Screening And Localization Of Neck Lymph Node Metastasis-ralated Genes In Nasopharyngeal Carcinoma

BackgroundNasopharyngeal carcinoma (NPC) is the most common head and neck cancer in southern China. The primary site of NPC is covert. It can not be found early and has a high malignant degree. Radiotherapy (RT) is the primary treatment for NPC. Although during the past decades the recovery rate of NPC has been increasing with the improvement of radiotherapy technology, metastasis is the biggest difficulty of curing NPC because most NPC patients had neck lymph nodes metastasis even distant metastasis at the first diagnosis. The mechanism of NPC early tissue infiltration and lymph node metastasis is still unclear. Contemporary international and domestic academics have realized that the occurrence and development of tumor is a complex process with multiple factors, genes and stages involved in. Many genetic studies have been carried out in order to explore the molecular biological mechanisms of NPC and to seek the breakthrough for the prevention and cure of tumor. In recent years a number of epigenetic studies have identified that gene methylation is associated with tumor, and more and more genes have been found to play important roles in the pathogenesis of NPC. However, the study of single gene methylation is difficult to reflect the macroscopic changes of epigenetics in NPC. High-throughput genome-wide methylation and expression profiling arrays have provided important technical support for multi-gene study. In this study, we use methylation and expression profiling arrays to screen the neck lymph node metastasis-ralated genes of NPC and to seek the ideal genetic markers for NPC so that it can provide new therapeutic target for gene therapy of NPC.Chapter Ⅰ The Preliminary Construction of Neck Lymph Node Metastasis-related Genes Methylation Profiles of Nasopharyngeal CarcinomaObjective:NimbleGen Human DNA Methylation Arrary was used to screen abnormal methylated genes in NPC and their relevance with neck lymph node metastasis in this study. Furthermore, we want to construct the neck lymph node metastasis-related gene methylation profiles in NPC and provide a theoretical basis for further study in the mechanism of its invasion and metastasis.Methods:Four paraffin specimens of stage Ⅰ NPC patients and another four fresh specimens of NPC patients with neck lymph node metastasis were collected. Genomic DNA (gDNA) were extracted from these eight tissue samples. The purified gDNA was quantified and quality assessed. The gDNA was sonicated to 200～1000bp with a Bioruptor sonicator before immunoprecipitation. The MeDIP-enriched DNA was amplified, purified and then labeled. The labeled DNA was used for array hybridization with NimbleGen Human DNA Methylation Arrary. Arrary hybridization signals were detected by using high-resolution microarray scanner. Several softwares such as GenePix, NimbleScan, Bioconductor packages Ringo, MEDME and SignalMap were used for data extraction, standardization, peak analysis and report.Results:All DNA samples passed the quality assessment, including Genomic DNA, Sheared DNA, MeDIP fold-enrichment, and labeled DNA.1134 methylated genes were found in all the four paraffin specimens of stage I NPC patients,835 in all the four fresh specimens of NPC patients with neck lymph node metastasis and 396 in all the 8 specimens.33 differential methylated genes located on 17 chromosomes were found in all the four fresh specimens of NPC patients with neck lymph node metastasis but negative in all the stage I NPC patients. In addition,153,658 and 2340 differential methylated genes were found in three of, two of, and one of the four fresh specimens of NPC patients with neck lymph node metastasis but negative in all the stage Ⅰ NPC patients.Conclusion:Thirty-three neck lymph node metastasis-ralated methylated genes of NPC had been screened out by genome-wide methylation arrays and the neck lymph node metastasis-related genes methylation profiles of NPC were preliminarily built. It provides new candidate genetic markers for the study of the mechanism of NPC’s tissue infiltration and lymph node metastasis and new research direction for preventing early metastasis and recurrence of NPC.Chapter Ⅱ Screening Differential Expressed Genes Related to Neck Lymph Node Metastasis by Gene Expression Profiling Array in Nasopharyngeal CarcinomaObjective:NimbleGen Whole Human Genome Expression Profiling Array was used to screen differential expressed genes relating to lymph node metastasis in early stage NPC tissue and the matching NPC tissue with neck lymph node metastasis in this study, so that we can construct differential expression profiles. Further, we explore the molecular mechanisms of NPC’s neck lymph node metastasis based on the biological analysis of differentially expressed genes.Methods:Total RNA were extracted by TRIzol method from 6 cases of early stage NPC tissue,8 cases of NPC tissue with neck lymph node metastasis and 6 cases of normal nasopharyngeal tissue. The RNA were purified and quality assessed. The purified RNA were used for double-strand cDNA synthesis. And then labeled the cDNA after its purification. The labeled cDNA was used for array hybridization and washing with NimbleGen Whole Human Genome Expression Profiling Array. After the scan, data collection and standardization of hybridized array, we screened out the differential expressed genes relating to lymph node metastasis between the two groups of NPC.Results:The extraction results of 20 samples of RNA were good, and their assessments were qualified. There were 6587 differentially expressed genes relating to lymph node metastasis in NPC.3359 genes’expression were up-regulated and 3228 were down-regulated. The up-regulated genes mainly located at Chromosome 1(11.10%), Chromosome 2(7.62%), Chromosome 17(6.28%) but rarely at Chromosome Y(0.21%), Chromosome 21 (0.86%), Chromosome 18(1.28%). The down-regulated genes mainly located at Chromosome 1(10.19%), Chromosome 19(8.89%), Chromosome 17(6.85%) but rarely at Chromosome Y(0.46%), Chromosome 18 (1.02%), Chromosome 13(1.39%).GO analysis showed that the up-regulated genes were involved in the biological process including cellular metabolic process (GO:0044237), organic substance metabolic process (GO:0071704), viral process (GO:0016032), cellular response to stress (GO:0033554), nitrogen compound metabolic process (GO:0006807); the molecular function including protein binding (GO:0005515), organic cyclic compound binding (GO:0097159), catalytic activity (GO:0003824), ion binding (GO:0043167), nucleic acid binding (GO:0003676) and the cellular component including intracellular part (GO:0044424), membrane-bounded organelle (GO.0043227), intracellular organelle (GO:0043229), cytoplasm (GO:0005737).The down-regulated genes were involved in the biological process including regulation of cellular process (GO:0050794), response to stimulus (GO:0050896), cell communication (GO:0007154), system development (GO:0048731), cell-cell signaling (GO:0007267); the molecular function including receptor activity (GO:0004872), transporter activity (GO:0005215), nucleic acid binding transcription factor activity (GO:0001071), channel activity (GO:0015267), G-protein coupled receptor binding (GO:0001664), receptor agonist activity (GO:0048018) and the cellular component including membrane part (GO:0044425), cell periphery (GO:0071944), extracellular space (GO:0005615), keratin filament (GO:0045095).KEGG pathway analysis showed that 1240 of the 3359 up-regulated genes were respectively involved in 87 biological pathways, such as viral carcinogenesis (hsa05203), Epstein-Barr virus infection (hsa05169), RNA transport (hsa03013) and 672 of the 3228 down-regulated were respectively involved in 33 biological pathways, such as cytokine-cytokine receptor interaction (hsa04060), transcriptional misregulation in cancer (hsa05202) and so on.Three negative-correlated differentially methylated genes (BAZ2B, CYP2J2 and ZNF628) were screened out by the combination of 33 neck lymph node metastasis-ralated methylated genes and 3228 down-regulated genes analysis. CYP2J2 was involved in two KEGG pathways:linoleic acid metabolism (hsa00591) and ovarian steroidogenesis (hsa04913). The three differentially methylated genes are mainly involved in regulation of transcription, component of nucleus, endoplasmic reticulum and microsome, and molecular function like DNA binding, protein binding and ion binding.Conclusion:3359 up-regulated and 3228 down-regulated neck lymph node metastasis-ralated differentially expressed genes of NPC had been screened out by genome-wide expression profiling arrays and the differentially expressed genes profiles were preliminarily built. Biological analysis was done on the differentially expressed genes, and it could provide theoretical basis for studying the molecular mechanism of neck lymph node metastasis in NPC. Three negative-correlated differentially methylated genes (BAZ2B, CYP2J2 and ZNF628) were screened out by the combination of 33 neck lymph node metastasis-ralated methylated genes and 3228 down-regulated genes analysis, it also could provide references for looking for the ideal genetic markers and new therapy targets of NPC.