The Role Of IL-6 In The Regulation Of HIF-1α In EOC Cells Via JAK/STAT3 Pathway Under Normoxia And Hypoxia

Objective: To investigate whether IL-6 regulates the transcriptional activity and expression of HIF-1α in human epithelial ovarian cancer cells via JAK2/STAT3 pathway under normoxic and hypoxic conditions,and promote tumor hypoxia tolerance and tumor formation.It provides a new idea for the diagnosis and treatment of EOC targeting IL-6 and related signal molecules.Methods: 1.Four human ovarian cancer cells(A2780,SKOV3,ES-2,OVCAR3)were routinely cultured,and the total RNA and protein were extracted respectively,and the expression levels of STAT3 and HIF-1α were detected by real-time quantitative PCR and Western Blotting.The expression level of STAT3 and HIF-1α m RNA was detected in the former,and the expression level of phosphorylated and total STAT3 and HIF-1α were detected in the latter.The levels of IL-6 in the four ovarian cancer cells were detected by enzyme-linked immunosorbent assay(ELISA).2.Human ovarian cancer cell lines A2780 and SKOV3,with significant difference in the secretory level of IL-6,were treated with different concentrations of IL-6(1 ng/ml,10 ng/ml,50 ng/ml)for different time(6 h,12 h,24 h,48 h).Total RNA was extracted from A2780 and SKOV3.The expression levels of STAT3 and HIF-1α were detected by real time quantitative PCR to determine the optimal concentration and time for exogenous IL-6 treatments.3.A2780 and SKOV3 cells were treated with IL-6(10 ng/ml and 50 ng/ml,respectively)for different time(5 min,15 min,30 min,2 h,6 h,24 h,48 h).Expression levels of phosphorylated and total STAT3 and HIF-1α proteins were detected with Western Blotting.In addition,pretreatment with AG490(50 μM),an inhibitor of JAK/STAT3 pathway,was preformed for 1 hour before adding IL-6,and then total RNA and protein were extracted from each group to detect the above indicators for reverse verification experiment.4.Under the conditions of normoxic(21%),hypoxia(1%)or Co Cl2 simulated hypoxia(A2780 cells: 50 μM,SKOV3 cells: 100 μM),the cells were tre ated with exogenous IL-6 for 12 h.A2780 and SKOV3 cells were also tra nsfected with Lipofectamine 2000 to establish cell lines with endogenous o ver-expression of(ss)IL-6 or anti-expression of(as)IL-6.Meanwhile,cont rol groups for hypoxia(1%)or simulated hypoxia were established.Total RNA and proteinwere extracted,quantitative q PCR reaction and Western Bl otting were used to detect the m RNA level of STAT3 and HIF-1α and prot ein levels of phosphorylated and total STAT3 and HIF-1α.5.Dual Luciferase Reporter Assay was used to detect the effect of IL-6 on the transcriptional activity of HIF-1α in A2780 and SKOV3 cells under normoxic and hypoxic conditions.Results: 1.The results showed that three molecules(IL-6,STAT3,HIF-1α)have different expression levels in the four ovarian cancer cells,in which IL-6 is hardly detected in A2780 cells,whereas in the other cell lines,especially SKOV3,IL-6 is highly expressed.The protein expression of phosphorylated STAT3 and HIF-1α are similar to that of IL-6.Therefore,considering these results,weselected A2780 cells(not secreting IL-6)and SKOV3 cells(high expression of IL-6)as the model of cells for this study.2.Ovarian cancer cells were treated with different concentrations of IL-6(1 ng/ml,10 ng/ml,50 ng/ml)at different time periods(6 h,12 h,24 h),determining the optimal concentrations of IL-6 were 50 ng/ml and 10 ng/ml in A2780 and SKOV3 cells,respectively.The results showed that IL-6 can significantly improve the expression levels of STAT3 and HIF-1α genes.3.IL-6(50 ng/ml)was used to treat A2780 cells for different time.We found that IL-6 had a significant effect on the expression of phosphorylated STAT3 and HIF-1α after treatment for 30 min and 12 h,while the effect of IL-6on the expression of STAT3 was not obvious.When IL-6 was applied to SKOV3 cells,the expression of phosphorylated STAT3 in IL-6 was continuously enhanced within 12 h and then dropped.The expression of HIF-1α was consistent with the expression of phosphorylated STAT3.Therefore,the optimal time of IL-6 treatment was finally determined as 12 h.4.Under normoxic condition,exogenous of IL-6 or endogenous transfection of IL-6 could significantly promote the m RNA level of STAT3 and HIF-1α and the protein expression of phosphorylated and total STAT3 and HIF-1α.5.In ovarian cancer cells,which could be weakened by AG490.Under the condition of hypoxia,this effect is more obvious.These results suggest that under normoxic and hypoxic conditions,IL-6 may activate STAT3,by regulating JAK/STAT3 signaling pathway and ultimately promote the expression of HIF-1α in ovarian cancer cells.6.Under normoxic condition,exogenous of IL-6 or endogenous transfection o f IL-6,detected by luciferase reporter gene assay,could significantly enhan cethe transcriptional activity of HIF-1α in ovarian cancer cells.Hypoxia als o showed the same effects.When combining with hypoxia,IL-6has a muc h more significant promoting effect on the transcriptional activity of HIF-1α.These results suggest that both IL-6 and hypoxia can significantly enha nce the transcriptional activity of HIF-1α in EOC cells,and there is a syn ergistic effect between them.Conclusion: In this study,we used human epithelial ovarian cancer cell lines and lipofectamine stably transfected clone screening cell lines for in vitro studies.The results showed that under normoxic and hypoxic conditions,IL-6 can regulate the expression and transcriptional activity of HIF-1α in ovarian cancer cells by activating the JAK/STAT3 pathway,which provides a new target for the diagnosis and treatment of EOC.