Profile Of Differentially Expressed Genes Related To Metastasis Of Nasopharyngeal Carcinoma And Functional Analysis In Northwest China

Objective:Metastasis is the main failure cause of nasopharyngeal carcinoma (NPC) aftertreatment, and its molecular mechanisms remain to be further explored. It has beenrecognized that metastasis is a progressive course involving multiple genes interactingwith eaeh other. Genome-wide gene screening was useful in identifying differentiallyexpressed genes involved in metastasis of NPC.To date, most research about differentiallyexpressed gene profling associated with metastasis of NPC derived from cell lines ratherthan tissue samples. In this study, we for the first time screened NPC metastasis relatedgenes in NPC tissue samples with or without metastasis in northwest China, and thenvalidate the gene expression by real time-PCR and immunohistochemistry. The objectiveof this study is to elucidate the comprehensive molecular mechanisms of nasopharyngealcarcinoma metastasis and provide new potential therapeutic targets.Methods:1. Using the Agilent4X44K Gene Expression Microarray chip with41.000individualgenes, we compared3pairs of cases of untreated nasopharyngeal carcinoma with otwithout metastasis in northwest China. Microarray analysis was performed in triplicate.We identified the differentially expressed genes whose expression difference was twice or more by using GoMiner software. KEGG software was used for the analysis of theinvolved pathway of the differential genes.2. Real time-PCR was used to validate the expression of DMBT1, MAZ, SFTPB,TCL1B, HSP90AA2and MYBPC1, whose expression showed significant difference.3. Western Blot was used to test the expression of MAZ,DMBT1,SFTPB in NPC cellline5-8F and6-10B.4. Immunohistochemistry was used to test the expression of DMBT1, MAZ, SFTPBat protein level in10cases of newly diagnosed NPC tissues with metastasis and40casesof NPC tissues without metastasis with the follow-up of3years.5．NPC cell lines5-8F and6-10B were transfected with MAZ siRNA and DMBT1siRNA respectively. Cell function related to metastasis was evaluated by scratch andtranswell assay.6. SPSS13.0software was used in the statistical analyses. Data was analyzed byStudent’s t-test and Wilcoxon test. p<0.05was considered as statistically significant.Results:1. Compared with NPC tissues without metastasis,4731differentially expressedgenes were detected in3cases of NPC with metastasis, with2314genes up-regulated2417down-regulated.2. Gene ontology analysis and Pathway analysis showed that: the up-regulated genesparticipated in501Biological Process,125Cellular Component,98Molecular Functionand35Pathway; and the down-regulated genes participated in333Biological Process,37Cellular Component,72Molecular Functionand29Pathway. Most of these genesparticipated in cellular and metabolic precesses, immune response, catalytic activity, andprotein binding.3. Six genes with altered progress/expression by Gene Chip analysis were chosen tobe verified using Real time-PCR. Compared with NPC without metastasis,3genes wereconfirmed to be significantly up-regulated (MAZ,HSP90AA2,MYBPC1)in metastastictissues.3genes were also confirmed to be significantly down-regulated (DMBT1，SFTPB，TCL1B).4. The different expressions of MAZ, DMBT1and SFTPB were validated byimmunohistochemistry， which was consistent with the results of Gene Chip.5．IN NPC cell lines5-8F，MAZ was confirmed to be significantly up-regulated,and in6-10B cell，DMBT1,SFTPB was confirmed to be significantly down-regulated byWestern Blot.6. The migration and invasion ability of5-8F cell line was significantly inhibited byknockdown of MAZ using siRNA. The migration and invasion ability of6-10B cell linewas significantly increased by DMBT1knowdown7. The expression of MAZ was significantly related well with lymph node and distantmetastasis (P<0.05), and the expression of MAZ was increased in WHOIII pathologygroup.The expression of DMBT1was significantly related to distant metastasis (P<0.05).and the expression of DMBT1was increased in WHOII pathology group (P<0.05)..Conclusion:Profound alterations of genes expression were observed between NPC tissue sampleswith or without metastasis in northwest China by microarray analysis. Expression of MAZ,DMBT1and SFTPB were confirmed by RT-PCR and immunohistochemistry. UsingsiRNA interference of MAZ gene expression in5-8F, migration and invasion abilitywere decreased significantly. The migration and invasion ability were increasedsignificantly by DMBT1knockdown in6-10B cell.We conclude that overexpression ofMAZ and downexpression of DMBT1plays an important role in NPC metastasis.