Single-cell and bulk RNA sequencing reveal heterogeneity and diagnostic markers in papillary thyroid carcinoma lymph-node metastasis

Abstract Purpose Papillary thyroid carcinoma (PTC) is characterized by lymph-node metastasis (LNM), which affects recurrence and prognosis. This study analyzed PTC LNM by single-cell RNA sequencing (scRNA-seq) data and bulk RNA sequencing (RNA-seq) to find diagnostic markers and therapeutic targets. Methods ScRNA-seq data were clustered and malignant cells were identified. Differentially expressed genes (DEGs) were identified in malignant cells of scRNA-seq and bulk RNA-seq, respectively. PTC LNM diagnostic model was constructed based on intersecting DEGs using glmnet package. Next, PTC samples from 66 patients were used to validate the two most significant genes in the diagnostic model, S100A2 and type 2 deiodinase (DIO2) by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and immunohistochemical (IHC). Further, the inhibitory effect of DIO2 on PTC cells was verified by cell biology behavior, western blot, cell cycle analysis, 5-ethynyl-2′-deoxyuridine (EdU) assay, and xenograft tumors. Results Heterogeneity of PTC LNM was demonstrated by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis. A total of 19 differential genes were used to construct the diagnostic model. S100A2 and DIO2 differ significantly at the RNA ( p < 0.01) and protein level in LNM patient tissues ( p < 0.001). And differed in PTC tissues with different pathologic typing ( p < 0.001). Further, EdU ( p < 0.001) and cell biology behavior revealed that PTC cells overexpressed DIO2 had reduced proliferative capacity. Cell cycle proteins were reduced and cells are more likely to be stuck in G2/M phase ( p < 0.001). Conclusions This study explored the heterogeneity of PTC LNM using scRNA-seq. By combining with bulk RNA-seq data, diagnostic markers were explored and the model was established. Clinical diagnostic efficacy of S100A2 and DIO2 was validated and the treatment potential of DIO2 was discovered.