| Objective: Esophageal squamous cell carcinoma(ESCC) is one of the highest incidence of tumors in china, about 300000 new cases appeared every year.Esophageal squamous cell carcinoma also has the characteristics of metastasis and recurrence,ESCC patients with the same age,sex,tumor size,pathological grade and the same clinical stage have different survival length that suggests that there are other factors to affect the prognosis of patients,for instance,the subtypes of ESCC independent of pathological classification. cDNA microarray method is commonly used for identifying differentially expressed genes and genotype the cancer.Different genes expressional files between Barrett esophagus and esophageal squamous cell carcinoma had been identified with cDNA microarray method,it was also confirmed that different genotypes of esophageal squamous cell carcinoma predict metastasis rate in Japan,while ESCC in china have not been genotyped. The aim of this study is to identify the sub-genotypes of ESCC which can be used to assess their relation to clinical characteristics and outcome.Because RNA expression levels in polysome is similar to proteins expression levels,we perform polysome cDNA microarray analysis for ESCC patients,polysome cDNA microarray is superior to cDNA microarray.Methods:1 Samples collection: 16 esophageal squamous cell carcinoma specimens were obtained ,including 9 Stage II and 7 Stage III , at the Fourth Hospital of Hebei Medical University from Jan 2009 to Mar 2009,and confirmed by pathological examination,and quickly frozen into liquid nitrogen,stored in -80℃. all the patients did not receive any therapy before surgery.2 PolysomeRNA extraction:200mg esophageal squamous cell carcinoma tissue was pulverized under liquid nitrogen and the powder lysed in 1ml of lysis buffer,supplemented with 1% deoxycholate.After lysing the cells by pipetting up and down several times,the nuclei were removed by centrifugation(12000g,10sec,at 4℃),the supernatant is supplement with 500 ul extraction buffer and centrifuged(12000g,5min,at 4℃)to remove mitochondria and membranous debris.The supernatant is layered onto 15%-40% sucrose gradient and then centrifuged in a SW41Ti rotor for 120min at 38000rpm at 4℃.Supernatant from top to bottom is collected by 12 tubes,1ml in each tube where 100ug proteinase K was added and RNAs were recovered by extraction with an equal volume of phenol-chloroform-isoamy alcohol,followed by ethanol precipitation.3 Probe labeling and Microarray test:RNA transcription using Superscri-PtⅡReverse Transcriptase, tumor RNA was labeled with Cy3-DUTP and Human Reference RNA was labeled with Cy5-DUTP, labeled probes hybridized with Agilent 44K human array,then the microarrays were scanned by GenePix4000A Scanner.4 Microarray analysis: the array data were analyzed with the unsupervised hierarchical clustering method. The difference between the subtype were identified with Analysis of Microarray(SAM)method.Resluts:1 Ultraviolet analysis and electrophoresis showed that high -qualitied polysomeRNA was obtained and ESCC ploysome cDNA microarray profiles were available.2 16 ESCC patients were divided into two groups based on the similarity of gene expression with Unsupervised Hierarchical Clustering method. 1,3,7 and 8 patients belong to A types, the others belong to B types.3 The differently expressed genes with total of 832 between A and B were identified with Significant Analysis of Microarray method at p=0.01 levels. these genes were involved in cell proliferation,cell apoptosis,cell signaling pathways,and JUN gene,XAF1gene,TNFSF4 gene and RASSF5 gene were associated with tumor genotype. Conclusion:1 we successfully extracted ESCC ploysomeRNA and obta-ined ploysome cDNA microarray profiles.2 There were Sub-genotypes of ESSC independent of pathological classific- ation which was association with tumor carcinogenesis and progress. |
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