| Summary: | 碩士 === 長庚大學 === 生物醫學研究所 === 97 === Nasopharyngeal carcinoma (NPC) is a common cancer in South East Asia. According to statistic analysis, the number of each million males who suffer from NPC each year in Taiwan is twelve times more than in Occident. Generally speaking, the major risk factor for NPC is EB virus. Studies have shown that about sixty-five percentage of NPC tissues express the EBV onco-protein, LMP1, suggesting that expression of LMP1 is strongly related to NPC. LMP1 has been shown to directly regulate the transcription factors (TF) NF-κB. Numerous studies have shown that NF-κB plays an important role in oncogenesis. Hence, finding out which genes are regulated by NF-κB is quite important to prevent and treat NPC.
First, we used real-time PCR (polymerase chain reaction) to quantify the expression levels of genes known to be involved in tumor formation in HEK293/LMP1 Tet-on cell line. We found 13 genes whose expression levels were significantly up-regulated upon the induction of LMP1 and twelve of them contain potential NF-κB binding element in their promoter regions. These twelve genes were selected as potential targets.
To find additional targets, we also analyzed the effect of LMP1 on global gene expression pattern using Affymetrix microarray. In HEK293 LMP1 Tet-on cells, we detected 1074 genes showing more than 1.5-fold up-regulation upon LMP1 induction. In LMP1-positive tissues, we found 1190 genes up-regulated compared to normal tissue. A total of 89 genes showed up-regulation in both cell and tissue samples. We selected 12 genes for validation and found one of these genes showed up-regulation in real-time PCR. This gene is also selected as potential target.
Subsequently, we conducted chromatin immunoprecipitation (ChIP) assay to verify whether any of these potential target genes can be regulated by NF-κB directly. ChIP assay detected an increase in NF-κB binding in the promoter regions of BIRC3, CCL20, CXCL10, CD40 and IL8 upon the induction of LMP1, indicating that these genes are regulated by LMP1 through NF-κB pathway.
This study will help to find potential biomarkers regulated by LMP1 through NF-κB. We hope this basic study will be useful for future clinical application.
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