Abstract
OBJECTIVE: To investigate the effects of low background radiation environments in deep underground settings on the biological behavior of NP69 human nasopharyngeal epithelial cells (NP69 cells) and the underlying molecular mechanisms. METHODS: A parallel control experimental design was adopted and NP69 cells were synchronously cultured in settings of three underground depths at the China in situ Deep-Underground Facility & Life Observatory (DeUFO)-ground level (DeUFO-0 m), 1000 m underground (DeUFO-1000 m), and 1500 m underground (DeUFO-1500 m). Changes in cell proliferation and migration capabilities were assessed using the Cell Counting Kit-8 (CCK-8) assay and scratch assay, respectively. High-throughput RNA sequencing (RNA-Seq) was performed to identify differentially expressed genes (DEGs). Functional annotation and pathway enrichment analysis of the DEGs were performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. RESULTS: CCK-8 assay revealed that, after 72 h of culture, the absorbance value of the DeUFO-0 m group was 1.35 times and 1.27 times those of the those of the DeUFO-1000 m and DeUFO-1500 m groups, respectively (both P < 0.0001). After 96 h of culture, the absorbance value of the DeUFO-0 m group was 1.52 times and 1.41 times those of the DeUFO-1000 m and DeUFO-1500 m groups, respectively (both P < 0.0001). Colony formation assays revealed that the number of cell colonies in the DeUFO-0 m group was 1.59 times and 1.27 times those in the DeUFO-1000 m group and DeUFO-1500 m group, respectively (both P < 0.001). The scratch assay revealed that the 36-hour wound healing rate of the DeUFO-0 m group was 2.22 times and 4.00 times those of the DeUFO-1000 m group and DeUFO-1500 m group, respectively (both P < 0.0001). Transwell assays revealed that the number of migrating cells in the DeUFO-0 m group was 2.08 times and 2.56 times those in the DeUFO-1000 m group and DeUFO-1500 m group, respectively (both P < 0.0001). Transcriptome sequencing analysis revealed consistent upregulation of CELF2, CELF4, CGB8, GRHL2, and DMRTA2 genes in the DeUFO-1000 m and DeUFO-1500 m groups. Pathway enrichment analysis indicated significant enrichment of extracellular matrix (ECM) remodeling-associated pathways and gene expression regulation pathways in the experimental groups (false discovery rate [FDR] < 0.05). CONCLUSION: The low background radiation environment in deep underground settings suppresses the proliferation and migration activities of NP69 cells by mediating ECM remodeling and post-transcriptional regulatory mechanisms through the regulation of target genes such as the CELF family. This study provides experimental evidence for establishing a dose-response relationship between environmental radiation and cellular effects.