Influence Of The Expression Of Epidermal Growth Factor Receptor By RNA Interference To Radiosensitivity In Nasopharyngeal Carcinoma Cell

Nasopharyngeal carcinoma (NPC) is a malignant cancer in the nasopharyngeal mucosa, which is high incidence in southern China and Southeast Asia. Although great progress in the investigation of NPC, such as radiotherapy and imaging diagnosis and so on, have been made in the late 90s, the cure rate of NPC has not been improved yet and 5-year survival rate still remained around 50%～60%.Radiotherapy is the most important treatment method for NPC because the majority of NPC is the poorly differentiated and high sensitivity of the radiation, and the primary tumor and neck lymphatic drainage area is included easily in the radiation field. Treatment failure of NPC is mainly because of local relapse and metastasis, the survival rate is decreased obviously. Radiotherapy is a regional treatment.A better local curative effect of radiotherapy not only can improve local contral rate, but also can affect survival rate and metastasis.Radiation therapy is a double-edged sword, it will result in a certain degree of radiation injury about normal tissue inevitably when oncology was cured by radiation therapy. Therefore, how to enhance tumor radiosensitivity is the key to improve the radiation therapy. Reaction of organizations to a certain amount of rays is known as the radiation sensitivity, radiosensitivity of tumor is a very complicated problem, which is decided by organize source, differentiation degree, pathological type and the bed of tumor, anemia and local infection, life index...etc. Radiosensitization is the most active area of research in radiation oncology community. The ideal radiation sensitizer should take advantage of differences between tumor cells and normal cells, which is non-toxic or low toxic for normal tissue when radiosensitization is achieved,. Oncologists have been plagued how to improve the efficacy of radiotherapy, to improve radiation sensitivity.Epidermal growth factor receptor (EGFR) is a sort of receptor tyrosine kinase (RTK), which are widely distributed on the surface of endothelial cell of normal mammal,5×104～10x104 receptors in every cell average. The phosphorylation of itself results in a series of signal cascade amplification and transmission after integrated with corresponding ligand. At last, it affects the expression of intranuclear genes and induces cell proliferation and differentiation. After Hendler had found the overexpression of EGFR in non-small cell lung cancer at the first time, the overexpression or amplification was detected in several solid tumor as breast, colorectal, ovarian, head and neck cancer, cerebroma, carcinoma of bladder and renal carcinoma.Many studies have shown radiation can activate EGFR, produce a series of biological effects and thus affect the efficacy of cancer radiotherapy. It is very complex mechanism that EGFR decrease radiosensitivity of tumor cells, maybe reasons as follow:1. EGFR can inhibit radiation-induced apoptosis of tumor cells; 2. radiation therapy-induced repopulation of tumor cells dependent on the EGFR; 3. That irradiation activate EGFR make its downstream gene express highly so that cell proliferation accelerate; 4. irradiation activate EGFR may induce tumor angiogenesis; 5. EGFR may contribute to tumor cells repair of radiotherapy-induced potentially lethal damage and sublethal injury, causing cells to resist radiation.It is reported that positive expression rates of EGFR as high as 80%～100% in NPC patients, more than 25% of patients of tumor cells EGFR-positive expressing showed poorly differentiated, low local control rate and distant metastasis, and the lower the degree of tumor differentiation, the higher the rate of EGFR expression; the more higher patients with lymph node metastasis. Clinical data have shown it is feasible and effective that EGFR monoclonal antibody—cetuximab combined with different chemotherapy in the treatment of head and neck cancer, but individual differences caused treatment failure as well as the formation of drug resistance still requires to seek to further treatment that EGFR has been looked as a target.RNA interference (RNAi) develops very quickly as a pragmatic molecular biology technology in recent years. That transient or stabile gene silencing be mediated by siRNA has a high specificity and no side effect. In practically, use of RNAi not only provides an economical, fast, and efficient technological means of inhibiting gene expression, but also likely to open up a new idea that gene function determine and gene therapy, etc.In recent years, athymus nude mice have become indispensable experimental animal models in the field of biomedical research. It can maintain their biological characteristics when human tumors are transplanted into immune-deficiency animals, which helps a lot to be used to investigate the drug sensitivity of human tumor. Human malignant tumors were transplanted into nude mice, which have the following characteristics:1. The original tumor histology construction or a variety of functions is remained when tumors were transplanted.2. The original structure of the cancer has disappeared that can reproduce after the organization of the human cancer tissue culture is transplanted in the kinds of nude mice.3. Metastasis could not be found nearly in transplanted tumors.In this study, RNA interference EGFR gene expression vector shRNA-EGFR is chemical synthesis in vitro, the human nasopharyngeal carcinoma cell xenografts were constructed in nude mice. shRNA-EGFR is injected nude mice xenografts by intratumoral injection and radiation sensitivity is observed so that it can provide experimental data and theoretical basis of cancer gene radiation therapy.Objective:To construct the EGFRshRNA expression vector and complete the transfection and identity, and detect the expression of EGFR after tranfection.Methods:1. shRNA-EGFR as well as various controls were transfected into CNE1,CNE2, human nasopharyngeal carcinoma cell line, in vitro via Lipofectamine 2000. There are 4 groups in experiment:The control group (without any treatment),Lipofectamine group,shNC group and shRNA-EGFR group.2. Real-time PCR was used to detect the level of EGFR mRNA.3. Western blotting was used to examine the expression of EGFR protein.Result:1. shRNA-EGFR as well as various controls can be effectively delivered into CNE1 cells via lipofectamine2000. 2. Expression of CNE1 and CNE2 EGFRmRNA was detected by real-time PCR:Expression of CNE1 EGFRmRNA was decreased significantly in shRNA-EGFR group in contrast to the control group,lipofectimne2000 group and shNC group. In statistics analysis, the difference of Expression of CNE1 cells EGFRmRNA was significant between shRNA-EGFR group and shNC group (P<0.001), so was between shRNA-EGFR group and lipofectimne2000 group (P<0.001). Expression of CNE1 cells EGFRmRNA was significant between shRNA-EGFR group and before transefection (P<0.001) Expression of CNE2 EGFRmRNA was decreased significantly in shRNA-EGFR group in contrast to the control group,lipofectimne2000 group and shNC group. In statistics analysis, the difference of Expression of CNE2 cells EGFRmRNA was significant between shRNA-EGFR group and shNC group(P<0.001), so was between shRNA-EGFR group and lipofectimne2000 group (P<0.001). Expression of CNE2 cells EGFRmRNA was significant between shRNA-EGFR group and before transefection (P<0.001).3. Western blotting showed that remarkable downregulation of protein expression in the CNE,CNE2 cells shRNA-EGFR was transfected.Conclusion:1. ShRNA expression vector was constructed and transfected successfully. EGFR expression of CNE1,CNE2 was inhibited by shRNA-EGFR in cells, it provided an experimental basis for application of the next step in human nasopharyngeal carcinoma xenograft.Objective:To study changes of radiosensitivity after RNA interference EGFR in nasopharyngeal carcinoma cell CNE1, CNE2 nude mice xenograft tumor.Method:1. Nasopharyngeal carcinoma in nude mice xenograft model was established.2. Tumors were divided into 4 groups randomly when the tumor size up to 6mm～8mm,8 each group, give the appropriate treatment:The control group (without any treatment), radiotherapy group (tumor was local irradiated by 20Gy), shRNA-EGFR treatment group (shRNA-EGFR plasmid was intratumoral direct injected) and shRNA-EGFR in combination with radiotherapy group (tumor was local irradiated by 20Gy at 24h after shRNA-EGFR plasmid was intratumoral direct injected).3. Tumor volume and weight was measured in athymic nude mice, difference of tumor volume and weight are observed between experimental groups.Results:1. Nasopharyngeal carcinoma xenograft was constructed successfully in nude mice; 2. Change of xenograft tumor volume:There was a significant difference of tumor volume in athymic nude mice between the group of shRNA-EGFR in combination with radiotherapy and the control group, the simple radiotherapy group or shRNA-EGFR group (P<0.01). After mice were sacrificed, tumors were removed and weighed:There was a significant difference of weight in athymic nude mice between the group of shRNA-EGFR in combination with radiotherapy and the control group, the simple radiotherapy group or shRNA-EGFR group (P<0.01).Conclusion:Expression of EGFR was inhibited after application of shRNA-EGFR expression vector in the xenograft tumor cells, in combination with treatment with X-ray irradiation has synergistic anti-tumor effect, it is able to inhibit tumor growth significantly, and shRNA-EGFR can enhance sensitivity of nasopharyngeal carcinoma radiotherapy.