Nasopharyngeal Myocardin Transcription Loss Of Living Mechanisms And Functional Study

Background:Nasopharyngeal carcinoma (NPC) is an serious health problem in southern China and south-eastern Asia while the disease is very rare in most parts of the world. The tumorgenesis of NPC is proposed to be a multistep process; besides its significant association with Epstein-Barr Virus infection, epigenetic inactivations of tumor suppressor genes (TSG) and other genes play indispensable roles in its carcinogenesis. Exploring novel genes that are targets of hypermethylation in NPC may provide further insights into the development of this unique malignancy and open the way to discover novel diagnostic and therapeutic strategies. To identify novel candidate TSGs in NPC, we performed a genome-wide screening for genes inactivated by promoter hypermethylation. By analyzing changes in global gene expression profiles in two NPC cell lines before and after treatment with the combine treatment of the a demethylating agent 5-aza-2O-deoxycytidine (5-aza-dC) and the histone deacetylase (HDAC) inhibitor TSA, we identified Myocardin was downregulated by promoter hypermethylation in NPC.Methods:Transcriptional expression levels of Myocardin in NPC cells and normal nasopharyngeal epithelia were evaluated by semiquantitave RT-PCR. Methylation status of Myocardin in NPC cells, primary tumors and normal nasopharyngeal epithelia were addressed by methylation specific PCR and bisulfate genomic sequencing. The complete CDS of Myocardin gene were coloned into the expression vector pCMV-3Tag3A and transfected into CNE2 cells. Stable transfectance of Myocardin were selected by geneticin. The properties of Myocardin as a tumor suppressor gene were address by cell proliferation assay and colony formation assay.Results: Myocardin mRNA expression were inactivated in four out of five NPC cell lines. Myocardin were aberrantly methylated in 80%(4/5) NPC cell lines and 73.8%(48/65) of NPC primary tumors, but not in any of the twelve normal nasopharyngeal tissues. Loss of Myocardin expression can be completely restored by the methyltransferase inhibitor 5-aza-dC in four NPC cell lines. Ectopic expression of Myocardin in the Myocardin-silenced and NPC cell line CNE2 shows that Myocardin could inhibit cell cycle progression, colony formation.Conclusions:Epigenetic inactivation of Myocardin is frequent and tumor specific event in NPC. Myocardin might be considering as a candidate tumor suppressor gene in NPC.