STAT3Mediates The Inhibition Of IL-6on Autonhagy

Objective:To investigate the effect of IL-6on starvation-induced autophagy and its mechanism.Methods:First, experimental cells were divided into three groups:control, starvation and starvation co-treatment with IL-6group. Then LC3protein levels were detected by immunoblot analysis and endogenous LC3puncta were analyzed by immunofiuorescence method. Autophagic vacuoles were observed by transmission electronic microscopy. Then the expression of main signal proteins were detected by western blot method to select the key signal molecule affecting autophagy. Third, inhibitor of the key signal molecule was used and mutant was constructed to ascertain its importance in the process of IL-6inhibiting autophagy. At the same time, Beclin1and Atg14were knockdown with Beclin1siRNA and Atg14siRNA seperately to analyze the mechanism of IL-6affecting autophagy.Results:(1) We found that in starved U937cells, the addition of IL-6reduced the ratio of LC3-Ⅱ/LC3-Ⅰ and the ratio decreased with increasing concentration of IL-6. The immunofluorescence LC3puncta decreased as well, indicating the inhibitory effect of IL-6on autophagy.(2) Phosphorylated protein levels of Akt, ERK1/2, STAT3were changed in cells under starvation condition. Although the addition of IL-6had little effect on Akt or ERK1/2, it promoted the phosphorylation of STAT3and activated STAT3signal pathway, increasing the expression of Bcl-2while decreasing the ratio of LC3-Ⅱ/LC3-Ⅰ. No obvious degradation of p62occurred in the process. Treatment of STAT3inhibitor LLL12reduced the activity of STAT3in cells, as well as increased the ratio of LC3-Ⅱ/LC3-Ⅰ and levels of Beclin1and VPS34. IL-6had no effect on autophagy in cells transfected with mutant STAT3Y705F.(4) In cells transfected with Beclin1siRNA and Atgl4siRNA, IL-6 had little impact on expression of autophagy-related proteins.Conclusion:Taken together,(1) IL-6inhibits autophagy and STAT3mediates the inhibition of IL-6on autophagy;(2) Beclin1and Atg14are involved in the process of IL-6inhibiting autophagy. Further discussion of the relationship between IL-6and autophagy will contribute to the research on diseases closely related to IL-6.