Single-cell RNA sequencing integrated with bulk RNA sequencing analysis of clock circadian regulator with prognostic and immune microenvironment in thyroid cancer.

Background: Disruption of circadian rhythm was found to be associated with immune infiltration and thyroid cancer. However, the role of clock circadian regulator (CLOCK) in the progression of thyroid cancer and its immune microenvironment remains largely unexplored. Therefore, our aim was to explore the role and potential mechanism of CLOCK in thyroid cancer.

Methods: Single cell sequencing analysis and bulk RNA sequencing analysis was used for LASSO regression and Kaplan-Meier survival estimates. Potential mechanism analysis were gained through KEGG/GO analysis, GSEA analysis and PPI network. In vivo and in vitro experiment was used for further validation.

Results: The result showed CLOCK protein was overexpressed in thyroid cancer compared with normal tissue in both thyroid specific mouse model and human sample. A prognostic model incorporating CLOCK and other related genes (FAT4, OR6K2, STK40, TMEM63A, HRCT1, SUPT5H, and OR2C3) was developed using LASSO regression. Functional assay and bioinformatics analysis indicated that CLOCK knockdown hindered tumor growth and the activity of MAPK signaling. Besides, analyses of gene enrichment, signaling pathways, and immune checkpoints suggested that CLOCK might inhibit immune infiltration within the tumor microenvironment. Confirmatory in vitro experiments and immunohistochemical assays in human samples further linked high CLOCK expression to reduced T cell cytotoxicity and infiltration.

Conclusion: These findings underscore the pivotal role of CLOCK in thyroid cancer prognosis and immune suppression, highlighting its potential as a target for therapeutic intervention and prognostic assessment in thyroid cancer management.