Functions Of H3K9me3 In Predicting The Prognosis And Regulating The Gene Expression In ACC

PURPOSES: To analyze the correlation between H3K9me3(H3 lysine 9 trimethylation, H3K9me3) and clinicopathologic characteristics and prognosis in salivary adenoid cystic carcinoma(ACC) and investigate how H3K9me3 regulated the expression of genes and involved in the biological behaviors in ACC.METHODS: H3K9me3, EDNRB and CHL1 expression was detected by immunohistochemistry(IHC) in 66 ACC samples. The correlation between H3K9me3, EDNRB and CHL1 expression, the clinicopathological characteristics and prognosis of ACC patients was analyzed. Ch IP on chip was used to analyze the combination between H3K9me3 and related gene in ACC. EDNRB and CHL1 protein expression was detected by Western Blot and IHC in 28 paired of ACC tissues and adjacent normal tissues. Ch IP-PCR was used to verify the relationship between H3K9me3 and the promoter regions of EDNRB and CHL1. In addition, the m RNA expression of EDNRB and CHL1 were detected before and after treated with Chaetocin. All the data were evaluated by SPSS 18.0.RESULTS: 1. The H3K9me3 expression is significantly higher in ACC tumor tissues than that in adjacent salivary gland(P<0.001). H3K9me3 expression is higher in solid tumor than that in Cribriform-tubular tumor(P=0.002). H3K9me3 expression is higher in patients with dis tant metastasis(P=0.001). Patients with higher H3K9me3 expression had worse overall survival(OS) and disease-free survival(DFS). The expression of H3K9me3 can be used as independent predictor for OS and DFS in ACC.2. Ch IP on chip indentified 877 and 1191 which might be correlated with H3K9me3 in SACC-83 and SACC-LM cells, respectively, and those results shared 551 identical genes. Those genes are involved in olfactory transduction and neuroactive ligand-receptor interaction pathways and biological process, such as G-protein coupled receptors and cell adhesion. EDNRB and CHL1 might be the most impossible and important genes regulated by H3K9 me 3 in ACC.3. The protein expression of EDNRB and CHL1 is higher in adjacent normal tissues than those in ACC tumor tissues. There is site-specificity for H3K9me3 combined with the promoter regions of EDNRB and CHL1, most of H3K9me3 binding to the upstream of transcription start site(TSS):-1069~-877 bp,-1332~-1111 bp and-373~-176 bp regions of EDNRB and-1997~-1803 bp and-239~-70 bp regions of CHL1. This might lead to the down-regulation of these two proteins in ACC. Treated with Chaetocin can restore the EDNRB and CHL1 m RNA levels.4. The EDNRB expression is higher in early stage patients with ACC than those with advanced stage tumors(P=0.039). There is a tendency between lower expression of CHL1 and lymph node metastases in ACC(P=0.062).CONCLUSIONS: The H3K9me3 expression is significantly higher in ACC tumor tissues than that in adjacent salivary gland. The expression of H3K9 me 3 can be used as independent predictor for OS and DFS in ACC. Most H3K9me3 combined with the upstream of TSS:-1069~-877 bp,-1332~-1111 bp and-373~-176 bp regions of EDNRB and-1997~-1803 bp and-239~-70 bp regions of CHL1 and can regulate the expression of those two genes. H3K9me3 might promote tumor growth and lymph node invasion through regulating EDNRB and CHL1 expression, and the correlation between H3K9me3 and histological patterns, distant metastases and prognosis might be because H3K9me3 can regulate other genes.