The circadian gene CRY2 is associated with breast cancer aggressiveness possibly via epigenomic modifications
Although the role of core circadian gene cryptochrome 2 (CRY2) in breast tumorigenesis has been demonstrated, the correlations of CRY2 with clinical parameters in breast cancer patients and its involvement in epigenetic processes such as DNA methylation remain relatively unexplored. In the current study, we first queried the Oncomine database and the Gene Expression-Based Outcome for Breast Cancer Online (GOBO) database to identify associations between CRY2 expression levels and clinical parameters in breast cancer patients. We then silenced CRY2 in vitro and performed a genome-wide methylation array to determine the epigenetic impact of CRY2 silencing. The Ingenuity Pathway Analysis software was used to further explore the genes exhibiting altered methylation identified using the array. We found that CRY2 was frequently down-regulated in breast cancer tissue compared to adjacent normal tissue or breast tissue from healthy controls. Lower CRY2 expression was associated with estrogen receptor (ER)-negativity (P < 0.0001), higher tumor grade (P < 0.0001), and shorter overall survival time in breast cancer patients (HR = 1.44, 95 % confidence interval (CI) 1.09–1.91). Genome-wide methylation analysis showed that a total of 515 CpG sites were hypermethylated following CRY2 knockdown, while 730 sites were hypomethylated. The pathway analysis revealed several cancer-relevant networks with genes exhibiting significantly altered methylation following CRY2 silencing. These findings suggest that the core circadian gene CRY2 is associated with breast cancer progression and prognosis, and that knockdown of CRY2 causes the epigenetic dysregulation of genes involved in cancer-relevant pathways, which provide further evidence supporting a role of the circadian system in breast tumorigenesis.
KeywordsCRY2 Breast cancer Biomarker DNA methylation Network analysis
This work was supported by the National Institutes of Environmental Health Sciences (NIEHS) grant ES018915.
Conflicts of interest
The authors declare that they have no competing interests.
YM carried out the DNA methylation assay, public data search and analysis, and drafted the manuscript. AF and DIJ participated in the data analysis and manuscript preparation. AEH constructed the expression vector and helped with the DNA methylation analysis. MJ and KC participated in the design of the study and helped the statistical analysis. YZ conceived the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
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