The Mechanism Of SNCG Regulating Mapk Pathway To Promote Malignant Progression Of Endometrial Carcinoma

Endometrial carcinoma is one of the most common malignancies in the female genital tract and affects women's health.In recent years,the incidence of endometrial carcinoma has increased significantly worldwide,showed younger tend.Although the treatment of endometrial carcinoma continues to improve,the treatment of advanced endometrial carcinoma is still difficult.The five-year survival rate is low while the recurrent rate is high.It is necessary to further explore the pathogenesis of endometrial carcinoma,find a reliable therapeutic target and improve the level of cancer prognosis.Our previous clinical study showed that the expression of SNCG protein in endometrial carcinoma was significantly higher than that in normal endometrial tissue.The expression of SNCG protein increased with lymph node metastasis,clinical stage and depth of myometrial invasion.The overall survival rate of SNCG positive group was significantly lower than that of negative group.Then the HEC-1A cell line with higher expression of SNCG protein was screened by Western Blot,and the best efficiency shRNA sequence that silenced the SNCG gene was screened by lentiviral vectors.We constructed a HCE-1A cell line that stably expresses SNCG silencing,which laid the foundation for cell model of the subsequent experiments.Subsequently,we found that the proliferation,migration,invasion and anti-apoptosis ability of SNCG gene silenced HEC-1A cells were significantly reduced,and the cell cycle arrested at G1 and G2/M phase in vitro.The mechanism of overexpression of SNCG in the malignant biological behavior of endometrial carcinoma needs to be studied.It has been shown that tumorigenesis is a complex multi-pathway molecular regulation process.Mitogen-activated protein kinase pathway is an important and widespread signal transduction system in organisms.MAPK pathway involves cell growth,development,division,differentiation and death.To further elucidate the mechanism of overexpression of SNCG-regulated MAPK pathway in promoting malignant biological behavior of endometrial carcinoma,we first construct subcutaneous xenograft model of human endometrial carcinoma in nude mice,and then detect the expression of MAPK related proteins in HEC-1A cells and xenograft tumor tissues of nude mice by Western Blot in vitro and in vivo.PART I ESTABLISHMENT OF A SUBCUTANEOUS XENOGRAFT TUMOR MODEL OF ENDOMETRIAL CARCINOMA IN NUDE MICE AND THE EFFECT OF SNCG GENE ON THE GROWTH OF XENOGRAFT TUMOR?Objective?To establish the subcutaneous xenograft tumor model of human endometrial carcinoma in nude mice,and observe the effect of stable silenced of SNCG gene on the growth of xenograft tumor of human endometrial carcinoma in nude mice.?Method?We developed the HEC-1A cells of experimental group with SNCG gene silenced and the HEC-1A cells of empty vector transfection group with lentiviral empty plasmid vector by cell culture techniques.The proliferative phase cells were injected subcutaneously into the right shoulder blade back of 20female nude mice to establish xenograft tumor model of human endometrial carcinoma in nude mice.Observing the growth of xenograft tumor,measuring the length(L)and width(W),and then calculating the volume(V=1/6?×L×W~2).After 4 weeks,all nude mice were killed to take out the subcutaneous xenograft tumor.The length and weight of xenograft tumors were measured.Finally,the xenograft tumors were preserved with liquid nitrogen.?Results?The mean rate of xenograft tumor growth in experimental group with SNCG gene silencing was much slower than that in empty vector transfection group(P<0.05).The mean volume of xenograft tumor in experimental group was(75.06±85.46)mm~3,which was lower than that in empty vector transfection group(P<0.05).The weight of xenograft tumor in experimental group was(0.106±0.105)g,which was lighter than that in empty vector transfection group(P<0.05).?Conclusion?SNCG gene silencing inhibits the growth of subcutaneous xenograft tumor of human endometrial carcinoma in nude mice.Xenograft tumor tissue laid the foundation for subsequent signal pathway experiments.PART II THE MECHANISM OF SNCG REGULATING MAPK PATHWAY TO PROMOTE MALIGNANT PROGRESSION OF ENDOMETRIAL CARCINOMA?Objective?To investigate the mechanism of SNCG regulating MAPK pathway to promote malignant progression of human endometrial carcinoma.?Method?The subjects were divided into four groups: experimental group of cell: sh RNA lentiviral vector SNCG after low expression of HEC-1A cells;empty vector transfection group of cell: transfected with lentiviral empty plasmid vector of HEC-1A cells;experimental group of xenograft tumor: xenograft tumor of human endometrial carcinoma in nude mice with SNCG gene silencing HEC-1A cell;empty vector transfection group of xenograft tumor: xenograft tumor of human endometrial carcinoma in nude mice with empty vector transfection HEC-1A cells.We detected the expression of ERK1/2?JNK1/2/3?P38?ERK5 and their phosphorylated protein by quantitative Western Blot.The rate was calculated by dividing phosphorylated protein by non-phosphorylated protein.?Results?There were no significant difference in the expression of ERK1+2 ?JNK1+2+3?P38 or ERK5 protein between SNCG gene silencing HEC-1A cells and empty vector transfection cells(P>0.05).The protein expression of p-ERK1+2 and p-ERK5 on SNCG gene silencing HEC-1A cells were lower than that of empty vector transfection cells,while the protein expression of p-JNK1+2+3 and p-P38 were higher,the difference were statistically significant(P<0.05).The rate of p-ERK1+2/ERK1+2 and p-ERK5/ERK5 on SNCG gene silencing HEC-1A cells were lower than that of empty vector transfection cells,while the rate of p-JNK1+2+3/JNK1+2+3 and p-P38/P38 were higher,the difference were statistically significant(P<0.05).There were no significant difference in the expression of ERK1+2 ?JNK1+2+3?P38 or ERK5 protein between SNCG gene silencing xenograft tumor and empty vector transfection xenograft tumor(P>0.05).The protein expression of p-ERK1+2 and p-ERK5 on SNCG gene silencing xenograft tumor were lower than that of empty vector transfection xenograft tumor,while the protein expression of p-JNK1+2+3 and p-P38 were higher,the difference were statistically significant(P<0.05).The rate of p-ERK1+2/ERK1+2 and p-ERK5/ ERK5 on SNCG gene silencing xenograft tumor were lower than that of empty vector transfection xenograft tumor,while the rate of p-JNK1+2+3/JNK1+2+3 and p-P38/P38 were higher,the difference were statistically significant(P<0.05).?Conclusion?SNCG gene can activate ERK1/2 and ERK5 signal pathway,while inhibit JNK and P38 signal pathway in endometrial carcinoma.