Effects Of STEAP1 On The Invasion And Metastasis Of Endometrial Cancer

BackgroundThe investigation from 2008 to now shows that endometrial cancer has become the female reproductive tract malignant tumor with the highest incidence in China,and has already far surpassed cervical cancer.In the United States,Europe and many other countries,endometrial cancer is the most pervasive malignant tumor in the females,and more than 75%of patients are perimenopause and postmenopausal women.According to the American cancer society,there were 63,230 new cases of endometrial cancer in 2018,and 11,350 women died from the disease.In recent years,with the rapid increase of new cases of endometrial cancer,screening,diagnostic criteria and surgical treatment for endometrial cancer have been widely used.However,there is still no accurate way to evaluate the prognosis of endometrial cancer.Therefore,it is urgent to find a biomarker that can evaluate the prognosis of endometrial cancer and carry out targeted therapy.As a member of the STEAP protein family,prostate transmembrane antigen 1 is mainly located in the plasma membrane and at the junction between cells.STEAP1 is involved in intercellular communication and has metal reductase activity,which may be related to the membrane transport function of secretion and endocytosis,cell apoptosis and survival.The STEAP family has been shown to be expressed in different kinds of malignancies,including prostate cancer,lung,colorectal,breast,and ewing’s sarcoma.STEAP1 is considered as an important diagnostic indicator and an effective therapeutic target.Therefore,the expression and clinical significance of STEAP1 in endometrial cancer are worth exploring.Objective1.To verify how STEAP1 is expressed in endometrial carcinoma tissues and cells.2.To investigate the effect of lentivirus transfection on the growth,proliferation and other cellular functions of endometrial cancer cells.3.To explore the effects of STEAP1 in the invasion and migration abilities of endometrial cancer.Methods1.Use Immunohistochemistry(IHC)to detect the expression of STEAP1 in normal endometrial tissues and endometrial carcinoma tissues.2.Use RT-qPCR and Western blotting to detect the expression of STEAP1 in endometrium.3.The expression of STEAP1 in human endometrial cancer cells KLE and Ishikawa was enhanced or inhibited by lentivirus mediated exogenous force,and the transfection effect was verified by RT-qPCR and Western blotting test.4.Immunocytochemical assay(ICC)was used to detect the expression of STEAP1 in endometrial cancer cell lines with different invasive abilities to exogenously enhance and inhibit the expression of STEAP1.5.Use Transwell test to detect the ability of invasion and migratioon of endometrial cancer cells after lentivirus mediated RNA interference,and to clarify the role of STEAP1 in the invasion and metastasis of endometrial cancer cells.6.Cell cycle was detected by flow cytometry and cell plate cloning experiment was carried out to detect the growth and proliferation ability of endometrial cancer cells after the transfection.7.A subcutaneous xenograft model of nude mouse endometrial carcinoma was established to prepare exogenous forced overexpression of STEAP1 cells and RNA interference silencing of STEAP1 cells.The successfully transfected cells were inoculated under the skin of nude mice,and the tumor growth rate and volume were recorded regularly.All nude mice will be killed 56 days later.8.Rt-qPCR and Western blotting were used to detect the expression of EMT-related genes in endometrial cancer cells before and after transfection.9.The expression of matrix protein metallase MMP2 and MMP9 before and after transfection was detected by gelatinase assay.Results1.The staining of STEAP1 in normal endometrial tissues was stronger than cancer tissues(P<0.05).The expression of STEAP1 in cancer was related to grade differentiation,clinical stage,infiltration and lymphatic metastasis(P<0.05),and the under-expression of STEAP1 was closely connected to bad prognosis.2.STEAP1 is highly expressed in normal endometrial cells but low in endometrial cancer cells,and is expressed differently in cells with different invasion and metastasis abilities,suggesting that STEAP1 may be related to the metastasis and invasion of endometrial cancer cells.3.Lentivirus transfection was observed under a high-connotation microscope,and both RT-qPCR and Western blotting were used to verify that the actual efficiency of transfection was run up to 85%.4.Immunocytochemical assays showed that STEAP1 expression was decreased in cancer cells after STEAP1 silencing and increased in cancer cells after STEAP1 overexpression.5.Downregulation of STEAP1 expression significantly promoted the invasion,metastasis and proliferation of endometrial cancer cells,while overexpression of STEAP1 significantly inhibited the invasion,metastasis and proliferation of endometrial cancer cells.6.In animal experiments,the tumor volume was larger and nodules were more in the group with high STEAP1 overexpression,while the tumor volume was smaller and smoother in the group with low STEAP1 down-expression,and the growth rate was slower than in the group with overexpression.7.Rt-qPCR,Western blotting and gelatinase assays showed that after RNA interference with STEAP1 up-regulated expression,E-cadherin expression was down-regulated in Ishikawa of endometrial cancer cells,while N-cadherin,Vimentin and some other genes like Snail,Slug,Twist and MMP2,9 were up-regulated.After STEAP1 overexpression,E-cadherin expression was increased in the endometrial cancer cells KLE,while N-cadherin,Vimentin,Snail,Slug,Twist,MMP2 and MMP9 were down-regulated and EMT process was obstructed.Conclusion1.Low expression of STEAP1 in endometrial cancer is associated with clinical malignant phenotype and poor prognosis.2.STEAP1 can be used as a therapeutic target for endometrial cancer and has the function of inhibiting the capacity of multiplication,invasion and migration of endometrial cancer.3.Up-regulated expression of STEAP1 can inhibit EMT progression in endometrial.