Identification Of Nasopharyngeal Carcinoma Molelular Markers In Different Stages And Construction Of Differentially Expressed Genes Crosstalk Networks

【Analysis of Nasopharyngeal Carcinoma Differentially Expressed Gene Profiling】We selected the H80s high-density chip to construct the nasopharyngeal carcinoma(NPC) differentially expressed gene profiling and found that a set of 6 unique genes may be the candidate molecular markers by supervised clustering and weighted voting class prediction models.Because it predicted NPC from non-cancerous tissues most accurately.Among the 6 genes,RB1,STMN1 and DSP were up-regulated in NPC,SERPINB6,STYL2 and AGTRL1 were down-regulated in NPC.In order to find out much more valuable information from the NPC differentially expressed gene profiling,we analyzed the NPC differentially expressed gene profiling data using significance analysis of microarray(SAM) once again and found that there were 758 notably differentially expressed genes in which 270 genes were up-regulated in NPC tissues and 488 genes were down-regulated in NPC tissues.Thus we constructed the NPC differentially expressed gene profiling.Using Ingenuity Pathway Analysis(IPA),Gene Set Enrichment Analysis(GSEA) and DAVID to analyze the NPC differentially expressed gene profiling data and found that cell cycle pathway and TGFβpathway correlated molecular markers were closely associated with NPC tumorigenesis.【Identification of Molecular Markers for Nasopharyngeal Carcinoma with Different Stages】To further study the relationship and effect between differentially expressed genes,cell cycle pathway and TGFβpathway correlated molecules and NPC tumorigenesis,and evaluate whether the differentially expressed genes could be as molecular targets for NPC.We made high density TMA with different stages of NPC to validate their expression level.It is important to screen and validate target genes of NPC with different stages and looking for biomarkers of early diagnosis, metastasis and prognosis for NPC.1.Construction of Nasopharyngeal Carcinoma Tissue Microarray with Different StagesThe tissue microarray(TMA) of NPC with different stages was made according to the patent technology(ZL 200410022818X).There are 896 dots on the TMA including normal and chronic inflammation nasopharyngeal epithelium(NPE),dysplastic NPE,adjacent epithelium of NPC,NPC(WHOⅠ,WHOⅡ,WHOⅢ;clinical stageⅠ,clinical stageⅡ, clinical stageⅢ,clinical stageⅣ;lymph node metastasis,without lymph node metastasis),normal NPE of NPC after radiation and relapse of NPC after radiation,lymphoma and laryngocarcinoma.The actual dots of TMA are 825 excluding lymphoma and laryngocarcinoma,and loss tissues.The 825 tissues included 141 cases normal and chronic inflammation NPE,98 cases dysplastic NPE,88 cases adjacent epithelium of NPC,434 cases NPC(15 cases WHOⅠ,402 cases WHOⅡ,17 cases WHOⅢ;32 cases clinical stageⅠ,155 cases clinical stageⅡ,156 cases clinical stageⅢ,91 cases clinical stageⅣ;283 cases with lymph node metastasis,151 cases without lymph node metastasis),64 cases normal NPE of NPC after radiation and relapse of NPC after radiation. 2.Identification of Nasopharyngeal Carcinoma Differentially Expressed GenesIt was identified that SPLUNC 1 was a molecular target for the early diagnosis of NPC.NGX6,LTF and AGTRL1 were a group of candidate biomarkers forcasting the lymph node metastasis of NPC.SPLUNC1, C1ORF102,AGTRL1 and SERPINB6 were a group of candidate biomarkers related to clinical progression.LTF,BRD7 and C1ORF102 were a group of perfect candidate biomarkers related to the prognosis of NPC among the 488 down-regulated genes in NPC using TMA.LTF, C1ORF102,SPLUNC1,BRD7,NAG7 and NGX6 were candidate suppressor genes of NPC which were cloned by our laboratory.AGTRL1 and SERPINB6 were candidate molecular markers of NPC by supervised clustering and weighted voting class prediction models by us before.The expression level of 27,p16,CDK4,RB1 and CDK8 which belong to cell cycle pathway was in accordance with cDNA microarray combine real-time quantitative RT-PCR with NPC TMA in different stages.We confirmed the aberrant activation of TGFβIIR,ACVRL1,INHBB,Axin2 and p300/CBP which belong to TGFβpathway using NPC TMA with different stages.This suggested that cell cycle pathway and TGFβpathway may play an important role in NPC tumorigenesis. We also confirmed that EBV infection is the main characteristic of NPC. The activated cell cycle pathway and TGFβpathway in NPC may be closely associated with EBV infection.3.Construction of Molecular Markers for Nasopharyngeal Carcinoma with Different Stages Using a series of large scale screening techniques from transcriptomics and TMA to identify differentially expressed genes and candidate NPC suppressor genes such as SPLUNC1,C1ORF102,BRD7, NAG7,NGX6 and cell cycle pathway and TGFβpathway members,we had preliminary constructed a molecular marker system in different stages of NPC:①SPLUNC1,p16,p27,and p19,a group of molecular targets for the early diagnosis of NPC.②LTF,NAG7,SPLUNC1,C1ORF102, CDK4,AGTRL1,INHBB and SERPINB6,a group of candidate biomarkers related to clinical progression.③NGX6,LTF,AGTRL1, INHBB and ACVRL1,a group of candidate biomarkers forcasting the lymph node metastasis of NPC.④LTF,BRD7,CDK4,Cyclin D1,RB1, EBER-1 and ACVRL1,a group of candidate biomarkers forcasting radiation sensitivity of NPC.⑤LTF,BRD7,C1ORF102,ACVRL1, INHBB,cyclin D1,a group of perfect candidate biomarkers related to the prognosis of NPC.The validation of biomarkers for NPC with different stages lay a substaintial foundation for molecular classification research of NPC.【Construction of Nasopharyngeal Carcinoma Differentially Expressed Genes Crosstalk Networks】The 758 differentially expressed genes were imported into IPA software to study the biological function and crosstalk networks associated with NPC tumorigenesis.The results showed that only 137 genes were in the network database and 127 genes had the functions or took part in some pathways.Among these networks,there were 9 notably changed networks.These networks involved in DNA replication and repair,cell cycle,cancer,gene expression,cell signaling and so on. SPLUNC1 participated in gene expression and cancer and regulated by retinoic acid(RA).We speculated that RA suppressed NPC cells growth maybe through regulating the expression of SPLUNC1.SERPINB6 participated in cell movement and immune response.