The Expressions Of FOXN1and CXCL13in Human Thymic Tissue And RNAi-related Research

Objectives:Thymoma is the most common tumor in mediastinum, with multiple histological subtype and a strong heterogeneity. Each subtype of thymoma could have the ability to invading adjacent tissue, even metastasizing. Although the pathogenesis is long and the therapeutic effect is not bad, thymoma is still a malignant tumor. Further, They associate more often than any other human tumors with various autoimmune diseases; myasthenia gravis is the commonest, occurring in10-50%of thymoma patients. FOXN1(as a transcription factor) and CXCL13(as a chemokine) are newly discovered, and relating with thymoma and myasthenia gravis (MG). FOXN1is mainly expressed in thymic epithelial cells and always positive in thymoma and thymic carcinoma. But the relation between FOXN1and thymoma is not clear. CXCL13is reported having relationship with some autoimmune diseases and tumor. But there is no report about thymoma. The aims of this research are to evaluate the expressions of FOXN1and CXCL13and their association with thymoma and to investigate the effect and mechanism of RNA interference on FOXN1/CXCL13expression in transfected thymic Thy0517cells.Methods and Results:In the first part, we firstly compare the changes of serum immunologic factor and T lymphocyte subtype groups in different thymic-related disease. We find that the concentrations of IgG, IgE and Oreactive protein among thymoma with MG and thymoma without MG patients. The helper T cells become over-activted and the suppressor T cells less, indicating the relationship of this changes with MG and the different mechanism between thymic hyperplasia and thymoma in MG. After this, immunohistochemistry and real-time PCR were used to detect FOXN1and CXCL13in MG/thymoma related and normal thymic tissue(n=88). The expressions of FOXN1and CXCL13in thymoma with and without MG are much higher than in normal and MG hyperplasia thymic tissue(P<0.05), indicating FOXN1and CXCL13may be marker for thymoma or participate in the development of thymoma. In the second part, western blotting and real-time PCR were used to compare the expression of FOXN1and CXCL13in human normal thymic epithelial cells (CRL7660) and human thymoma cells (Thy0517). The expression of FOXN1and CXCL13is much higher in Thy0517than in CRL7660and then Thy0517was chosen to do the next RNAi. The RNAi plasmid for FOXN1and CXCL13gene were constructed, Thy0517cells transfected with the plasmids, western blotting and real-time PCR were used to determine the mRNA and protein levels of FOXN1and CXCL13and MTT test was used to test the proliferative ability of Thy0517cells after RNAi. DNA sequencing confirmed the plasmid for FOXN1and CXCL13were successfully constructed. Real-time PCR and western blot assay showed that in FOXNl RNAi cells, the expressions of FOXNl and CXCL13were both lower than control cells while only the expression of CXCL13was lower than control cells in CXCL13RNAi cells. MTT test showed that the proliferative ability of FOXN1RNAi cells and CXCL13RNAi cells was significantly reduced with48h after transfection. These results showed that FOXN1is an upstream gene and regulating the expression of CXCL13gene and both two gene can promote the proliferation of thymoma cells and this action is partial.Conclusions:The expression of FOXN1and CXCL13are much higher in thymoma and thymoma cell line than normal thymic tissue and normal TECs, indicating FOXN1and CXCL13as a biomarker of thymoma or participating in the mechanism of thymoma. The later RNAi showed that the down-regulation of FOXNl and CXCL13could equally inhibit the ability of proliferating of thymoma cells, and confirming FOXN1is the upstream control gene of CXCL13. All these provide the in vitro evidence for further research of down-regulating the expression of FOXN1and CXCL13in vivo.