A Preliminary Study On The Role And Mechanism Of Junctional Adhesion Molecule B (JAM-2) In Breast Cancer

PART?.STUDY OF JAM2 EXPRESSION IN BREAST CANCER TISSUES AND ITS CLINICAL RELEVANCEObjective: In this study we explored the expression levels of JAM2 in multiple databases,clinical specimens and breast cancer cell lines and analyzed the diagnostic and prognostic value of its expression in breast cancer and explored whether JAM2 expression receives regulation by DNA methylation.Methods: We explored JAM2 expression in breast cancer in multiple databases including CCLE,GTEx,METABRIC and TCGA and analyzed its diagnostic and prognostic value in breast cancer and verified the stratification effect of JAM2 expression on overall survival of breast cancer patients using META analysis.Five pairs of extracted breast cancer and paired paracancer samples were validated using western blot to detect differences in JAM2 protein expression between the two groups of samples.Changes in JAM2 m RNA levels were detected using RT-q PCR after specific inhibition of DNA methyltransferase using 5-azacytidine in breast cancer cell lines.Results: 1.With the dual validation of database and clinical samples,we found that JAM2 expression was significantly decreased in breast cancer tissues,and the area under the curve(AUC)associated with JAM2 expression in normal and breast cancer tissues was analyzed by two algorithms,respectively,with AUCs of 0.929 and 0.887 for the TCGA cohort by logistic regression and random forest algorithm,indicating that JAM2 has good clinical diagnostic value,and the m RNA expression of JAM2 was an independent prognostic factor for breast cancer by combining univariate and multifactorial analysis after clinical indices(HR=1.88,P=0.004).2.Pearson correlation analysis of JAM2 m RNA levels and three DNA methyltransferases showed that the expression of JAM2 was significantly negatively correlated with the expression of DNMT1(r =-0.12,P < 0.01),DNMT3B(r =-0.14,P < 0.01)and EHMT2(r =-0.19,P < 0.01),so we used 5-azacytidine to specifically inhibit DNA methyltransferases in breast cancer cell lines MCF7 and MDA-MB231,respectively.There was a significant increase in JAM2 m RNA levels in the JAM2-treated group compared with the control group,indicating that JAM2 low expression in breast cancer received regulation of DNA methylation.Conclusions: 1.JAM2 was significantly downregulated in human breast cancer compared with normal tissue and likely received regulation by DNA methylation.2.JAM2 m RNA expression was an independent prognostic factor in breast cancer by univariate and multifactorial analysis(HR=1.88,P=0.004)and was validated by Meta-analysis in several databases,suggesting that JAM2 could be used as an independent predictor of prognosis.PART? STUDY OF THE FUNCTIONS AND MECHANISMS ASSOCIATED WITH JAM2 IN BREAST CANCERObjective: To investigate the role and mechanism of JAM2 in breast cancer on breast cancer cell invasion,migration and chemotherapeutic drug sensitivity.Methods: RT-q PCR and Western Blot techniques were used to detect the changes of related markers at molecular and protein levels,scratch assay and Transwall to detect cell migration and invasion,flow cytometry to detect cell cycle and apoptosis,and CCK-8 assay to detect cell viability and chemotherapeutic drug sensitivity.RNA-seq data of JAM2 overexpression were analyzed by gene microarray and bioinformatics methods,and we investigated the potential effect of JAM2 on the tumor microenvironment by using machine learning algorithms for drug sensitivity prediction and finally by using liquid phase microarray technology.Results: In the tumor database,JAM2 expression was found to be significantly lower in distant recurrent tumors than in primary focal tumors.Scratch assay showed that JAM2 overexpression significantly inhibited cell migration of MB231,BT549,MCF7 and T47 D,and the effect of JAM2 overexpression on the invasion and migration ability of breast cancer cells was further verified in the subsequent Transwall assay.Western Blot results also showed that EMT-related protein Vimentin,EZH2 was decreased after JAM2 overexpression.Followed by an increase in the level of EMT process inhibitor protein E-cadherin protein.By the combination of RNA-seq sequencing and machine learning algorithm,we predicted the chemotherapeutic drugs that might be sensitized after treatment with JAM2 overexpression,and we found that JAM2 overexpression could sensitize breast cancer cells to Doxorubicin by experimental validation,and we found in the follow-up experiments that this might be related to the fact that JAM2 overexpression could increase BAX and inhibit BCL-2 thus leading to apoptosis of breast cancer cells.Then we found that JAM2 could inhibit FN1 and inhibit the process of EMT by increasing the expression of ATF3 through Western Blot experiments.Finally,by liquid phase microarray technology,we found more tumor chemokines CXCL9/CXCL10 in the cell supernatant of JAM2 overexpressing cell lines compared to the null control,so this could be a potential mechanism for JAM2 to affect the tumor immune microenvironment.Conclusions: 1.JAM2 inhibits invasion and migration of breast cancer cells by suppressing EMT-related pathways;2.JAM2 overexpression increases BAX and inhibits BCL-2 leading to apoptosis in breast cancer cells,thereby sensitizing MB231 cells to doxorubicin;3.JAM2 inhibits FN1 by increasing ATF3 expression,and JAM2 can increase the expression of chemokines CXCL9/CXCL10,thus affecting the immune microenvironment.PART?.STUDY OF THE RELATIONSHIP BETWEEN JAM2 AND IMMUNE-RELATED RISK SCORESObjective: Patients with breast cancer show significant heterogeneity in overall survival.Current assessment models are insufficient to accurately predict patient prognosis and lack models to predict treatment response.We therefore wanted to construct a model that could accurately determine the prognosis and treatment response of breast cancer patients with an immune risk score and to discuss the relationship between JAM2 and the immune risk score.Methods: We evaluated the relationship between various immune cells and breast cancer and confirmed the association between immune infiltration and breast cancer progression.Different bioinformatics and statistical approaches were combined to construct a robust immune infiltration-associated gene tag for predicting prognosis and response to immunotherapy and chemotherapy.In addition,a new nomogram was constructed based on gene tags and clinicopathological features to improve risk stratification and to quantify risk assessment in individual patients.RESULTS: We constructed an immune infiltration-associated gene tag consisting of 15 genes.Patients' immune infiltration-related risk score(IRS)can be assessed based on gene expression.higher IRS indicates poorer prognosis and less sensitivity to immunotherapy.However,several chemotherapeutic agents are predicted to be more promising for patients with higher IRS.We also found a significant negative correlation between JAM2 and IRS,suggesting that patients with high JAM2 expression have a better prognosis and better sensitivity to immunotherapy.Finally,we constructed a new nomogram map containing clinicopathological features,IRS and JAM2 to improve personalized management and precise treatment of breast cancer patients.Conclusions: Our study may help to optimize survival risk stratification and personalized management of breast cancer.