Expression And Biological Roles Of MIF And CD74 In Cervical Squamous Cell Carcinoma

Cervical cancer is the second most common malignant tumor in women worldwide, and it is the most common carcinoma of genital tract of women in our courtry. Although cervical carcinogenesis requires a long time to transform, and experiences the course of precancerous lesions, there would be a considerable patients at early-stage to be die because of invasion and metastasis. Angiogenesis runs throughout the most part processes of tumor pathogenesis and development. Studies have demonstrated that angiogenesis is an independent prognostic factor in cervical cancer. Thus, it would be very necessary to explore the relative regulatory factors of angiogenesis and carcinogenesis and development of cervical carcinoma.MIF (macrophage migration inhibitory factor) is a cytokine, which can inhibit the random migration of macrophages. MIF has multiple biological functions, including catalytic activity, lymph immune, endocrine regulation, pro-inflammatory action and so on. Normally, content of MIF in human serum is low. In addition to its important roles in immune and inflammatory response, it also plays an important role in cell proliferation, differentiation, angiogenesis and tumor invasion and metastasis. Overexpression of MIF has been found in many kinds of carcinoma. The possible mechanisms of MIF involves in carcinogenesis and development include inactivation of tumor suppression gene p53, inhibition Rb, promotion angiogenesis. MIF secreted from infiltrative lymphocytes can increase the production of MMP-9 and interleukin-8, and promote the invasion and metastasis of nasopharyngeal cancer cells. Moreover, MIF secreted by tumor cells may increase the secretion of VEGF and IL-8, and promote the angiogenesis and tumor growth.CD74 is a transmembrane glycoprotein, which assists in MHC classⅡrelated exogenous antigen presentation. CD74 is a high affinity receptor for MIF. In addition to its functions in immune system, recent evidence demonstrated that CD74 protein is up-regulated in multiple cancer cells, indicating its roles in tumorigenesis and angiogenesis One study concluded that CD74 is a useful prognostic indicator for pancreatic cancer treated with multimodal therapy. However, the detailed function of CD74 in tumorigenesis remains poorly understood. There are lagre quantity of task to explain it.In present, the mechanism of MIF and CD74 in cervical cancer remains unclear. Examining the expression and localization of CD74 and its ligand, MIF in cervical cancer tissues and evaluated the possible biological roles including angiogenesis and cell proliferation on the cervical cancer cells. To approach their roles in angiogenesis, carcinogenesis and progression in cervical cancer, so as to provide experimental evidence on searching a novel therapeutic targets of cervical carcinomaObjective:To explore the expression of MIF and CD74 in cervical squamous cell carcinoma and the possible roles on the angiogenesis, proliferation, invasion of cervical cancer cells.Methods:MIF and CD74 expression were assessed by immunohistochemistry in normal cervical tissues, cervical intraepithelial neoplasia (CINs) and cervical squamous cell carcinoma(SCC) The correlations of MIF and CD74 with clinicopathologicla factors in SCC are also evaluated; CD34 staining was used for counting microvessel density (MVD) in different cervical tissues and we also evaluated the relationship of MVD and MIF/CD74 in SCC. Semi-quantitative RT-PCR and Western blot were used to detect mRNA and protein levels of MIF and CD74 in fresh normal and malignant cervical tissues and cervical squamous cancer cell lines SiHa and C-33A. Immunocytochemistry was used to detect the expression of MIF and CD74 proteins in SiHa and C-33A cells. The mRNA level and protein concentration of vascular endothelial growth factor (VEGF) and IL-8 in the conditioned media of cervical cancer cells treated with rhMIF, MIF-specific inhibitor-ISO-1 and anti-CD74 monoclonal antibody was analyzed by RT-PCR and ELISA, respectively. Cell proliferation of cells treated with rhMIF and anti-CD74 monoclonal antibody was evaluated with CCK-8 kit. Cell scratch method and Boyden chamber detected the influences of rhMIF and anti-CD74 monoclonal antibody on the migration and invasion in SiHa cells.Results:1. The expression rates of both MIF and CD74 were gradually increased in parallel with cervical epithelial lesions severity; MIF and CD74 were primarily localized in the cytoplasm and occasionally on the membrane or nuclei of cervical epithelial cells; MIF expression was correlated with the depth of stromal infiltration (χ2 test, P=0.023). However, no statistically significant correlation was found between CD74 expression and any clinicopathological variables.2. CD34 stainting can clearly distinguish the blood vessles and MVD amount was significantly increased with the gradual progression of cervical lesions; Correlation analysis demonstrated the positive relationship of MIF expression with CD74, MIF/CD74 expression with MVD amount in cervical cancer tissues.3. The expression of MIF and CD74 at mRNA and protein levels in fresh snap-frozen cervical cancer specimens was higher than adjacent normal cervix using RT-PCR and Western blot. Protein and mRNA levels of MIF and CD74 were abundantly expressed in cervical squamous cells in vitro.4. Immunocytochemistry detected that MIF and CD74 proteins were expressed in SiHa and C-33A cells and they mainly localized in the cytoplasm, occasionally on the membranes.5. CCK-8 was used to detect the cell proliferation, which was increased treated with rhMIF in dose-time dependent manner, and the effect could be partially inhibited with anti-CD74 monoclonal antibody.6. Stimulating SiHa and C-33A cells with rhMIF significantly promoted the secretion of VEGF and IL-8 in a dose-dependent manner; ISO-1 and anti-CD74 antibody can partially inhibited MIF-induced VEGF and IL-8 production.7. Exogenous MIF increased the migration and invasion of SiHa cell in vitro. However, CD74 don't take part in the migratory and metastatic courses.Conclusions:1. Expression of MIF and CD74 was gradually increased with the severity of cervical cancer, and mRNA and protein expression levels of MIF and CD74 in SCC were significantly higher than adjacent normal tissues. All the results indicate that MIF and CD74 play important roles in carcinogenesis and development in cervical cancer.2. Expression of MIF and CD74 is closely correlated with MVD in cervical cancer tissues. MIF may promote the secretion of VEGF and IL-8, and anti-CD74 mAb can partially inhibited the expression and secretion of pro-angiogenic factors. This demonstrate that MIF and CD74 can promote the angiogenesis of cervical cancer.3. MIF promoted the proliferation of cervical cancer cells in dose-time dependent manner by binding to CD74.4. MIF can increase the abilities to migrate and invade in SiHa cells.