Mechanism Of Abnormal Expression Of ZNF403 Genes In Laryngeal Squamous Cell Carcinoma

Laryngeal squamous cell carcinoma (LSCC) is a common upper respiratory cancer, accounting for 1%-8.4% of systemic cancer and 93%-99% of pathological type of laryngeal carcinoma. Its development is a multi-step process, involving abnormality in a number of related genes, such as: Ras c-Myc EGFR Cyclin D1and p53.Cyclin D1 is overexpression in a variety of solid tumors and is an important symbol in a variety of head and neck squamous cell carcinomas. Studies have shown that cyclin D1 expression in Laryngeal cancer is significantly higher than that in normal mucosa. The survival rate and pathological type has obvious relevance with expression level of cyclin D1. Cyclin D1 is an important protein involved in the G1/S transition phase, which can promote cells from G1 to S phase normally. Over expression of cyclin D1 will shorten the cells in G1 phase, leading to inadequate cell differentiation. Study have confirmed that Differentiation inducing factor-3(DIF-3), induces phosphorylation of cyclin D1 and reduces its expression. DIF-3 alias ZNF403 inhibits transcription and translation level of cyclin D1. DIF-3 is the most effective anti-tumor member in DIF family. ZNF403 is located on a high-frequency loss of heterozygosity region 17q12-17q21.1, encoding a 697 amino acids protein with a finger-like domain. The preliminary experimental study have shown ZNF403 mRNA are down in most laryngeal cancer tissues. Therefore ZNF403 abnormality may be closely related to the occurrence of laryngeal cancer.1. Analysis of ZNF403 mRNA expression in a variety of tumor cell lines and cancer tissuesIn order to analyze whether ZNF403 was associated with laryngeal cancer, we selected tumor cell lines, which had deletion, mutation or amplification on chromosome 17q, and 30 cases of laryngeal cancer and matched normal tissues. Semi-quantitative RT-PCR was performed to detect ZNF403 mRNA expression. The results showed that the expression of ZNF403 in laryngeal carcinoma cell line Hep-2 was much lower than others. ZNF403 was downregulated in 18 cases of laryngeal cancer tissues, and was lack in another two cases of laryngeal cancer and matched normal tissues. There were no relationship between ZNF403 expression and age and TNM stage (P> 0.05).2. Analysis of mechanisms of ZNF403 down-regulation in Laryngeal carcinoma cell line Hep-2 and cancer tissuesTo analyze the mechanism of ZNF403 downregulation, we first selected three STS (WI22201. WI16428, and RH78009) on chromosome 17q12 and detected the loss of heterozygosity (LOH) of 30 cases of laryngeal cancer and matching normal adjacent tissue using PCR-denaturant polyacrylamide gel electrophoresis method(Genetic status provides NO information if the allele is homozygote). The results showed that LOH was dectected in 3 cases at WI16428(3/28), 2 at RH78009(2/29) and none at WI22201. Homozygous deletion at WI22201 was found in 1 case. It suggested that LOH was not the main mechanism of downregulation of ZNF403.Next, bioinformatics analysis and luciferase reporter assay was used to confirmed the promoter region of ZNF403 gene in the upstream (-1370bp--607bp). Then, we determined CpG island methylated status of ZNF403 gene promoter in 30 cases of laryngeal cancer and adjacent tissues. The results showed: CpG island methylation rate in cancer tissue is 83% (25/30), and 46.7% (14/30) in matching tissues. ZNF403 gene promoter methylation was significantly higher in cancer tissues than the adjacent tissues. The differences are statistically significant (p<0.001). It suggested there was relevance between ZNF403 downregulation in cancer tissue and promoter DNA methylated status.To further analysis the revalence between ZNF403 gene promoter hypermethylation and ZNF403 mRNA expression, Hep-2 cells were treated with methyltransferase inhibitor, 5-Aza. RT-PCR and methylation-specific PCR was performed to detection ZNF403 mRNA expression and gene promoter methylation status. The results showed: ZNF403 expression gradually increased in Hep-2 cells. At the same time, ZNF403 gene CpG island methylation was inhibited after Hep-2 cells were treated with 5-Aza. In summary, the present study showed that the expression of ZNF403 in cancer tissues was significantly reduced. Although the loss of heterozygosity have played a certain role in this gene inactivation, but promoter CpG island methylation is the main mechanism.