| Nasopharyngeal carcinoma(nasopharyngeal carcinoma,NPC) is a common malignancy in the head and neck with significant regional and ethnic distribution. Southern China regions, including Guangdong, Guangxi, Hunan and Fujian, are among the pandemic areas of nasopharyngeal carcinoma. Pathogenesis of nasopharyngeal carcinoma has not been fully understood, etiology and pathogenesis studies suggested that the causal factors may include EB virus infection, genetic factors, chemical carcinogens, and disturbance in oncogenes and tumor suppressor genes. Due to the specific anatomical structure and position of nasopharyngeal carcinoma, radiation therapy has become the preferred treatment for this disease. In recent years, with the advances in radiotherapy,5-year survival of nasopharyngeal carcinoma patients has greatly improved. However, local recurrence and distant metastasis still impede prognosis of nasopharyngeal carcinoma patients, and limit further improvement in patients’survival. Therefore, it would be of great clinical significance to explore the role of metastasis-related genes in nasopharyngeal carcinoma.KAI1 (Kang Ai 1) gene was first identified by Dong et al in 1995 which specifically inhibit tumor metastasis. KAI1 is located on human chromosome 11p11.2, encoding a 267-amino acid protein, which is a member of the transmembrane 4 super family. TM4SF protein has now been proved to inhibit tumor metastasis through extensive glycosylation of cell membrane proteins and specific localization that enhance cell-cell and cell-matrix adhesion and thus play a significant role in both tumor invasion and metastasis. It is proved that KAI1/CD82 are closely related to metastasis and invasion of a variety of tumors.Endothelial progenitor cells are first isolated from human peripheral blood by Asahara et al in 1997. They are a subpopulation of hematopoietic stem cells involved in angiogenesis, and play a major role in angiogenesis during early embryonic development. Studies have shown that circulating EPCs can be specifically attracted to the tumor microvasculature system, and participate in tumor angiogenesis. EPCs are also very good carriers that allow exogenous genes to be expressed in local organs.In this study, we first determined mRNA and protein expression of KAI1/CD82 in human nasopharyngeal carcinoma tissue and non-neoplasia nasopharyngeal tissue, and preliminarily analyzed its clinical significance in nasopharyngeal carcinoma. Then we established a xenograft mouse model of nasopharyngeal carcinoma, and infected umbilical vein-derived EPCs with KAIl/CD82-expressing lenti-virus to get KAI1/CD82-overexpressing EPCs, which were then injected into the tumor-bearing mice through the tail vein, to observe its effect on tumor size and metastasis. Through these experiments, we seek to clarify the role of KAI1/CD82 in metastasis of nasopharyngeal carcinoma, and to evaluate the clinical value of KAI1/CD82-EPCs in prevention of nasopharyngeal carcinoma. This study mainly includes the following two aspects:Part I Expression of KAI1/CD82 in Nasopharyngeal Carcinoma and Its Clinical SignificanceObjective:To determine KAI1/CD82 protein expression in nasopharyngeal carcinoma and non-neoplastic tissue through S-P immunohistochemistry, and its mRNA expression by RT-PCR, so as to analyze the correlation of abnormal expression in nasopharyngeal carcinoma with age, gender, histological typing, T staging, lymph-node staging of the patients. To provide new ideas for improving the efficacy of nasopharyngeal carcinoma and preventing tumor invasion and metastasis.Method:1. KAI1/CD82 protein expression in 142 cases of nasopharyngeal carcinoma and 50 samples of non-neoplasia nasopharyngeal tissue was determined by S-P immunohistochemistry, and its correlation with clinicopathological indicators of nasopharyngeal carcinoma were analyzed.2. KAI1/CD82 mRNA expression in 68 cases of nasopharyngeal carcinoma and 23 samples of non-neoplasia nasopharyngeal tissue was determined by RT-PCR, and its correlation with clinicopathological indicators of nasopharyngeal carcinoma were analyzed.Results:1. Rate of positive KAI1/CD82 protein expression was 45.1%(64/142) in the nasopharyngeal carcinoma samples, significantly lower than that in non-neoplasia nasopharyngeal tissue (70.0%,35/50) (P=0.002).2. Rate of positive KAI1/CD82 mRNA expression was 45.6%(31/68) in the nasopharyngeal carcinoma samples, significantly lower than that in non-neoplasia nasopharyngeal tissue (73.9%,17/23)(P=0.019).3. KAI1/CD82 protein expression in nasopharyngeal carcinoma showed no significant correlation with age, gender, pathological typing and T staging of the patients, but was significantly correlated with lymph node metastasis (N staging). Rate of KAI1/CD82 expression was significantly lower in patients with lymph node metastasis (NO) than those without [40.2%(49/122) vs.75.0%(15/20), P<0.01]. And KAI1/CD82 protein expression decreased as N staging progressed (NO:75.0%, N1:47.2%, N2:40.6%, N3:27.3%).4. KAI1/CD82 mRNA expression in nasopharyngeal carcinoma showed no significant correlation with age, gender, pathological typing and T staging of the patients, but was significantly correlated with lymph node metastasis (N staging). Rate of KAI1/CD82 expression was significantly lower in patients with lymph node metastasis (NO) than those without [40.0%(24/60) vs.87.5%(7/8), P<0.05]. And KAI1/CD82 protein expression decreased as N staging progressed (NO:87.5%, Nl: 50.0%, N2:40.6%, N3:20.0%).Conclusion:1. KAI1/CD82 is highly expressed in non-neoplasia nasopharyngeal tissues, but lowly expressed or absent in nasopharyngeal carcinoma tissue, suggesting a correlation between KAI1/CD82 expression and occurrence and development of nasopharyngeal carcinoma.2. Low expression of KAI1/CD82 is correlated with lymph-node metastasis of nasopharyngeal carcinoma, suggesting an inhibitory effect of KAI1/CD82 on lymph-node metastasis of nasopharyngeal carcinoma.Part II Construction of a Xenograft Mouse Model of Human Nasopharyngeal Carcinoma and The Effect of KAI1/CD82-expressing EPCs on Lung Metastasis of This Xenograft Mouse ModelObjective:1. To clarify the role of KAI1/CD82 in metastasis of nasopharyngeal carcinoma2. To evaluate the clinical efficacy of KAI1/CD82-expressing EPCs in prevention of nasopharyngeal carcinomaMethod:1. Umbilical vein-derived EPCs were infected with KAI1/CD82-expressing lenti-virus to get a KAI1/CD82-overexpressing EPC cell line (KAI1/CD82-EPC)2. A xenograft mouse model of human nasopharyngeal carcinoma was established, and KAI1/CD82-EPCs were injected through the tail vein.3. The effect of the KAIl/CD82-EPCs on growth and metastasis of the xenograft was observed.Results:1. KAI1/CD82 gene was successfully transfected into the EPCs, which was confirmed by western blot.2. The xenograft mouse model of human nasopharyngeal carcinoma was successfully established.3. Time required for tumor formation was 14.70±3.81,15.05±3.85,14.20±3.55 days respectively for the EPC, EPC-NC, and KAI1/CD82-EPC groups, with no significant difference among the three groups.4. Weight of the xenograft was 1.388±0.204g,1.487±0.223g,1.485±0.234g respectively for the EPC, EPC-NC, and KAI1/CD82-EPC groups, with no significant difference (P=0.274).5. Rate of lung metastasis was 55%,45% and 10% for the EPC, EPC-NC, and KAI1/CD82-EPC groups, and the difference was significant (P=0.005). Number of metastatic lesions was 34.27±5.35,38.44±9.63,17,50±3.54 for the three groups, and the difference was also significant (P=0.007).6. Immunohistochemistry indicated positive KAI1/CD82 expression in metastatic lesion of the KAI1/CD82-EPC group, but no KAI1/CD82 expression in the EPC group or EPC-NC group.Conclusion:1. A stable xenograft mouse model of human nasopharyngeal carcinoma was successfully established.2. KAI1/CD82-expressing EPCs inhibits lung metastasis of the xenograft mouse model of human nasopharyngeal carcinoma. |
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