| evidence-clockprocrc-derivation | Circadian framework derivation | GSE10644-derived circadian reference mapped into the GSE166555 CRC single-cell cohort, followed by KNN meta-cell aggregation and high-dimensional co-expression analysis | A CRC-focused circadian transcriptional architecture was derived from a 906-gene colon circadian reference and a 94,683-cell single-cell framework. primary tissue samples: 13 CRC and 12 normal mucosal samples; post-QC cells: 94,683; cellular structure: 19 clusters; 9 major cell types; initial circadian reference: 906 genes; FDR <0.01 and R² ≥0.7 in the source temporal analysis; meta-cell sparsity setting: K=50 gave the reported optimal sparsity reduction; co-expression modules: 9; selected CRC-associated modules: M1, M3, M4 and M6; CTD signature: 559 genes across the four selected modules. | Public single-cell / systems-biology derivation | Cell counts are not participant counts. | Results: single-cell CRC landscape and high-dimensional circadian co-expression network, Fig. 1A-I and Supplementary Fig. 2; Methods: Single-cell RNA sequencing, Bulk RNA sequencing, and microarray chip cohort processing; Expression of the circadian gene set in the colorectum; High-dimensional co-expression network analysis; Supplementary Tables 2-3 | KF1 |
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| evidence-score-construction | ClockProCRC scoring and circadian robustness | ClockProCRC development and cross-species circadian robustness evaluation | ClockProCRC used expression-matched random-background correction and tracked circadian perturbation across multiple independent human and animal datasets. expression stratification: 100 expression bins; random-background sampling: 1,000 iterations with expression-matched feature selection; single-cell CRD grouping: 75th-percentile cutoff; bulk-RNA grouping: cohort median commonly used; article-reported robustness datasets: 16 independent public datasets; cross-species phase alignment: mouse Zeitgeber time shifted by 12 h before alignment with human/primate rhythms; interpretation: higher ClockProCRC values represented greater endogenous circadian dysregulation in the article's framework; score gene-set scope: The scoring Methods refers to circadian rhythm disorder genes (CRGs). The evidence page does not assume that the separate 559-gene CTD network signature is identical to the final ClockProCRC score input.. | Computational scoring and cross-species public-dataset evaluation | Circadian perturbation robustness, not human dosing-time equivalence. | Results: ClockProCRC score detection and quantification of endogenous circadian disorder states, Fig. 2B-I and Supplementary Fig. 3A; Methods: Development of ClockProCRC scoring system; Detection of the biological clock rhythm of the ClockProCRC score; Assessment of Cross-species Rhythmic Module Concordance | KF2 |
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| evidence-clinical-association | Clinical/progression association | Clinical-association and cross-cohort evaluation of ClockProCRC | Higher ClockProCRC values were associated with CRC progression-related states and poorer survival in the analyzed retrospective/public cohorts. survival cohort: GSE17537, n=55; survival association: higher ClockProCRC associated with lower overall-survival probability; log-rank P<0.01; RCS reference: ClockProCRC 0.0197 corresponded to ln(HR)=0 / HR=1 in the displayed spline model; progression trajectory: scores increased across normal mucosa → adenoma/polyp → high/severe dysplasia → CRC → colorectal liver metastasis; GSE68468 progression comparison: P<0.0001; multi-cohort robustness: >1,400 CRC samples across 10 independent cohorts. | Retrospective/public cohort association | Association is not prospective clinical utility. | Results: Analysis of clinical significance of ClockProCRC score, Fig. 3A-I; Methods: Analysis of the evaluation and clinical correlation of circadian rhythm disorder scores | KF3 |
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| evidence-single-cell-state | Tumor-microenvironment state | Single-cell analysis in GSE200997 and GSE196964 using ClockProCRC state assignment, Ro/e enrichment and NICHES signaling analysis | ClockProCRC-defined circadian states were associated with distinct single-cell composition and intercellular-communication patterns in CRC. single-cell evaluation cohorts: GSE200997 and GSE196964; intensified-CRD enrichment pattern: epithelial and plasma-cell populations were preferentially enriched in higher-disruption states; more stable-rhythm pattern: fibroblast and endothelial populations were preferentially enriched in lower-disruption/stable states; communication pattern: higher-disruption states showed stronger epithelial/plasma interaction weights and altered pathway-level cell-cell signaling; pathway pattern: progression from alleviate to intensify states was associated with reduced apoptotic-pathway activity and increased pro-inflammatory communication. | Single-cell association | Single-cell state association, not a causal human mechanism. | Results: Dissecting the circadian rhythm disorder of colorectal cancer from single-cell resolution, Fig. 4A-G and Supplementary Fig. 3C-E; Methods: Single-cell RNA data analysis; NICHES ligand-receptor signaling analysis | KF4 |
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| evidence-syne1-network | NFATC2-SYNE1 regulatory prioritization | Dysregsig-based gene-regulatory-network analysis of CTD genes followed by cross-dataset SYNE1 contextualization | Gene-regulatory-network analysis prioritized SYNE1 within a CRC-associated circadian dysregulation axis, with NFATC2-SYNE1 regulation differing between CRC and normal mucosa. genetic-algorithm iterations: 100; recurrent regulatory axes: 61; NFATC2→SYNE1 regulatory strength in CRC: 0.607; 95% CI 0.412-0.802; NFATC2→SYNE1 regulatory strength in normal mucosa: -0.102; 95% CI -0.058-0.262; circadian context: SYNE1 showed time-dependent or perturbation-associated expression changes across independent circadian datasets including GSE43073, GSE35026, GSE56931 and GSE30411; metastasis-associated expression: SYNE1 expression was higher in colorectal liver metastasis than primary CRC in the reported analyses (Wilcoxon P=0.035 and P=0.048). | Computational regulatory-network inference plus cross-dataset context | Does not establish a human therapeutic target. | Results: Key circadian and night regulatory genes screening and their effects on circadian rhythm disorder in colorectal cancer, Fig. 5A-G and Supplementary Figs. 3F, 4A-E, 5F-G; Methods: Construction of the circadian rhythm gene regulation network (GRN) | KF5 |
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| evidence-syne1-functional | SYNE1/CLOCK functional perturbation | HCT116 and SW480 CRC-cell experiments using SYNE1 or CLOCK siRNA, cell-line RNA sequencing, qRT-PCR, functional assays and melatonin rescue conditions | SYNE1 and CLOCK perturbation experiments linked the computational circadian state to CRC-cell migration, invasion, proliferation-associated phenotypes and epithelial/mesenchymal marker changes. cell models: HCT116 and SW480; SYNE1 perturbation: two specific siRNAs were used for knockdown; Transwell result: SYNE1 knockdown reduced migration and invasion; 3 independent biological replicates with 4 random microscopic fields averaged per experiment; wound-healing result: SYNE1 knockdown reduced migration; colony-formation result: SYNE1 knockdown reduced colony formation in both cell lines; epithelial/mesenchymal markers: E-cadherin and ZO-1 increased, while N-cadherin decreased after SYNE1 knockdown; CLOCK perturbation: CLOCK knockdown increased ClockProCRC; melatonin treatment partially restored the molecular state; SYNE1 rhythmicity: si-NC P=2.5×10^-4; si-CLOCK P=0.0704; si-CLOCK+melatonin P=3.4×10^-3; SYNE1 ROC scope: AUC=0.915 for discriminating CLOCK-knockdown experimental conditions, not for diagnosing CRC in patients; melatonin condition: 0.1 mM for 24 h before RNA collection in the cell experiment. | In-vitro functional evidence | No in-vivo or human treatment-efficacy conclusion. | Results: SYNE1 functional and circadian-perturbation experiments, Fig. 6A-N and related supplementary figures; Methods: Cell lines; Cell transfection with siRNA and plasmids; Bulk RNA-seq; qRT-PCR; Transwell assay and scratch assay; Western blot analysis; Cell cloning formation experiment | KF6 |
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