Study On The Effect And Mechanism Of MAGE-A3 Gene On The Proliferation,Invasion And Migration Of Cervical Cancer

Background and objectiveAlthough most of the causes of cervical cancer are high-risk HPV infection,not all of the infected people will have canceration,and the mechanism of its occurrence and development is still unclear.At present,the median total survival time of advanced cervical cancer is only 16.8 months,and the total 5-year survival rate of all stages of development is 68%.Invasion and metastasis are closely related to poor prognosis and are the main causes of death.Reducing the incidence rate and mortality of cervical cancer has been the focus of scholars in various countries.Therefore,it is very important to find the tumor specific antigen and related antigen of cervical cancer in clinic,which is of great significance to understand the occurrence,development and prognosis of cervical cancer,as well as lay the foundation for the individual treatment and biological targeted treatment of advanced cervical cancer.In our previous research,we searched the gene expression omnibus,We found that among the genes highly expressed in cervical cancer,the gene of melanoma associated antigen A3(MAGE-A3)was not expressed in other normal tissues except placenta and testis,but it was highly expressed in a variety of malignant tumors.To test our hypothesis,we searched the Cancer Genome Atlas(TCGA)database and tissue genome expression,GTEX)database to predict the relationship between MAGE-A3 gene and the prognosis of cervical cancer;in order to study the relationship between MAGE-A3 gene and the occurrence and development of cervical cancer,we detected the expression of MAGE-A3 in cervical squamous intraepithelial lesions,cervical cancer tissues and normal cervical tissues.The expression of MAGE-A3 in cervical cancer cell line was detected.Then the overexpression of MAGE-A3 cell line was constructed by transfecting cervical cancer cells with overexpression plasmid,and MAGE-A3 silent cell line was constructed by siRNA transfection.A series of cell function experiments were carried out to observe whether the expression level of MAGE-A3 had an impact on the proliferation,migration and invasion of cervical cancer cells.In addition,we further explored the possible mechanism of MAGE-A3 affecting the pathogenesis of cervical cancer,and verified the results of in vitro cytology experiment by constructing the tumor forming experiment of immunodeficient mice in vivo.Methods:Chapter 1,The expression of MAGE-A3 in 40 cases of cervical squamous intraepithelial lesions(CIN?),90 cases of cervical cancer and 40 cases of normal cervical tissue was detected by immunohistochemistry,PCR and Western blot,and the relationship between MAGE-A3 expression and clinicopathological characteristics and prognosis was analyzed.Chapter 2,The human cervical epithelial immortalized cell line end/E6E7 and cervical cancer cell lines HeLa,SiHa and c33a were selected as the research objects.The expression of MAGE-A3 in cervical cancer cell lines was detected by qRT-PCR and Western blot.Chapter3,the overexpression cell line of MAGE-A3 was constructed by pcDNA3.1-MAGE-A3 transfection.The silencing cell line of MAGE-A3 was constructed by si-MAGE-A3 transfection.The transfection effect was identified by QR-PCR and Western blot.The effects of MAGE-A3 on the proliferation of cervical cancer cells was observed by CCK8 and Plate cloning test.The effect of MAGE-A3 on invasion and migration of cervical cancer cells was observed by scratch test and Transwell test.Chapter4,In the overexpression and silencing cell lines of MAGE-A3,QRT-PCR and Western blot were used to detect the effect of MAGE-A3 on EMT markers in cervical cancer cells.The expression of Wnt pathway related proteins was detected in MAGE-A3 overexpression and silencing cell lines,respectively.Chapter5,HeLa cells,SiHa cells and transfected cells were transplanted into the subcutaneous of nude mice.The tumor volume was measured every 7 days after injection.After 28 days of injection,the tumor was isolated from the nude mice and the tumor volume was observed.The expression of EMT markers was detected by immunohistochemistry.Results:Chapterl,Compared with normal cervical tissue,MAGE-A3 was highly expressed in cervical cancer;the overall survival rate of MAGE-A3 in the high expression group was lower than that in the low expression group,and was related to clinical stage,grade,lymph node metastasis and HPV infection,with significant difference(P<0.01).Chapter2,Compared with end1/E6E7,MAGE-A3 was highly expressed in cervical cancer cells,the difference was statistically significant(P<0.05),and the expression of MAGE-A3 was the highest in HeLa and the lowest in SiHa.Chapter3,pcDNA3.1-MAGE-A3 transfected SiHa cells significantly up regulated the expression of MAGE-A3.Si-MAGE-A3 transfected HeLa cells effectively silenced the expression of MAGE-A3.The results of CCK8 assay showed that the OD value of HeLa cells transfected with si-MAGE-A3 was significantly lower at 48h,72h and 96h than that of si-con group.The number of clones in si-MAGE-A3 group was also less than that in si-con group.The OD value of overexpressed MAGE-A3 SiHa cells was significantly higher than that of the control group.Compared with the control group,the overexpression of MAGE-A3 in SiHa cells significantly increased the number of clones.Chapter4,After down regulating MAGE-A3 in HeLa cells,the protein expression level of N-cadherin and vimentin decreased,and E-cadherin increased significantly.while after up regulating MAGE-A3 in SiHa cells,the protein expression level of N-cadherin and vimentin increased,and E-cadherin decreased significantly.The downregulation of MAGE-A3 significantly reduced the expression of ?-Catenin,c-myc and CyclinD1 in HeLa cells.Overexpression of MAGE-A3 significantly increased the levels of ?-Catenin,c-myc and CyclinDl in SiHa cells.Chapter5,The tumor growth rate of si-con group was faster than that of si-MAGE-A3 group,and the tumor growth rate of SiHa cell MAGE-A3 group was faster than that of vector group(P<0.05).In the tumor tissues of silenced MAGE-A3 group,the expression of E-cadherin increased,and vimentin protein decreased.In the overexpression MAGE-A3 group,the expression of E-cadherin protein decreased,and vimentin protein increased.Conclusion:MAGE-A3 gene is involved in the development of cervical cancer,which is highly expressed in cervical cancer tissues and cells,and negatively related to the overall survival of patients,indicating that the expression of MAGE-A3 can indicate the prognosis of patients.it can promote the proliferation,invasion and migration of cervical cancer cells,and may promote EMT and affect the biological behavior of cervical cancer cells by activating Wnt signaling pathway.It may be able to pass through in vivo Hyperemt regulates the growth of cervical cancer.MAGE-A3 gene is up-regulated in cervical cancer cells and may regulate Wnt signaling pathway and EMT process.The overexpression of MAGE-A3 in cervical cancer may be involved in the process of cervical cancer,which is of great clinical value for further understanding the biological behavior and prognosis of cervical cancer.