The Gene Therapy Of Nasopharyngeal Carcinoma Based On MicroRNA

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in the Southern of China, and it ranks the first in head-neck cancer. The place where NPC originates from is covert and it is difficult to be discovered. The nasopharynx is neighbor to nose cavity, nasal sinuses and encephalic structure, so clinic symptoms appear late, and early signs are multiple. There is the highest incidence of distant metastasis for NPC among head and neck cancer. For the reasons stated above, NPC is easy to be misdiagnosed or fail to be diagnosed, and it's curative effect is not satisfactory. The maximum age incidence is the fouth to fifth decade. It brings great suffering to patient's family and society. It is very important to research early diagnosis and therapy of NPC.Nowaday, the major therapeutic modality of NPC is radiotherapy. The chemotherapy only is used in the patients who aren't sensitive to radiotherapy or the patients who have recurrence tumors after previous radiotherapy, or patients who are found lately. As an adjunctive therapeutic modality, operation only is limited to the special conditions. In recent years, some authors attempt to use photodynamic therapy and microwave hyperthermia therapy to treat NPC, but those methods also are regarded as adjunctive therapy.With the development of DNA recombination, the gene therapy is developed fastly. The gene therapy based on the pathogenesis of carcinoma has become popular. Former researchers mostly used antisense oligodeoxynucleotides (ASODN) to treat NPC, and acquired some achievements. Recently as application of RNA interference (RNAi) in mammal, it provides a new way of gene therapy for carcinoma.Nowaday, siRNA is used as effective molecule to apply to gene therapy for carcinoma. But an other small RNA——microRNA (miRNA) has attracted researcher's great attention. MicroRNA belongs to a class of non-coding RNAs that act as endogenous triggers of the RNA interference pathway. It can induce RISC to cleave the target gene mRNA though the classic RNAi pathway. It also can suppress translation of target gene, and can direct rapid deadenylation of targe mRNAs to lead to a rapid mRNA decay. MiRNA has been regarded as the biggest family of genome regulators.The purpose of this study is to construct recombination plasmids which express precursor of miRNA——pre-miRNA by simulating natural miRNA, and to attempt to regulate the expression of cancer-related genes——hTERT and VEGF, and to determine gene therapy in NPC. The experiment is divided into three sections:Section one: To construct the plasmid which regulates target genes, and t(?) s(?)reen effective plasmid, and to confirm efficient positions in which miRNA direct against hTERT gene and VEGF gene, and to observe regulative effect of candidate plasmid. These studies will provide the foundation for follow-up experiments in vitro and in vivo. For each gene, three candidate plasmids are constructed and one effective vector is picked up, namely: pcDNA6.2-GW/EmGFP-miR-hT2035, pcDNA6.2-GW/EmGFP-miR-V1025. We find that the loci from 2035 to 2055 are effective to regulate hTERT gene, and the loci from 1025 to 1045 are effective to regulate VEGF gene. After the plasmids are transfected CNE-2 cell, the levels of mRNA of hTERT and VEGF gene are down-regulated to 77.3% and 79.3% respectively, the expression of protein of two genes are also down-regulated to 73.2% and 80.2% respectively, miRNA regulation of hTERT gene results in inhibition of tumor cell proliferation and cell detention at G0～G1 stage and cell apoptosis.Section two: To observe inhibitory effect of CNE-2 cell by regulating expression of hTERT and VEGF gene, by chaining miR-hTERT and miR-VEGF, we construct another plasmid: pcDNA6.2-GW/EmGFP-miR- hTERT-VEGF. Four plasmids, including the miRNA plasmid above and pcDNA6.2-GW/EmGFP-miR-hTERT and pcDNA6.2-GW/EmGFP-miR-VEGF and negative control plasmid, are used to transferred to CNE-2 cell line separately, and to generate stable cell lines that constitutively express miRNA by antibiotics selection. The effects of stable transfection of the miRNA plasmids are observed by qRT-PCR, Western blotting, soft agar colonies formation assay. Result find that four cell lines express stably miRNA, and miRNA regulate expression of targe gene in stable cells.Section three: In vivo, the test of nude mice is carried. Four stable cell lines and wild type CNE-2 cell line are inoculated to subcutis of nude mice, animal models are established. The tumor volume and tumor morphology are observed, and the protein expression is detected by immunohistochemistry. Result find that in addition to negative plasmid, all three plasmids can inhibite growth of the transplanted tumor of nude mice.Conclusions:(1)We construct successfully the recombination plasmid vectors which target hTERT gene and VEGF gene; the plasmid vectors which express miRNA can down-regulate the expression of hTERT gene and VEGF gene efficiently; the miRNA of hTERT gene loci from 2035 to 2055 is effective to down-regulate hTERT gene, the miRNA of VEGF gene loci from 1025 to 1045 is effective to down-regulate VEGF gene;(2)In vitro miRNA plasmids of hTERT and of VEGF can regulate effectively expression of hTERT and VEGF genes, and down-regulate the mRNA and protein level of hTERT and VEGF, and detent the CNE-2 cell at G0-G1 stage, and induce cell apoptosis.(3)In vivo, we establish animal model of NPC in nude mice; the result proved that all of three recombination plasmids which regulate hTERT,VEGF,hTERT and VEGF respectively can efficiently inhibit the growth of the transplanted tumor in mude mice. All results prompt that it might be effective for gene therapy of nasopharyngeal carcinoma.