The Relationship Between Promoter Methylation And Expression Of 14-3-3σ, Annexin A1 And SCCA1 Genes In Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma(NPC)is one of the most common malignant tumors in southern China.However,the mechanism underlying the pathogenesis of NPC remains unclear.DNA methylation is one of the epigenetics mechanisms of gene expression regulation.And promoter methylation of tumor suppression genes plays a critical role in carcinogenesis.Therefore,screening for methylation silenced genes may help to reveal the pathogenesis of NPC,which has important theoretical and clinical value.Our previous study has found that the expression of 14-3-3σ,Annexin A1 and SCCA1 proteins were downregulated in NPC by laser capture microdissection combined with proteomics,but the mechanisms of their downregulation need to be elucidated.To explore whether the downregulation of 14-3-3σ,Annexin A1 and SCCA1 in NPC is caused by promoter methylation,four NPC cell lines (CNE1,CNE2,5-8F,6-10B),one immortalized human nasopharyngeal epithelial cell line NP69,75 cases of NPC biopsy tissues and 25 cases of normal nasopharyngeal mucosal biopsy tissues were enrolled for the current study.Methylation specific PCR(MSP)was used to examine methylation levels of 14-3-3σ,Annexin A1 and SCCA1 gene promoters; Semi-quantitative RT-PCR,immunohistochemistry and western blotting were performed to assess the expression of 14-3-3σ,Annexin A1 and SCCA1 at mRNA and protein level,respectively.And demethylation agent 5-aza-2dC of 0,0.1,1,5 and 10μmol/L was used to treat NPC cell lines,and then MTT assay was used to detect the cell growth,and flow cytometry was adopted to detec the cell cycle distribution and apoptosis.The main results of the current study as following:(1)High frequent promoter methylation was found for 14-3-3σ(84%VS 28%),Annexin A1 (92%VS 12%)and SCCA1(87%VS 22%)genes in NPC tissues when compared with normal nasopharyngeal mucosal tissues;(2)In NPC tissues,complete methylation of 14-3-3σ,Annexin A1 and SCCA1 gene promoters was accompanied with expression loss of their mRNA and proteins,and partial methylation of 14-3-3σ,Annexin A1 and SCCA1 gene promoters was accompanied with significant downregulation of their mRNA and proteins.Methylation of 14-3-3σ,Annexin A1 and SCCA1 gene promoters was negatively correlated with their protein expression levels;(3)Methylation of 14-3-3σ,Annexin A1 and SCCA1 promoter were positively correlated with NPC lymphonode metastasis;(4) Methylation with different degree,s of 14-3-3σ,Annexin A1 and SCCA1 gene promoters was detected in all of the four NPC cell lines but not in NP69 cell line,and the methylation levels were related with the differentiated degree and metastatic potential of NPC cell lines;(5)The expression levels of 14-3-3σ,Annexin A1 and SCCA1 were significantly lower in the four NPC cell lines than those in NP69;(6)5-aza-2dC could inhibit methylation of 14-3-3σ,Annexin A1 and SCCA1 gene promoters in a dose-dependent manner,and upregulate the expression of their mRNA and proteins in the four NPC cell lines;(7)5-aza-2dC could inhibit cell proliferation,promote cell apoptosis,and blocks cell cycle progress in a dose-dependent manner in all of the four NPC cell lines.The results indicate:(1)Promoters of 14-3-3σ,Annexin A1 and SCCA1 genes are high frequently methylated in NPC;(2)The methylation might be reasons for decreased or lossed expressions of 14-3-3σ,Annexin A1 and SCCA1 genes;(3)14-3-3σ,Annexin A1 and SCCA1 are novel methylation-silenced genes in NPC;(4)The methylation of 14-3-3σ,AnnexinA1 and SCCA1 gene promoters was associated with lymphonode metastasis in NPC,indicating that methylaton of 14-3-3σ,Annexin A1 and SCCA1 gene promoters might be a biomarker for NPC metastasis;(5)5-aza-2dC could decrease the methylation levels of 14-3-3σ,AnnexinA1 and SCCA1 gene promoters, upregulate their expressions,inhibit cell proliferation,and promote cell apoptosis in NPC.