Research Paper Volume 13, Issue 24 pp 26161—26179

Coupling of serum CK20 and hyper-methylated CLIP4 as promising biomarker for colorectal cancer diagnosis: from bioinformatics screening to clinical validation

Zhongjian Liu1,2, *, , Hui Tang1,2, *, , Wen Zhang1,2, , Jinli Wang1,2, , Lilan Wan1,2, , Xisha Li1,2, , Yuping Ji3, , Na Kong3, , Yanfang Zhang3, , Jiangang Wang3, , Zhang Fan3, , Qiang Guo1,2, ,

  • 1 Department of Gastroenterology, The First People’s Hospital of Yunnan Province, Kunming, China
  • 2 The Affiliated Hospital of Kunming University of Science and Technology, Kunming, China
  • 3 Department of Gastroenterology, The Third People’s Hospital of Yunnan Province, Kunming, China
* Equal contribution

Received: September 7, 2021       Accepted: December 13, 2021       Published: December 29, 2021
How to Cite

Copyright: © 2021 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Colorectal cancer (CRC) is one of the most common and lethal malignancies. The identification of minimally invasive and precise biomarkers is an urgent need for the early diagnosis of CRC. Through bioinformatics analysis of 395 CRC tissues and 63 CRC cell lines, CK18, CK20, de-methylated HPDL and hyper-methylated CLIP4 were identified as candidate serum biomarkers. Then, a training cohort consisting of 60 CRC, 30 colorectal adenomas (CA) and 33 healthy controls and a validation cohort consisting of 60 CRC, 30 CA and 30 healthy controls were enrolled. In the training cohort, enzyme-linked immunosorbent assay (ELISA) showed that CK18 and CK20 were all significantly higher in CRC and CA. CK18 diagnosed CRC with 46.67% sensitivity and 87.3% specificity; CK20 diagnosed CRC with 28.33% sensitivity and 90.47% specificity. Methylation-specific PCR (MSP) indicated that de-methylated HPDL and hyper-methylated CLIP4 were significantly detected in CRC and CA. De-methylated HPDL diagnosed CRC with 36.67% sensitivity and 93.65% specificity and hyper-methylated CLIP4 with 73.33% sensitivity and 84.13% specificity. Random combined analysis suggested that CK20/hyper-methylated CLIP4 diagnosed CRC with 91.67% sensitivity and 82.54% specificity. In the validation cohort, CK20 diagnosed CRC with 36.7% sensitivity and 88.3% specificity and hyper-methylated CLIP4 with 80% sensitivity and 85% specificity. CK20/hyper-methylated CLIP4 diagnosed CRC with 95% sensitivity and 81.7% specificity. Compared with serum biomarkers reported before, CK20/hyper-methylated CLIP4 possessed the potential to be a new effective and precise diagnostic biomarker for CRC.


ASCL2: Achaete-Scute Family BHLH Transcription Factor 2; CA: Colorectal adenomas; CA125: Carbohydrate antigen 125; CA19-9: Carbohydrate antigen 19-9; CCLE: Cancer cell line encyclopedia; CEA (CEACAM5): Carcinoembryonic antigen; cfDNA: Cell free DNA; CK8 (KRT8): Cytokeratin 8; CK18 (KRT18): Cytokeratin 18; CK20 (KRT20): Cytokeratin 20; CLIP4: CAP-Gly Domain Containing Linker Protein Family Member 4; CRC: Colorectal cancer; DAC: 5’-Aza-2’-deoxycytiding; ELISA: Enzyme-linked immunosorbent assay; EPCAM: Epithelial Cell Adhesion Molecule; FERMT1: FERM Domain Containing Kindlin 1; GAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase; GEPIA: Gene Expression Profiling Interactive; GTEx: Genotype-Tissue Expression; HNF4G: Hepatocyte Nuclear Factor 4 Gamma; HPA: Human Protein Atlas; HPDL: 4-Hydroxyphenylpyruvate Dioxygenase Like; KCNE3: Potassium Voltage-Gated Channel Subfamily E Regulatory Subunit 3; LGR5: Leucine Rich Repeat Containing G Protein-Coupled Receptor 5; MSP: Methylation-specific PCR; MUC13: Mucin 13; SLC12A2: Solute Carrier Family 12 Member 2; TCGA: The Cancer Genome Atlas.