / mTOR signaling pathway is involved in this process.