The Differential Gene Expression Profile Of Allergic Rhinitis(AR) And AR With Asthma

The prevalence of allergic disorders such as allergic rhinitis(AR)has increased so drastically over the past two-three decades accompanied by growth of medical science and economic development throughout the world.AR has been a global health problem.To help increase worldwide awareness of allergy related problems,The World Health Organization (WHO)and The World Allergy Organization(WAO)has declared July 8 as World Allergy Day in 2005 years.Asthma is the most severe complication of AR and.also has a strong impact on patients.quality of life.Surveys report that up to 80 percent of asthmatic patients have co-existing allergic rhinitis,while up to 40 percent of allergic rhinitis patients have asthma,the coexistence of sinusitis and asthma,the presence of rhinitis as a risk factor for developing asthma,further emphasize this link and together lead to the operative definition of United Airways Disease(UAD).Recently,extensive research on transcriptional regulation for genes of allergy disorders such as AR,asthma has established that mutil-factor regulation or space-time interaction between multiple gene and biomolecule exist in the development of AR to asthma. However,traditional gene analysis could only study one or a few genes at a time,and is costly and time-consuming,hardly explaining the whole process accurately and completely.The recent development of DNA microarray technology,a type of high-density analysis for gene expression, has opened a new era in medical sciences.It can analyze the expression of many genes simultaneously.Affymetrix oligonucleotide microarray is the most popular one.To clarity molecule and gene regulation mechanisms of progression of AR toward asthma,we screen the differential expression gene profile of nasal mucosa of seasonal allergic rhinitis(SAR)and SAR with asthma using oligonucleotide microarray(Affymetrix HG-U133-plus2),analyze the changes of expressions gene profile with software GeneSpring and carry out data mining by integrating GeneCard database,Pubmed-NLM database and differential genes.The main results are as following:PartⅠ.Construction of genechip and screening differential gene expression profile of seasonal allergic rhinitis(SAR)and SAR with asthma with DNA microarray technique1.Select study objects and the extraction and purification of total RNA in inferior turbinate mucosaSAR and SAR with asthma patients were recruited strictly according to 1997 Haikou AR diagnostic criteria and bronchial astham diagnostic criteria respectively.The total RNA of SAR and SAR with asthma samples were extracted by a TRIZOL method.The integrity of the purified RNA was determined with 1:2%denaturing agarose gel electrophoresis.The results show that all study patients were atopy,two bands of ribosomal RNA 28S,18S could be found with denaturing agarose gel electrophoresis.The extraction RNA exhibit OD260/OD 280 ratio about 2.0.These results indicate that RNA of SAR and SAR with asthma inferior turbinate mucosa samples have been extracted successfully.2.Screening differential gene expression profile of SAR and SAR with asthma with Affymetrix microarray techniqueTotal RNA extracted from inferior turbinate mucosa samples was reverse-transcribed into cDNA,and then transcribed into biotin-labeled cRNA and fragmented.The cRNA fragments was hybridized with the GeneChip Human U133-plus2.0 array slides.The hybridization slides were scanned and analyzed with the Microarray Suite Software version 5.0.Some different gene was verified by RT-PCR.Our study showed that 22002(40.2%)out of total 54675 probe sets is present,31820(58.2%)absent,853(1.6%)marginal.The average present signal is 495.8.The probe sets number of signal increase,decrease and no change is 1917(3.5%), 2726(5.0%),49637(90.8%),respectively.Signal Log Ratio,which estimates the magnitude and direction of change of a transcript when two arrays are compared,of at least 1.0 or-1.0(that indicates an increase or decrease of the transcript level by 2 fold change)and Change p-value, which measures the probability that the expression levels of a probe set in two different arrays are the same or not,≤0.05(that means the expression level in the experiment array is higher than that of the baseline array)were choosen to select differentially expressed genes.1900 genes were differentially expressed at least 2-fold in which 849 genes were up-regulated and 1051 genes were down-regulated in nasal mucosa of SAR with asthma patients compared with that in SAR patients.PartⅡBioinformatics analysis of differential genes in SAR with asthma samples compared with SAR samples3.Gene ontology(GO)annotations and pathway analysis of differential genes in SAR with asthma samples compared with SAR samplesThe GO annotations of differential genes(above 2,4 fold,respectively)in SAR with asthma samples compared with SAR samples was performed using GeneSpring software based on Gene Ontology database;the pathway of differential genes(above 2 fold)in SAR with asthma samples compared with SAR samples was analyzed using database such as KEGG,GenMapp.In addition, the top 10 changed genes in the genechip were analysed detailedly and completely. Ontology(GO)classification and TreeView analysis,the differentially expressed genes were further divided into three groups:1 biological process,molecular function,cellular component profile.The changed genes(above 2 fold)in the SAR and SAR with asthma samples were involved in cell metabolism,gene transcription,cell proliferation,signal transduction,immune response,enzyme activity,transmembrane receptor activity,cytoskeletal protein binding,and so on.Pathway(only includes≥20 genes)analysis is as follow:Cytokine-cytokine receptor interaction,40 genes;Focal adhesion,28 genes;Cell adhesion molecules(CAMs),26 genes; Regulation of actin cytoskeleton,26 genes;Cell Communication,24 genes; Gap junction,23 genes;MAPK signaling pathway,23 genes;Calcium signaling pathway,21 genes;Leukocyte transendothelial migration,21 genes;Purine metabolism,20 genes.Among the top 10 changed genes,AKAP14 genes expressed in tissues containing axoneme-based organelles(cilia and/or flagella).Highly expressed in airway cilia was down-regulated 100 folds (SLR=-6.7);TEKT1 gene was structural component of ciliary and flagellar microtubules and discovered down-regulated 130 folds(SLR=-7.2).RAF1 gene participated MAPK signaling pathway which connected with allergic asthma.4.the squamous cell carcinoma antigens genes could as relevant biomarkers of the progression of allergic rhinitis toward asthmaA comprehensive data and literature mining was performed to differential expression genes using Pubmed-NLM and GeneCard database.Results show in GeneCard database,354 genes was related with asthma,45 genes was related with allergic rhinitis,13 genes was related with allergic asthma.SCCA1(SERPINB3)and SCCA2(SERPINB4)were highly expressed in nasal mucosa of SAR with asthma patients,compared with SAR patients at mRNA lever.SCCA1 and SCCA2 was further identified as an important sign of the progression of allergic rhinitis toward asthma.by integrating array data with array-independent literature mining.The result of the study indicate that in the occurrence and development of SAR complicating asthma,there are multiple genetic expression and regulation having changed.It may be useful for clarifying the mechanism of the occurrence and development of AR complicating asthma,and finding some unknown signal pathway to study the different genes between them.In addition,building the different gene expression map of adult SAR and SAR complicating asthma,and elucidating the related signal transduction gradually will provide important data for establishing gene network theory of AR related desease and gene expression database of adult SAR and SAR complicating asthma.To evaluate the validity and value of the result of our gene array,we should consider thoroughly not only the quality and quantity of the target sample,the technique of detection and verification of the array,and the strategy and method of producing data,but also the selectivity, space-time,and no ultimacy of gene expression.Because the level of mRNA is not equal to the level of the expressed protein(the level of the expressed protein is only represent that the protein is existing,and its quantity is different),the final effect of the gene expression is to determine the function of the coded protein.In a word,we should have a entire,reasonable and objective explanation and recognition.Further more,some results of our study will need large sample and multilevel clinical experiment to verify.