The Study On The Mechanism Of TAMs Promoting The Progression Of Endometrial Carcinoma By Down-regulating PTEN And Activating The PI3K/AKT/mTOR Signaling Pathway

Objective: Endometrial carcinoma is one of the three malignant tumors of female reproductive tract.The incidence of endometrial cancer is second only to cervical cancer.Now the incidence of endometrial carcinoma is increasing year by year worldwide.It has overtaken cervical cancer in some countries and become the most common malignancy of female reproductive tract.The incidence of endometrial cancer is becoming younger and poses a serious threat to women's health.Despite the advances in diagnosis and treatment,mortality rates are still high in developing countries.The mortality of endometrial cancer is on the rise.At present,the main treatment method for endometrial cancer is surgery.Although surgical methods and techniques have been greatly improved,many patients are still at risk of recurrence,local or distant metastasis after surgery.The prognosis is poor.In patients with advanced stage or relapsed metastasis,the 5-year survival rate is less than 30%.However,many patients are already in advanced stage when they go to the clinic.Radiotherapy and/or chemotherapy are often the primary treatment options for patients with advanced endometrial cancer and recurrent metastasis.However,the radiotherapy and/or chemotherapy have significant side effects,increasing patient suffering and reducing patients' quality of life.Therefore,the study of factors affecting the occurrence and development of endometrial cancer from the perspective of molecular biology has become the focus of the research,in order to provide valuable information for the diagnosis,treatment and prognosis of endometrial cancer.With the deepening of the research,it has been found that the tumor microenvironment is of great significance in the occurrence and development of tumors.The tumor microenvironment refers to the surrounding external environment on which the tumor lives,including tumor associated macrophage,stromal cells,endothelial cells,fibroblasts,white blood cells,cytokines,blood vessels,hypoxia,etc.All of these make up the stroma of the tumor.Most of the stroma of the tumor is composed of tumor associated macrophages(TAMs).The effect it is significant on the tumor microenvironment.There are two main types of TAMs: M1 and M2.M1 is the classical activated type with strong antigen-presenting function,which can activate the body's anti-tumor immune functionand has anti-tumor activity.M2-type's antigen presentation is weak.It can inhibit inflammatory and immune responses,thereby promot tumor growth.TAMs has similar phenotypic and functional characteristics to M2-type macrophages.Abnormal amounts of TAMs can lead to severe immunosuppression,which is related to the occurrence,development and poor prognosis of a variety of tumors.Therefore,we speculate that abnormal amounts of TAMs may also exist in endometrial cancer tissues.The study on the distribution of TAMs in endometrial carcinoma tissues is helpful to further understand the mechanism of endometrial carcinoma.Phosphatase and tension homologue(phosphatase and tensin homolog,PTEN)is a kind of important tumor-suppressor genes.It already got the attention of many scholars.It is related to cell growth,proliferation,apoptosis and adhesion.It has been studied that it is the gene with the highest mutation rate in endometrial cancer,and is most closely related to endometrial cancer.It has been regarded as the guardian gene of endometrial cancer.Discovered in 1997,it is located at 10q23.3 of the human chromosome and consists of 9exons and 8 introns,with a length of about 200 KB.It is a missing phosphatase and tonin homologous gene on chromosome 10.The expression of PTEN gene is closely related to the occurrence,development and invasion of tumors.The relationship between PTEN and TAMs in endometrial cancer is still unclear.Therefore,this study attempted to preliminarily explore the relationship between PTEN and TAMs in terms of histology and cytology.Studies have found that the occurrence and the development of endometrial cancer is closely related to the overactivation of PI3K/AKT/mTOR signaling pathway.Upon activation,PI3 K binds to the PH domain of intracellular AKT and activates AKT.Activated AKT is transferred from the cell membrane to the nucleus and cytoplasm,where phosphorylation regulates downstream m TOR.mTOR regulates cell survival,growth and reproduction.It is necessary for cell initiation of translation signals and the transition from G0/G1 phase to S phase.It is the center of regulating cell division and anabolism.This signaling pathway is one of the most important signaling pathways for the transmission of extracellular signals to the nucleus.It plays an important role in cell's metabolism,survival,proliferation,cycle regulation,apoptosis and microangiogenesis.It is closely related to the occurrence,development and metastasis of tumors.Theoccurrence and development of tumor is the product of tumor cells and their interaction with the surrounding microenvironment.What is the mechanism by which TAMs acts as the most important component of the tumor microenvironment? Is it related to the PI3K/AKT/mTOR signaling pathway? It is unclear.Therefore,this study intends to preliminarily explore the relationship between TAMs and PI3K/AKT/mTOR signaling pathway through cytological experiments,in order to provide valuable information for the study of the mechanism of endometrial cancer.Methods: The density and distribution of CD163 positive macrophages in different parts and adjacent normal tissues of 35 cases of endometrial carcinoma were detected by immunohistochemistry.We analyze the correlation with vascular endothelial growth factor(VEGF),clinicopathological parameters and microvascular density(MVD).The expression of CD163 labeled TAMs and PTEN in endometrial carcinoma tissues and adjacent normal tissues was detected by immunohistochemistry and we analyze the clinical significance.After 72 hours of treatment with PMA(48h)and IL-4(24h),the expression of CD163 markers in THP-1 cells was analyzed by flow cytometry.Endometrial carcinoma RL95-2 cells were co-cultured with PTEN overexpression plasmid to establish a PTEN overexpression endometrial cancer cell line.TAMs cells were co-cultured with endometrial cancer cells and PTEN overexpressed endometrial cancer cells in vitro.The effects of TAMs and PTEN on the proliferation and invasion of endometrial cancer cells were evaluated by CCK-8,invasion and migration experiments.The induced TAMs were co-cultured non-contact with endometrial cancer cells(RL95-2),PTEN expression of endometrial cancer cells,PTEN expression negative control endometrial cancer cells,and endometrial cancer cells added with PI3 K inhibitor(LY294002)and mTOR inhibitor(Rapamycin)respectively.CCK-8,proliferation assay,invasion assay and apoptosis assay were used to detect the effect of TAMs on biological learning of endometrial cancer cells.The induced TAMs were co-cultured non-contact with endometrial cancer cells(RL95-2),PTEN expression of endometrial cancer cells,PTEN expression negative control endometrial cancer cells,and endometrial cancer cells added with PI3 K inhibitor(LY294002)and mTOR inhibitor(Rapamycin)respectively.Western blot and RT-PCR were used to detect the expressions of key proteins and mRNA of endometrial cancer cells(RL95-2).Results:1.Tumor-associated macrophages(TAMs)were mostly distributed at the edges of carcinoma nests,tumor stroma and invasive margin,and their density was higher than that of normal endometrial tissues(P<0.01).2.The correlation between TAMs density and clinicopathological parameters of endometrial carcinoma showed that the density of TAMs at the edge of endometrial carcinoma was correlated with FIGO stage,histological grade,muscle layer infiltration and lymph node metastasis of endometrial carcinoma(P<0.05).The TAMs density in the stroma of endometrial carcinoma was correlated with histological grade,invasive margin and lymph node metastasis.There was no correlation between the density of carcinoma nests and FIGO stage,histological grade,invasive margin,lymph node metastasis,age,body mass index,menopause or tumor size(P>0.05).There was no correlation between the density of TAMs at the edge of cancer nests and age,body mass index,menopause or tumor size(P>0.05).3.The correlation analysis between TAMs density and VEGF showed that there was a significant positive correlation between TAMs density and VEGF in the tumor stroma and invasive margin(P<0.05).4.Pearson correlation analysis between TAMs density and MVD showed that there was a positive correlation between TAMs density and MVD in the stroma of cancer and the infiltrating edge of tumor(Pearson correlation coefficients were 0.57 and 0.51 respectively,and P values were 0.018 and 0.011).There was no obvious correlation between the density of TAMs and MVD in carcinoma nests.(Pearson correlation coefficient was 0.17,P=0.353).5.There was a significant correlation between CD163 positive cell's density and PTEN protein expression in endometrial carcinoma stroma(P<0.05).The increased density of CD163 positive cells was correlated with the loss of PTEN protein expression.6.The expression of CD163 marker in THP-1 cells was significantly increased after induction by flow cytometry,that is,THP-1 cells were successfully induced to be tumor-associated macrophag.7.After co-culture of endometrial carcinoma RL95-2 cells with PTEN overexpression plasmid,high level of PTENmRNA and protein could be detected by real-time PCR and Western blot.A stable PTEN overexpression RL95-2 cell line was successfully established.The results of CCK-8experiment,invasion experiment and migration experiment showed that TAMs could promote the proliferation,invasion and migration of RL95-2 cells.Overexpression of PTEN can restrain the multiplication,invasion and migration of RL95-2 endometrialcancer cells induced by TAMs.8.The results of CCK-8 experiment,invasion experiment,migration experiment and apoptosis experiment showed that TAMs could promote the proliferation,invasion and migration of RL95-2 cells.Inhibiting apoptosis of RL95-2cells,PTEN overexpression,PI3 K inhibitor(LY294002)and mTOR inhibitor(Rapamycin)can inhibit proliferation,invasion and migration of RL95-2 cells induced by TAMs,and promote apoptosis of RL95-2 cells.9.Western blot results showed that the protein contents of p-PI3 K,p-AKT and p-m TOR were significantly increased after co-culture of RL95-2 cells with TAMs,which was significantly different from the control group(P<0.01).PTEN overexpression group,PI3 K inhibitor(LY294002)group,mTOR inhibitor(Rapamycin)group all decreased p-PI3 K,p-AKT,p-mTOR protein content,the difference was significant(P<0.01).Real-time PCR showed no significant difference in PI3 K,AKT and mTORmRNA levels in each group.Conclusion:1.The relationship between clinicopathological parameters and the density of TAMs in different parts of endometrial carcinoma tissues was different.TAMs density in tumor stroma and invasive margin is higher,and is closely related to VEGF and MVD.2.The increased cell density of TAMs was associated with the loss of PTEN protein expression.In endometrial cancer TAMs is able to accelerate the hyperplasia,invasion and migration.Overexpression of PTEN can inhibit the proliferation,invasion and migration of RL95-2 endometrial cancer cells induced by TAMs.3.TAMs activates the PI3K/AKT/mTOR signaling pathway by reducing PTEN,thereby promoting the progression of endometrial cancer.Overexpression of PTEN,PI3 K inhibitor(LY294002)and mTOR inhibitor(Rapamycin)inhibited the progression of endometrial cancer by inhibiting the PI3K/AKT/m TOR signaling pathway.