Perturbation of the circadian clock contributes to radiotherapy-induced salivary gland hypofunction

Salivary gland hypofunction is a common adverse effect during and after radiotherapy of head and neck cancers, resulting in the dry mouth syndrome called xerostomia. Previous studies suggested that the functionality of the salivary gland is under the regulation of the circadian clock, however, the extent and scope of this regulation remains unexplored. Here, we profiled the diurnal fluctuation of gene expression in the mouse submandibular salivary gland. We further analyzed the regulatory role of key circadian transcription factors Bmal1, Nr1d1 (Rev-erba), and Dbp, which revealed a wide range of potential down-stream target genes. The circadian clock was disrupted upon irradiation, as revealed by gene expression analysis. We propose that the mechanism of salivary gland hypofunction in radiotherapy involves perturbation of the circadian clock. Overall design: For RNA-seq, we euthanized mice aged 6-8 weeks using the neck dislocation method, cut the skin along the neck to expose the submandibular gland, and then remove the gland tissue. Total RNAs were extracted using the Trizol method. Library construction and sequencing were performed on the DNBSEQ-T7 platform at the Geneplus Co, Shenzhen, China. Final data were presented as FPKM value for each gene. Tissue samples were collected at ZT3, ZT9 and ZT15 (10:00 am, 4:00 pm, and 10:00 pm). For Cut-taq, the mouse submandibular gland was minced and digested with Worthington Collagenase I at 0.2% concentration for 25-30 minutes at 37°C. The sample was filtered through a Falcon 40um filter, and the remaining tissue was incubated with 1x Gibco Trypsin/EDTA at 37°C for 20 minutes. After filtration, the sample was resuspended in PBS and centrifuged at 500 g for 5 minutes to obtain the supernatant. Standard operations were performed according to the instructions ofTD901-TD902 from Hyperactive In-Situ Chip Library Prep Kit for IIIumina. The 150 bp paired-end DNA sequencing was performed on the Illumina NovaSeq 6000 system (Haplox Genomics Center, Jiangxi, China).