Assay for transposase-accessible chromatin with high-throughput sequencing reveals radioresistance-related genes in oral squamous cell carcinoma cells.

Radiotherapy is commonly used to treat oral squamous cell carcinoma (OSCC), and radioresistance is a critical factor resulting in poor outcomes. Several genes have been reported to be therapeutic targets for radioresistance; however, the involvement of chromatin accessibility in radioresistance has not been clarified in OSCC cells. Accordingly, in this study, we evaluated chromatin accessibility in radioresistant (HSC-3) and radiosensitive (KOSC-2) cells, identified from nine OSCC cell lines using clonogenic survival assays after irradiation. Chromatin accessibility in radioresistant OSCC cells was assessed using assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Gene expression was evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and immunoblot analysis. Viability was assessed by MTS assay. We found 1273 peaks (open chromatin regions by ATAC-seq) related to 8 Gy irradiation in HSC-3 but not KOSC-2 cells, among which 235 genes located around the chromatin open peaks were identified by ChIPpeakAnno analysis. Subsequently, 12 genes were selected as signal transduction-related genes by Gene Ontology analysis, and gene expression was confirmed by RT-qPCR. Among these genes, adenylate cyclase 2 (ADCY2) was significantly upregulated after treatment with irradiation in HSC-3 but not KOSC-2 cells. To further evaluate ADCY2 function in radioresistant cells, we performed ADCY2 knockdown by transfection of HSC-3 cells with small interfering RNA (siADCY2). Cell viability after irradiation was significantly decreased in siADCY2-transfected cells compared with that in control cells. These results suggested that ADCY2 expression was related to the open chromatin region in radioresistant OSCC cells and that ADCY2 may have therapeutic efficacy when used in combination with radiotherapy in patients with OSCC.