The Establishment Of The MicroRNA Differential Expression Profiles Related To Radioresistence Of The Nasopharyngeal Carcinoma

Background:Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern China with the highest incidence and mortality rate in the world. Radiotherapy is the primary treatment for this disease. Although the radiotherapeutic equipments and technique are improved continuously, the radiotherapeutic efficacy of NPC remains unfavorable. The main reason for the failure of NPC radiotherapy is that there are a certain percentage of radioresisitant cells in the NPC tissue with higher radioresistance, which is a complex process involving a great number of genes, factors and mechanism. The previous studies showed that radioresistance is the results of the increasing expression of the DNA repair protein, affects of cellar hyposia, angiogenesis and autophagy. But so far the exact mechanism has not been clear.MicroRNAs (miRNAs) is a class of endogenous non-coding RNA. Via mechanisms such as binding to target genes mRNA3’ untranslated region (3’UTR), miRNA inhibits translation, which therefore regulates transcription of target genes and thus affect target protein expression. MiRNAs are involved in the regulation of a variety of malignant behaviors of tumor cell, such as proliferation, apoptosis, migration and invasion. Previous studies have demonstrated that miRNA may be involved in the regulation of tumor radiation sensitivity. The screening of differentially expressed miRNAs in radio-resistant cells or tissues and the study of the gene expression mechanism regulated by miRNA may provide us a new inspiration and basis to improve radiotherapy and curative effects of nasopharyngeal carcinoma.Objective:(1) To establish the radio-resistant cell line of the nasopharyngeal carcinoma CNE-2-Rs via exposion to a series of increasing X-ray doses, so as to supply comparable pair cells for investigating the mechanism of radio-resistance.(2) Screening of differentially expressed miRNAs using Illumina Hiseq2000platform. Using bioinformatics methods to analyze differentially expressed miRNAs and their target genes, trying to identify the possible biological functions and pathways.Methods:(1) Parental cell line CNE-2was exposed to a series of increasing X-ray doses and screened out the radio-resistant cell line CNE-2-Rs. Survival rates after exposed to irradiation were measured by CCK-8assays. The relative radio-sensitivities of tumor cells were assessed by standard colony formation assays. The time courses of cell cycle distribution before and after irradiation were investigated by flow cytometry (FCM).(2) Screening of differentially expressed miRNAs using Illumina Hiseq2000platform.Using bioinformatics methods to analyze differentially expressed miRNAs and their target genes trying to identify the possible biological functions and pathways via GO and KEGG analysis.Results:(1) After exposed to irradiation respectively, the survival rate in CNE-2-Rs was higher than that in CNE-2by CCK-8assays. The Multiplication capacity of CNE-2-Rs cells was stronger than that of CNE-2cells. By using the colony forming assay, multi-target single-hit and linear quadratic model, the parameters of CNE-2and CNE-2-Rs were obtained which demonstrated that the radio resistance of CNE-2-Rs cells was stronger than that of CNE-2cells. The CNE-2-Rs subline showed higher percentage of cells in S phase (p<0.05)(2) We identified192differentially expressed miRNAs (107up-regulated,85down-regulated, p<0.05), inwhich50miRNAs Fold change>1(36 up-regulated,14down-regulated, p<0.05). There are21miRNA clusters and/or miRNA families had two or more differentially expressed miRNAs. GO enrichment analysis:function annotations of target genes of the14down-regulated miRNAs enriched in28biological processes (p<0.05) including biopolymer modification, protein modification process, and others. No significant biological process is enriched for the36up-regulated miRNAs. KEGG analysis:target genes of the14down-regulated miRNAs enriched in4pathways (Pathways in cancer, Purine metabolism, Wnt signaling pathway, Axon guidance, q<0.05). No significant enrichment is observed for the target genes of the36up-regulated miRNAs.Conclusions:(1) The application of the increasing X-ray doses is a method through which we can induce the cell line with radio-resistance. Compared with the parental cell line, CEN-2-Rs has a higher radio-resistance.(2) We established the profile of differentially expressed miRNAs related to radio-resistance in nasopharyngeal carcinoma. The radio-resistance may be attributed to several miRNAs in same miRNA cluster/family. Bioinformatics analyses provide us possible biological processes and pathways for further study,such as Wnt singnaling pathway.