结直肠癌（CRC）是一种异质性疾病，WNT和TGFβ介导的致癌途径的激活在该病理中经常发生。然而，迄今为止，关于昼夜节律时钟与CRC病因和分层的关联的报道有限。本研究旨在评估独立的CRC队列中重要昼夜节律标记物PER2、PER3和NR1D1的表达及其与CRC相关途径的关联。利用现有的GEO（GSE39582）和TCGA数据集进行基因表达分析。采用定量实时聚合酶链反应法量化PER2、PER3和NR1D1在FFPE（福尔马林固定石蜡嵌入）CRC组织样本中的表达水平。此外，还评估了WNT和TGFβ途径激活的肿瘤中昼夜节律标记物的富集。在GEO（P<0.0001）和TCGA结肠和直肠腺癌数据集（P<0.05）中，肿瘤与对照样本中PER3的显著下调。GEO数据集分析表明，结肠腺癌中PER2和NR1D1显著上调（P<0.01），并在FFPE验证队列中通过qRT-PCR在CRC肿瘤样本与对照组中得到确认。结肠腺癌中NR1D1的高表达与不良预后有关。相反，PER3在肿瘤中显著下调（P<0.001），并与高级CRC肿瘤相比低级肿瘤有关。WNT途径激活的肿瘤PER3显著降低，PER2略微上调（<0.0001）。有趣的是，PER3和NR1D1的差异表达与TGFβ1表达的肿瘤显著相关（P<0.0001）。此外，MYC扩增的肿瘤PER3水平下降。因此，CRC中PER3的低表达和NR1D1高表达患者的不良生存揭示了昼夜节律抑制循环中的基因在CRC中的失调，因此指出分离癌症中昼夜节律通路的重要性。

Colorectal cancer (CRC) is a heterogeneous disease and activation of WNT and TGFβ mediated oncogenic pathways is frequently observed in this pathology. However, to date, limited reports have been published addressing the association of circadian clock with CRC pathogenesis and stratification. The current study aims at assessing the expression of important circadian markers, PER2, PER3 and NR1D1, in independent CRC cohorts and their associations with CRC-related pathways.Gene expression analysis was performed using available GEO (GSE39582) and TCGA datasets. Quantitative real time polymerase chain reaction was used to quantify the expression levels of PER2, PER3 and NRID1 in FFPE (formalin fixed paraffin embedded) CRC tissue samples. Furthermore, enrichment of circadian markers in WNT and TGFβ pathways-activated tumors was assessed.Statistically significant downregulation of PER3 was found in tumor versus control samples in GEO (P<0.0001) and TCGA colon and rectal adenocarcinoma datasets (P<0.05). Analysis of GEO dataset revealed a statistically significant upregulation of PER2 (P<0.01), and NR1D1 in colon adenocarcinoma, which was confirmed by qRT-PCR in CRC tumor samples versus controls in FFPE validation cohort. Higher expression of NR1D1 was associated with poor prognosis in colon adenocarcinoma. Contrastingly, PER3 was significantly downregulated in tumors (P<0.001) compared to controls and was associated with high-grade CRC tumors versus low-grade tumors. Tumors with WNT pathway activation had significantly low PER3 and slightly upregulated PER2 (<0.0001) expression. Interestingly, differential expression of PER3 and NR1D1 was significantly correlated with TGFβ1-expressing tumors (P<0.0001). Moreover, MYC- amplified tumors exhibited decreased PER3 levels.Thus, low PER3 expression in CRC and poor survival of patients with NR1D1-high tumors reveal that genes in the suppressor loop of circadian rhythm are dysregulated in CRC, hence pointing out to the importance of dissecting the circadian pathway in cancer.