The Application Of SiRNA In The Treatment Of Nasopharyngeal Carcinoma By Inhibiting The Expression Of HTERT Gene And VEGF Gene

Nasopharyngeal carcinoma (NPC) is the most common carcinoma of the head-neck in our country. The incidence of NPC in China is the highest of the world. The incidence of NPC in Guangdong, Guangxi, Hunan, Fujian and Jiangxi province is higher than the other provinces' in China. The place NPC arises from is covert and it is difficult to be found. Those places are often neighbor to nose, nasal sinuses and basilaris cranii. The early signs of NPC are multiple. NPC often have early metastases. NPC is easy to be misdiagnosed and missed diagnosed. The maximum age incidence is in the forth decade and the fifth decade. NPC strikes the patients and their family greatly. The researches of the early diagnosis and therapy of NPC are the emphases of the otolaryngologists' researches in our country.The etiology of NPC is not clear up to now. Doctors think it is related with the infection of EB virus, environment pollutions and genetic factors. The major therapy of NPC is radiotherapy. The chemotherapy only use in the patients who isn't sensitive with radiotherapy or the patients in recurrence as the adjunctive therapy, and it also used in the patients of later period as the alleviative treatment. The NPC patients rarely have the chance to be operated, because, the place of NPC arises from is difficulty to reach and NPC's early metastasis. Operation can only be used in some special conditions as a adjunctive therapy. Scientists made many attempts to get better therapeutic effects to the NPC patients especially the patients who are insensitive to the radioactive ray, who are in recurrence and who have residual tumor in recent years. They paid more attention to the biotherapy of NPC such as immunotherapy, gene therapy, anti-vascular therapy, immunomodulator therapy and the therapy of biological response modifier. Although there are many achievements in these areas, it is still difficulty to use in clinic medicine and we still need to work hard to get more efficient therapeutic methods. Now the gene therapies based on the pathogenesis and the biology characters of carcinomas are popular. The application of RNAi in mammal provides a new way of gene therapy of carcinomas.The length of telomere is related to the proliferation and life-span of cells. Telomerase can prevent telomeres shortening with cell division and stabilize the terminal of chromosome. Cells can be immortalized by the activation of telomerase. The study of the relations between telomerase and the carcinomas is helpful to our research. Most of the carcinomas including NPC can be detected with the active of telomerase. And the activity of telomerase in normal tissue and benign tumors is negative. As the activity subunit of telomerase, hTERT gene can be a good target of NPC's gene therapy.VEGF is the only mitogen which can act especially on the endothelial cells. VEGF is related with the recurrence of NPC. The lymph nodes' metastases and distant metastates are also related with it. VEGF and VEGF receptor express highly in most carcinomas. Scientists find there are some relations between the expression of VEGF and the patients' life-span and distant metastases. The carcinoma patients with higher expression of VEGF have the lower life-span and more chances of distant metastases. The NPC patients with higher VEGF expression have more chances of lymph nodes metastases. Now more and more attention is paid to the tumor's anti-bloodvessel newborn therapy. The co-express of VEGF and VEGF receptor and the establishment of paracrine/autocrine mechanism provide new target of the gene therapy of carcinoma. The inhibitation of the interaction of VEGF and VEGF receptor can prevent the newborn of carcinoma bloodvessel, the proliferation and the metastases of carcinomas. Now, more and more researchers of otolaryngology study the relations between VEGF and NPC and they wish it can help to the treatment of NPC.RNAi is a new technique discovered in recent years and it is useful in the researches of gene express, gene control and gene function. It works by the short interference RNAs' specificity silence of the homology genes. The siRNAs combinate with corresponding mRNA and made it degradation, then the translation of mRNA is terminated. RNAi present in every biology. It is economic and works quickly. At the same time, RNAi is of high efficiency and works in high specificity. RNAi can be used to study the function of genes, and it also can be used in carcinomas' gene therapy.Our research will study the gene therapy of NPC by the application of RNA interference by inhibiting the expression of hTERT gene and VEGF gene. We divided our research in three parts. The first, we will observe the RNA interference in NPC. Our objects are to confirm the efficient positions of RNAi of hTERT gene and VEGF gene and observe the result after RNAi. We design the siRNA by software of online web, synthesize the siRNA by transcription in vitro, and instant transfect to CNE-2 cells by liposome.Then we observe the results by semiquantitative RT-PCR, Western Blot, FCM and TRAP to find the efficient RNAi positions of those two genes. We find two efficient RNAi positions of hTERT gene and two of VEGF gene. The levels of mRNA of those two genes were down-regulated by 89%～92% and 80%～95% separately. And the expression of protein of hTERT gene and VEGF gene were down-regulated in different degree too. The efficient RNAi of hTERT gene resulted the cells' detention at G₀～G₁ stage, and different degrees of cell apoptosis were induced by different siRNAs. As the second part of our researches, we constructed three plasmids of siRNA which can work stablely in carcinoma cells with the information from part one. The plasmids were all constructed on the base of pGenesil-1 Vector. pGenesil-hTERT was constructed to silence hTERT gene. pGenesil-VEGF was constructed to silence VEGF gene. pGenesil-hTERT-VEGF was constructed to silence both hTERT gene and VEGF gene. We transfected this plasmids into CNE-2 cells and observe the RNA interference effect of them. These three plasmids were constructed successfully and they can synthesize siRNAs in CNE-2 cells stably. The plasmids we constructed can silence corresponding gene in CNE-2 cells effectively. The last part of our research is the in vivo RNAi test of nude mice. We expected the test in mice can help the application in human body in future. We transplanted CNE-2 cells to nude mice and established the animal model of human NPC. We transfected plasmid pGenesil-hTERT, pGenesil-VEGF and pGenesil-hTERT-VEGF to nude mice respectively. The tumor volume and tumor morphology were observed. The Apoptosis Index was calculated. And we observed the protein express by immunohistochemisty. We found these three plasmids all can restrain the proliferation of the transplanted tumor in nude mice and induced CNE-2 cell's apoptosis. pGenesil-hTERT-VEGF is the most effective plasmid in this three plasmids.At last, we come to some conclusions: (1) The siRNA synthesized in vitro can silence corresponding gene in NPC cells and their are RNAi in NPC. (2) The siRNA of hTERT gene from position 335 to 357 and from position 2324 to 2346 are effective to silence hTERT gene. The siRNA of VEGF gene from position 1130 to 1152 and from position 1504 to 1526 are effective to silence VEGF gene. (3) The effective siRNAs of hTERT gene can slow down the cell growth and restrain the proliferation. It also can down regulate the mRNA level of hTERT gene and depress the express of hTERT protein. It can result the cells detented at G₀～G₁ stage, and induce cell apoptosis. (4) The effective siRNAs of VEGF can down regulate the mRNA level of VEGF gene and depress the express of VEGF protein. But it will not restrain the proliferation of carcimoma cells and will not induce cell apoptosis. (5) We constructed three plasmids by pGenesil-1 vector and they all can silence corresponding genes stably in NPC cells. (6) We established animal model of human nasopharyngeal carcinoma in nude mouse. (7) The in vivo test proved that pGenesil-hTERT, pGenesil-VEGF and pGenesil-hTERT-VEGF all can inhibite the growth of the transplanted tumor in nude mice and induce the apoptosis of cells of NPC. They are all can be used to the gene therapy of nasopharyngeal carcinoma. (8) It is the first time to combinate hTERT gene and VEGF gene to one plasmid--pGenesil-hTERT-VEGF and the plasmid silenced these two genes successfully in the gene therapy of NPC. We had proved the silence effect of pGenesil-hTERT-VEGF is strong than pGenesil-hTERT and pGenesil-VEGF. Our results certified the feasibility and availability of multiple genes combinated therapy. It will provide a new approach to gene therapy of other genes, and it also the biggest innovation of our researches.