The Effect Of Metformin On Apoptosis By Inhibiting IL-6-activated STAT3/PD-L1 Signaling Pathway In KLE Endometrial Carcinoma

BackgroundEndometrial cancer is a kind of female reproductive system tumor,that often occurs in perimenopausal and postmenopausal women.The common symptom is abnormal vaginal bleeding after menopause.Its exact pathogenesis is not very clear up to now.According to the relationship between endometrial cancer and estrogen,it is traditionally divided into two types,estrogen-dependent(type I)and non-estrogen-dependent(type II).In recent years,with the changes of people’s lifestyles and the improvement of screening methods,the incidence of endometrial cancer has increased year by year.And more and more studies show that the occurrence and development of endometrial cancer are closely related to inflammation and immunity.The occurrence and development of endometrial cancer cannot be separated from the tumor microenvironment(TME).The tumor microenvironment is a complex pathological environment,composed of tumor cells,stromal cells,immune cells,and cytokines,all of which play a significant role in the proliferation,invasion,metastasis,and angiogenesis in tumor cells.IL-6(interleukin-6)is an important inflammatory factor that can promote cell growth and inhibit apoptosis.Tumor cells and immune cells in the tumor microenvironment can secrete a large amount of IL-6,which plays an important role in the tumorigenesis and progression.Studies have shown that IL-6 can activate the JAK(Janus kinase)/STAT3(signal transduction and activator of transcription 3)signaling pathway,which in turn regulates a series of downstream molecules to regulate cellular outcomes.Activated phosphorylated STAT3 enters the nucleus as a dimer and binds to the PD-L1(programmed death ligand-1)promoter,thereby promoting PD-L1 expression.PD-L1 is an immunosuppressive molecule that can be expressed on the cell membranes of a variety of immune and non-immune cells.Studies have shown that PD-L1 is highly expressed in a variety of tumor cells.PD-L1 can interact with its receptor PD-1,which inhibits T cell function in the tumor microenvironment,thus promoting the immune escape of tumor cells.PD-L1 can also activate signaling pathways in tumor cells,although the specific mechanism is still unknown.Previous studies have shown that PD-L1 can activate mTOR(mammalian target of rapamycin)to promote mRNA translation and increase the expression of some cytokines,hormones and so on.Surgery has always been the traditional treatment of endometrial cancer.But for some young patients,retaining the uterus is their urgent requirement,which poses a challenge to the traditional treatment.Metformin has been a hot topic about tumor in recent years,and it was reported that metformin can reduce the risk of cancer in diabetic patients.A study found that metformin can inhibit the phosphorylation of STAT3 induced by the high concentration glucose,thereby inhibiting related downstream anti-apoptotic proteins and promoting apoptosis.Metformin also controls cell proliferation and growth through a variety of pathways,such as AMPK(AMP-activated protein kinase),PI3K(phosohatidylinositol 3-kinase)/AKT(protein-serine-threonine kinase)pathway and so on.Therefore,we propose the hypothesis that IL-6 can activate the STAT3/PD-L1 pathway to inhibit apoptosis and promote growth in endometrial cancer cells cultured in vitro.But silencing STAT3 and PD-L1 expression with siRNA can promote cell apoptosis.And the ability of IL-6 can be reversed by the application of metformin,which can inhibit the STAT3/PD-L1 pathway,thereby promoting apoptosisMethodsEndometrial cancer KLE cells were cultured and divided into different groups.Except the control group,they were treatled respectively with IL-6,IL-6+siRNA STAT3,IL-6+siRNA PD-L1,and IL-6+metformin in different concentrations(1,3,5 mmol/l).Western blotting were used to localize and quantitatively analyze the expression of STAT3,phospho-STAT3,PD-L1,BCL-2,and BAX in different groups of cells,then analyze the differences between them.Results1.In endometrial cancer KLE cells,IL-6 can promote the expression of phosphorylated STAT3,PD-L1,BCL-2,reduce BAX expression,thus increasing activity of tumor cell.2.Silencing the expression of STAT3 with siRNA can inhibit PD-L1 expression,and suppressing PD-L1 expression with siRNA,both can promote apoptosis of tumor cell.3.In endometrial cancer KLE cells,metformin decreases the expression of phospho-STAT3,PD-L1,and BCL-2,promotes BAX expression,thus promoting apoptosis,and this change is concentration-dependentConclusion1.In endometrial cancer KLE cells,IL-6 can inhibit tumor cell apoptosis by activating the STAT3/PD-L1 pathway;and silencing STAT3 and PD-L1 expression expectively by siRNA can promote apoptosis.The results implies that reducing the inflammatory response,or using siRNA to inhibit the expression of target molecules,all of which can change cell outcomes.And if they are combined with targeted monoclonal antibody therapy may increase the clinical treatment effect.It also suggests that PD-L1 can not only generate immunosuppression by interacting with PD-1,but also activate intracellular signaling pathways to affect cell behavior,although this specific intrinsic activation pathway is currently unknown.2.Metformin inhibits the STAT3/PD-L1 pathway in a concentration-dependent manner,inhibits BCL-2 expression,promotes BAX expression,and thus promoting apoptosis.Metformin plays a significant role in the development and progression of endometrial cancer by this signaling pathway,providing great theoretical basis for clinical treatment of endometrial cancer with metformin.