Metformin Inhibits IL– 6 Activated The JAK/STAT3 Signaling Pathway Inducing Epithelial–Mesenchymal Transition Of Hela

Objective: Investigating the effects of Metformin and Human recombinant IL-6 on Epithelial-Mesenchymal Transition of cervical cancer cells through JAK/STAT3 signaling pathway,and analyzing its mechanisms.Methods: 1.In vitro,the Hela cells maintained in a humidified at 37°C under an atmosphere of 5% CO2;2.Hela cells have been treated by different drug concentrations IL- 6, metformin at different times, and OD value by CCK8,and select the relative proliferation(IL- 6), the proliferation inhibition(metformin) effective drug concentrations and the treated times.3.The experiment contains four groups, i.e.,the contorlled group, the IL-6 group, the Metformin group, the IL-6+Metformin group, according to the results of CCK8 with drug treatment Hela cells;4.Observe their morphological differences of Hela cells by microscope;5.Observe the differences of apoptosis and nerosis cells by AO/EB double fluorescent staining;6.The changes of the expression of p-STAT3, Twist and E-cadherin by Western Blot;7.The changes of the expression of Twist m RNA and E-cadherin m RNA by RT-PCR.Results: 1.The results of CCK8 demonstrate that the effective drug concentrations are 50ng/ml,10mmol/L,and the treated times is 48 h by treated with IL-6 and metformin.2.Results demonstrate that in the Controlled group Hela cells exhibited obvious epithelial cells with a distinctive cobblestone appearance and closely arranged; the IL- 6 group cells intercellular broadened, present fibroblast-like shapes and arranged loosely that are characteristics of mesenchymal-like morphology; the Metformin group cells present cobblestone appearance and form clusters with more floating dead cells;the IL- 6 + Metformin group cells show cobblestone appearance or fibroblast-like shapes and the nucleus osteoporosis, while the number of dead cells is increased significantly with forming floating clusters;3.AO/EB double fluorescent staining suggests that the controlled group,IL-6 and Metformin group have a small amount of cell apoptosis, Metformin group have more cells apoptosis compare to the controlled group,while the IL-6 and Metformin group almost all of apoptosis cells and form floating.Statistic indicates that IL-6 and Metformin group apoptosis and necrosis rate remarkable than the others in the four groups, the difference are statistically significant(p<0.05);4.Western Blot indicates that in IL-6 group the levels of p- STAT3(1.54±0.41 VS 1.00±0.00), Twist(1.61±0.08 VS 1.00±0.00) expression significantly increas compare d to the controlled group;while E-cadherin(0.63±0.02 VS 1.00±0.00) expression decreases, note that the difference are statistically significant compared to the controlled group(p < 0.05); In the Metformin group the levels of p- STAT3(0.94±0.03 VS 1.0±0.00), Twist(0.97±0.003 VS 1.00±0.00) and E- cadherin(1.03±0.0242 VS 1.00±0.00) expression has no remarkbale changes compared to the controlled group, while there are no statistically significant differences from the controlled group(p > 0.05); In the IL- 6 + metformin group the expression of p- STAT3(0.56±0.06 VS 1.00±0.00), Twist(0.34±0.12 VS 1.00±0.00), E cadherin(0.44±0.01 VS 1.00±0.00) decrease compared to the controlled group, and the differences are statistically significant compared to the controlled group(p < 0.05).5.RT-PCR suggests that the expression levels of Twist m RNA in the IL-6 group increase and E-cadherin m RNA decrease compared to the controlled group, and the difference are statistically significant(1.63±0.04 VS 1.00±0.00,0.56±0.06 VS 1.00±0.00;p<0.05). Both the expression levels of Twist m RNA and E-cadherin m RNA are decrease in the IL-6+Metformin group compared to the controlled group, the difference are statistically significant(0.45±0.02 VS 1.00±0.00,0.26±0.10 VS 1.00±0.00;p<0.05). There is no significant difference of Twist m RNA and E-cadherin m RNA expression levels between the Metformin group and the controlled group(0.94±0.06 VS 1.00±0.00,1.05±0.34 VS 1.00±0.00;p>0.05).Conclusions: 1. IL-6 can induce EMT of Hela cells;2. Metformin can inhibit IL-6 induced EMT of Hela cells through the JAK/STAT3 Signaling Pathway.