Identification of novel methylated DNA marker ZNF569 for head and neck squamous cell carcinoma

Abstract

Aberrant DNA methylation pattern plays an indispensable role in the initiation and development of head and neck squamous cell carcinoma (HNSCC). It is well recognized that lymph node metastasis is closely with unfavorable prognosis of HNSCC. Therefore, exploring the methylation events accounting for the lymph node metastasis of HNSCC is very important for improving the clinical outcome of HNSCC. Methylation data, RNA-seq data and clinical data were downloaded from The Cancer Genome Atlas (TCGA) and processed using the R package TCGA-Assembler. MethylMix was use for data analysis by integrating both methylation and gene expression data on HNSCC patients with lymph node metastasis and without lymph node metastasis. Pathway analysis was performed on significantly altered genes using ConsensusPathDB. The role of our interested gene zinc figure protein 569 (ZNF569) in HNSCC was further evaluated. Our results identified many novel hypermethylated/hypomethylated genes that might be closely associated with the lymph node metastasis of HNSCC. Pathway analysis revealed that increase in methylation of genes involved in generic transcription pathway including zinc figure proteins. ZNF569 was hypermethylated in HNSCC tissues especially those with lymph node metastasis. In addition, the expression levels of ZNF569 mRNA and protein were significantly lower in HNSCC tissues and cell lines compared to their respective controls. Moreover, overexpression of ZNF569 inhibited the proliferation, migration and invasion of HNSCC cells. HNSCC patients with lower ZNF569 expression suffered a significantly shorter overall survival than those with higher ZNF569 expression. In conclusion, we have identified many novel differentially methylated genes that might be important for the lymph node metastasis of HNSCC. In addition, ZNF569 might play a tumor suppressive role in carcinogenesis of HNSCC.

Keywords: Carcinogenesis; Head and neck squamous cell carcinoma; Methylation; Zinc figure protein 569.

Figure 1

The top methylated DNA markers (MDMs) associated with lymph node metastasis of HNSCC. (A) The top ten hypermethylated genes associated with lymph node metastasis of HNSCC; (B) The top hypomethylated genes associated with lymph node metastasis of HNSCC.

Figure 2

Heatmaps and pathway analysis of the altered MDMs. (A) Heatmap of hypermethylated genes associated with lymph node metastasis of HNSCC. The green/red color indicates relatively low/high methylation level. Each column represented an independent biological sample and each row indicates a gene; (B) Heatmap of hypomethylated genes associated with lymph node metastasis of HNSCC; (C) Pathway analysis of the hypermethylated genes.

Figure 3

ZNF569 was downregulated in HNSCC tissues and cell lines. (A-B) The expression level of ZNF569 protein was significantly downregulated in HNSCC tissues compared with the adjacent normal tissues (P=0.0016); (C) The expression level of ZNF569 mRNA was significantly downregulated in HNSCC tissues compared with the adjacent normal tissues (P<0.001); (D) Similarly, ZNF569 levels was found to be downregulated in cancer tissues compared to normal tissues in various independent cohort study such as GSE25099 (P<0.001), GSE37991 (P<0.001) and TCGA (P=0.0727). (E-F) The levels of ZNF569 mRNA and protein were remarkably lower in HNSCC cell lines compared to control cell line (P<0.001).

Figure 4

The construction of ZNF-569 overexpression lentiviruses. (A) The lentiviruses we constructed were able to infect HNSCC cell lines with high efficiency; (B-C) The expression levels of ZNF569 mRNA and protein were significantly higher in cancer cells that infected with ZNF-569 overexpression lentiviruses compared to those infected with the control lentiviruses (**P<0.01, ***P<0.001).

Figure 5

ZNF569 overexpression inhibited the proliferation of HNSCC cell lines. (A-B) The OD values were significantly lower in ZNF569 overexpression cancer cells compared to the control cells (**P<0.01, ***P<0.001); (C-D) The percentage of EdU positive cells were lower in ZNF569 overexpression cancer cells compared to the control cells (**P<0.01).

Figure 6

ZNF569 overexpression suppressed the migration and invasion of HNSCC cells. (A-B) The wound widths were larger in ZNF569 overexpression cancer cells compared to the control cells (**P<0.01); (C-D) The number of cancer cells that invaded through the membrane was lower in cancer cells from ZNF569 overexpression group (**P<0.01).

Figure 7

Low ZNF569 expression was associated with unfavorable overall survival. HNSCC patients with lower ZNF569 expression had a significantly worse long-term overall survival than those with higher ZNF569 expression (P=0.0059).