HOX Gene Expression Profile And Transcription Regulation Mechanism In Nasopharyngeal Carcinoma

Homeotic(HOX) genes, one category of highly conservative transcription factors belonging to the Homeobox family, control the tissue and organ development at the transcription regulation level. Many researches indicated that aberrant HOX expression profiles are closely related to abnormal embryonic development and disease such as malignant tumor.Nasopharyngeal carcinoma(NPC) is a rare maliganacy in most parts of this world but of high incidence in south China and southeast Asia. One of the main causes of NPC is Epstein-Barr Virus(EBV) whose coding protein latent membrane protein1(LMP1) is proved to function as oncoprotein. We use NPC cells as tumor model, by gene expression array and qRT-PCR to investigate HOX profile and identified some HOX genes such as HOXA5,HOXB3, HOXB13, HOXC8, were abnormally down-regulated in EBV LMP1positive cells compared with LMP1negative cells. Immunohisochemistry analysis of NPC tissue from23patients showed that those HOX genes are also low expressed in LMP1positive tissue, the same as cellular level. Moreover, dual luciferase reporter assay proved that LMP1can significantly reduce HOXB13and HOXC8promoter activies in a dose-dependent pattern, which confroms the suppression role of LMP1to HOX genes.The regulation mechanism of HOX gene involves with multiple aspects, and transcription regulation is one of the most direct ways. RNA Polymerase Ⅱ(Pol Ⅱ) is indispensable during the formation of mRNA in eukaryotic transcription. In human genome,50%genes are transcriptional activated,20%are silenced without Pol Ⅱ occupation the other30%are silenced either, but their proximal promoter region are occupied by Pol Ⅱ calling Pol Ⅱ stalling. We detected the Pol Ⅱ occupancy around the transcription start site(TSS) of HOXA5, HOXB3, HOXB13HOXC8in LMP1positive and negative NPC cells respectively using ChIP assay, found that Pol Ⅱ occupancy is significantly higher in LMP1positive cells. Combine with the down-regulated facts of these genes, we come to the conclusion that HOXA5, HOXB3, HOXB13and HOXC8are Pol Ⅱ stalled in LMP1positive NPC cells.Irradiation is the main treatment for NPC at this time. It’s interesting that many researches showed that irradiation induced DNA damage response(DDR) is usually coupled with DNA demethylation, and5hmC involves in this kind of demethylation process which is associated with gene activation. Accordingly, using CNE1-LMP1cell line, we established a DDR model and further confirmed by γ H2Ax immunofluorescence and comet assay. Through multiple techniques such as qRT-PCR, Pol Ⅱ ChIP, MeDIP and PvuRts1Ⅰ digestion, we found that stalled HOXA5, HOXB3, HOXB13, HOXC8reactivated, stalled Pol Ⅱ released from TSS region, both5mC and5hmC level reduced but DNA demethylase TET2up-regulated after irradiation treatment. These results suggest that TET2and5hmC may involve into DNA demethylation, affecting Pol Ⅱ release and HOX reactivation.Bioinformatic analysis of60tumor cell lines(CellMiner, NCI60), we know that HOXA5, HOXB3, HOXB13and HOXC8are significantly associated with variety of energy metabolism genes, indicating HOX gene may have important role in cancer energy metabolism. So we choose HOXC8as an example for studying the influences of HOX gene may conduct in NPC. HOXC8expression vector was contructed and exogenously expressed in CNE1-LMP1, resulting reduced cell proliferation, lactate formation or glucose consumption. Moreover, key genes like HK2and GLUT1in glycolysis pathway was repressed, but glycolysis negative regulator TIGAR expression was promoted, suggesting HOXC8may inhibit glycolysis. Meanwhile, Oxidative phosphorylation enzyme SCO2was up-regulated, indicating tricarboxylic acid cycle(TCA) was enhanced. Then, we checked the expression level of some important TCA enzymes including2-hydroxyglutaric acid dehydrogenase(2-HGDH), succinate dehydrogenase D(SDHD), α-ketoglutarate dehydrogenase(α-KGDH) consisting OGDH, DLST and DLD subunit, fumarate hydratase(FH) and aconitase(ACO1,ACO2). Result showed that HOXC8can significantly promot these gene expression in a dose-dependent manner. These results indicate that HOXC8may directly target to TCA cycle related enzymes.In this study, we investigated the relationship between HOX expression profile and NPC, found some HOX genes were aberrantly silenced by Pol Ⅱ stalling in LMP1positve. Irradiation induced DDR may influent HOX reactivation through TET2and5hmC mediated DNA demethylation. In addition, HOX may play important roles in NPC energy metabolism.