Nobuchi Takafumi, Saito Tomoaki, Kasamatsu Atsushi, Kawasaki Kohei, Nozaki Ryunosuke, Kase Yutaro, Iyoda Manabu, Saito Masayoshi, Uno Takashi, Uzawa Katsuhiro
Department of Oral Science, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan.
Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, 1-8-1Inohana, Chuo-ku, Chiba, 260-8670, Japan.
Biochem Biophys Res Commun. 2022 Mar 15;597:115-121. doi: 10.1016/j.bbrc.2022.01.122. Epub 2022 Feb 1.
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.
放射疗法常用于治疗口腔鳞状细胞癌（OSCC），而放射抗性是导致治疗效果不佳的关键因素。据报道，有几个基因是放射抗性的治疗靶点；然而，在OSCC细胞中，染色质可及性与放射抗性之间的关系尚未明确。因此，在本研究中，我们评估了从9种OSCC细胞系中通过照射后的克隆形成存活试验鉴定出的放射抗性（HSC-3）和放射敏感（KOSC-2）细胞中的染色质可及性。使用转座酶可及染色质高通量测序（ATAC-seq）评估放射抗性OSCC细胞中的染色质可及性。通过定量逆转录聚合酶链反应（RT-qPCR）和免疫印迹分析评估基因表达。通过MTS试验评估细胞活力。我们在HSC-3细胞中发现了1273个与8 Gy照射相关的峰（通过ATAC-seq确定的开放染色质区域），而KOSC-2细胞中未发现，其中通过ChIPpeakAnno分析确定了位于染色质开放峰周围的235个基因。随后，通过基因本体分析选择了12个基因作为信号转导相关基因，并通过RT-qPCR确认了基因表达。在这些基因中，腺苷酸环化酶2（ADCY2）在HSC-3细胞接受照射处理后显著上调，而KOSC-2细胞中未上调。为了进一步评估ADCY2在放射抗性细胞中的功能，我们通过用小干扰RNA（siADCY2）转染HSC-3细胞来敲低ADCY2。与对照细胞相比，siADCY2转染细胞照射后的细胞活力显著降低。这些结果表明，ADCY2表达与放射抗性OSCC细胞中的开放染色质区域相关，并且ADCY2与放疗联合使用时可能对OSCC患者具有治疗效果。
