The Role Of BCAP31 In The Pathogenesis Of Cervical Cancer And The Underlying Molecular Mechanism

Background: Cervical cancer is one of the most common malignant tumor in gynecology,the incidence of which is increasing year by year,and there is a tendency to young people for women patients.At present,the pathogenesis of cervical cancer is still unclear,and the clinical treatment is mainly based on surgical resection and chemotherapy,which brings great harm to cervical cancer patients,especially the young women.Therefore,the early diagnosis,prevention,reasonable and effective treatment of cervical cancer are of great importance.Until now,it is widely accepted that there is a close link between cervical cancer and persistent infection caused by Human Papillomavirus(HPV).HPV infection can activate the downstream mechanisms,leading to the replication and infection of HPV virus and the malignant proliferation of cervical epithelial cells,which is a major cause of cervical cancer.However,the key molecules and downstream signaling pathways regulated by HPV infection are obscure,limiting the timely early screening and the development and application of targeted biological therapy of cervical cancer.Therefore,itis of great importance to analyze the critical mechanisms after HPV infection,and to search for efficient and specific target molecules for the diagnosis and targeted therapy of cervical cancer.BCAP31 is an important member of the B cell receptor related protein family,mainly located in the endoplasmic reticulum,and responsible for the transport of a variety of proteins to the Golgi apparatus.BCAP31,as a carrier protein,plays an important role in the transportion of membrane proteins,the identification and degradation of misfolded proteins,and the regulation of Caspase dependent apoptosis.Our group previously had identified the BCAP31 as a novel cancer-testis antigen by using the original method of spermatogenic cells specific monoclonal antibody-defined cancer/testis antigen(SADA),immunoprecipitation,and mass spectrometry analysis.After analysis of the protein expression pattern by tissue microarray,we found that BCAP31 is not expressed in human normal tissues(except testis tissue),but highly expressed in the tumor tissues of cervical,ovarian,lung,breast,liver,esophagus,colon and rectum.Especially in cervical cancer tissues,the expression intensity and positive rate of BCAP31 was up to 87.5%,suggesting that BCAP31 may act as a new cancer-testis antigens involving in the development of cervical cancer.In this paper,we firstly verified the relationship between BCAP31 and development and metastasis of cervical cancer,and further explore the regulated functions of BCAP31 on the proliferation,migration,and invasion of cervical epithelial cells which give rise to the progression of cervical cancer and the underlying molecular mechanism.This research will deepen the understanding of the pathogenesis of cervical cancer,and might provide a new target for the efficient treatment of cervical cancer.Objectives: 1.To evaluate the expression of BCAP31 in cervical cancer tissues,and analyze the correlation between its expression level and the occurrence,development,and metastasis of cervical cancer;2.To investigate the effect of BCAP31 on the biological behavior of cervical cancer cell lines;3.To study the possible molecular mechanism of BCAP31 on the biological behavior ofcervical cancer cells,and to clarify the important role of BCAP31 in the development of cervical cancer.Methods: 1.Collection of different pathological grading of cervical cancer tissue samples and the clinical data;Western blot,Real-time PCR and immunohistochemical staining are employed to analyze the expression of BCAP31 in cervical cancer tissue;Statistically analysis of the correlation between the expression of BCAP31 and the tumor size,clinical stage,pathological grading,occurrence of distant metastasis,survival of patients,and other clinical indicators;2.BCAP31 specific si RNA was transfected to evaluate the effect of BCAP31 on the biological behavior of cervical cancer cells,such as cell growth,cell cycle,apoptosis,migration,and invasion;3.A stable hela cell line with low expression of BCAP31 was constructed for the xenograft model in nude mice.And tumor size,tumor metastasis,and survival of mice were observed to value influence of BCAP31 on the occurrence and development of cervical cancer;4.Co-precipitation and laser confocal method were used to identify the targeted molecules interacting with BCAP31,and the changes of downstream molecules and pathways were analyzed to reveal the mechanism of BCAP31 in the occurrence and development of cervical cancer.Result: 1.Real-time PCR and Western blot analysis showed that BCAP31 was significantly higher in cervical cancer with tumor proliferation,while was only slightly expressed in the surrounding normal tissue;2.The immunohistochemistry staining of 161 cases of cervical cancer tissues revealed that 135 cases showed high expression of BCAP31,accounting for 83.6% of the total cases;the correlation analysis indicated that the high expression of BCAP31 was closely related with the lymph node metastasis,tumor differentiation,and clinical stage of cervical cancer;3.Kaplan-Meier survival analysis showed that the survival time of patients with high expression of BCAP31 was significantly shorter;4.The study of cell function in vitro showed that interference the expression of BCAP31 in cervical cancer cell line significantly inhibited the proliferation,migration,and invasion ability of cervical cancer cell line;5.Western blot results and cycle analysis showed that interference the expression of BCAP31 in cervical cancer cell line dramatically inhibit the expression of cyclin Cyclin D,Cyclin E and Cyclin E2,meanwhile the cells were arrest in G1 phase;6.Xenograft mouse model were constructed with stable BCAP31-interfered Hela cells and the down-regulation of BCAP31 expression decreased the tumorigenic ability of Hela cells,prolonged the mouse survival and inhibited the liver metastasis in this model.7.The results of immunoprecipitation and Western blots showed that BCAP31 could regulate the expressions and subcellular localizations of Drebrin,M-RIP,SPECC1 and Nexilin;8.Further functional analysis showed that BCAP31 could regulate the expression of Drebrin and its subcellular localization to affect the assembly of F-actin inner the cell,thus regulating the invasion and migration of cervical cancer cells.Conclusion: In this study,we for the first time defined the association between the cancer-testis antigen BCAP31 and the occurrence and development of cervical cancer,reveals the regulated functions of BCAP31 in proliferation,migration,and invasion of cervical cancer cell and the molecular mechanism,and further enriches the study of the pathogenesis of cervical cancer.At the same time,interference targeting BCAP31 may be a new strategy for the treatment of cervical cancer.