Study Of Deoxyribozyme Targeting FOLR1Sensitizing Paclitaxel In Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma is a malignant epithelial cancers.Variety of factors have led to the occurrence and development of nasopharyngeal carcinoma. Southern China is a high incidence of nasopharyngeal carcinoma. At present, the treatment of nasopharyngeal cancer is radiation therapy, supplemented with chemotherapy and traditional Chinese medicine treatment. However,5-year survival of nasopharyngeal carcinoma is about50%. Although the problem of chemotherapy to improve treatment of nasopharyngeal carcinoma and improve survival remains controversial, clinical trial results show that chemotherapy combined with radiotherapy can significantly improve the prognosis of advanced nasopharyngeal carcinoma [1,2], Therefore, chemotherapy is still one of the important means in the comprehensive treatment of nasopharyngeal carcinoma.However, the long-term use of chemotherapy drugs often leads to drug resistance, resulting in the failure of chemotherapy.Lack of specificity of chemotherapy drug, conventional chemotherapy often leads to a larger side effects and serious impact on the quality of life of patients, The study of reversal of drug resistance and the elimination of chemotherapy side effects have important clinical significance.Targeted therapy is one of the better cancer treatment.It avoids the greater toxicity of conventional chemotherapy because of the lack of specificity.Few studies of folate receptor alpha as a new target for therapy in the treatment of nasopharyngeal carcinoma,this article will discuss the clinical significance of the folate receptor alpha as a new target for therapy of nasopharyngeal cancer and the reversal of drug resistance. Part I The aberrant expression of FOLR1and its clinical significance in nasopharyngeal carcinomaObjects:To study the folate receptor a expression in normal nasopharyngeal cells and nasopharyngeal carcinoma cells and to explore its relationship between pathological features and clinical stage.Methods:The expression of folate receptor a is observed in normal nasopharyngeal epithelial cells NP69,three nasopharyngeal carcinoma cells(CNE-1, HNE-2,5-8F),and paclitaxel resistant-nasophar yngeal carcinoma cells (CNE-1/Taxo,HNE-2/Taxol,5-8F/Taxol) by fluorescence quantitative RT-PCR, confocal laser,and Wester n-blot. In order to analyze the relationship between the folate receptor a expression and the clinicopathological features of nasopharyngeal carcinoma Immunohistochemistry was used to detected72cases of nasopharyngeal carcinoma tissues and10cases of normal nasopharyngeal tissues.Results:The expression of folate receptor a is positive in the three nasopharyngeal carcinoma cells, but in NP69normal Nasophary-ngeal epithelial cells is negative. Its positive expression rate was86.11%(68/72) in Nasopharyngeal carcinoma tissues, while the folate receptor a expression in normal nasopharyngeal mucosa is unexpressed (0/10), there is no relationship between folate receptor a expression and gender, age;but the expression in clinical stage III-IV cases is obvious higher than the case of the Ⅰ-Ⅱ period.Conclusions:The folate receptor a is not expressed in normal nasopharyngeal cells and tissues, but it is widely and highly expressed in nasopharyngeal carcinoma cells and tissues, and it is a positive correlation between with the clinical stage, which is laid the foundation for a new target for therapy of nasopharyngeal carcinoma Part II Design and selection of FOLRl10-23deoxyribozymesObjects:todesign,build, andscreeningtargetedthe10-23type deoxyribozyme of the specific FOLR1gene, close, cut and catalytic the mRNA of nasopharyngeal carcinoma FOLR1gene from people and low the protein expression levels of target genes,achieve the molecular enzymes in genge therapy for nasopharyngeal carcinoma and the reversal of drug resistance.Methods:Briefly, a region of10-23model DRz(DRzA, DRzB, DRzC DRzD, DRzE) was designed according to FOLR1mRNA and computer prediction. Realtime-PCR and western blotting were used to detect the effects of DRzs on the expression of (mRNA and protein).Results:Five type of10-23deoxyribozyme (DRzA, DRzB, DRzC, DRzD, DRzE) and a negative control (ODNs) were designed,which were transfected into nasopharyngeal carcinoma paclitaxel-resistant cell (CNE-1/Taxol).24h later, Realtime-PCR and western blotting were used to detect the effects of DRzs.Results show that the strongest inhibition of FOLR1gene is DRzE, ODNs can not inhabit it.Conclusions:At the mRNA and protein levels, DRzE inhibition of target genes is better than other deoxyribozymes,which laid the foundation for the subsequent experiments. Part III The chang of paclitaxel sensitivity in resistant cells after the the expression in FOLR1gene inhibited by deoxyribozyme.Objects:To obseve the chang of paclitaxel sensitivity in resistant cells after the deoxyribozyme targeted inhibition the expression in FOLR1genes.Methods:The expression of FOLR1gene inhibited by DRzE CCK-8assay was observed the changes of the resistant cells (CNE-1/Taxol) to paclitaxel, Annexin flow cytometry was used to detcet cell apoptosis.Results:the expression of FOLR1gene inhibited by DRzE, the sensitivity of CNE-1/Taxol to paclitaxel was significantly enhanced, the sensitivity increased by about44%, while there is no significant changes in the negative control group(ODNs group).it can obvious induce the cells apoptosis by paclitaxel (IC30:5ng/ml),and the apoptosis rate increases from6.09±2.37%to23.19±2.01%.Conclusions:deoxyribozyme DRzE targeting to FOLR1can significantly increase the sensitivity of CNE-1/Taxol to paclitaxel.