Identification of colon tumor marker NKD1 via integrated bioinformatics analysis and experimental validation

Abstract

Background Colorectal cancer is an important death‐related disease in the worldwide. However, specific colon cancer tumor markers currently used for diagnosis and treatment are few. The purpose of this study is to screen the potential colon cancer markers by bioinformatics and verify the results with experiments. Methods Gene expression data were downloaded from two different databases: TCGA database and GEO datasets, which were then analyzed by two different methods (difference analysis and WGCNA method). Venn and PPI analysis obtained the potential core genes, which were then performed the GO enrichment and KEGG pathway analysis. Expressions levels of NKD1 in colon carcinoma tissues were further confirmed by immunohistochemical staining and western blot assays. Moreover, the function was measured by MTT, clone formation, and tumor transplantation experiments. Importantly, co‐immunoprecipitation, immunofluorescence, and protein stability assays were further performed to explore the underlying mechanism of NKD1 promoting cell proliferation. Results Nine potential core genes highly expressed in colon cancer samples were screened out by bioinformatics analysis. NKD1, one of the hub genes, highly expressed in the colon carcinoma tissues could enhance the proliferation of colon cancer cells. Mechanism research demonstrated that NKD1 was essential for the combination between Wnt signalosome (DVL) and β‐catenin, and that NKD1 knockout remarkably decreased the β‐catenin expression. Immunofluorescence assays further implied that NKD1 knockout significantly inhibited β‐catenin nuclear accumulation. Importantly, the stability of β‐catenin proteins was maintained by NKD1 in the colon cancer cells. Conclusion We believe that NKD1 well expressed in the colorectal carcinoma tissues can enhance the proliferation of colon cancer cells. Furthermore, the functions that NKD1 may have in colon cancer cells should be different from that NKD1 has played in the zebrafish. Thus, NKD1 could be a specific colorectal cancer marker.

Let‐7b‐5p inhibits colon cancer progression by prohibiting APC ubiquitination degradation and the Wnt pathway by targeting NKD1

“…GAPDH and U6 were used as internal controls for mRNA and miRNA, respectively. 5 The primer sequences are shown in Table S2 .…”

“…Our previous study demonstrated that NKD1 could promote colon cancer cell proliferation. 5 The molecular mechanism by which NKD1 regulates colon cancer progression remains unclear.…”

Clinical Significance of NKD Inhibitor of WNT Signaling Pathway 1 (NKD1) in Glioblastoma

“…All cells were cultured at 37 °C in a humidifed atmosphere containing 5% CO2. Cells were transiently transfected with pcDNA3-NKD1 plasmids (YSY Biotech, Nanjing, China) using pcDNA3-vector as negative control [18] by Lipo3000 reagent (Termo Fisher Scientifcs, Pittsburgh, USA).…”

“…Furthermore, NKD1 was elevated in specifc mouse models of intestinal tumors comparing to healthy tissues, which represents a biomarker of tumor growth [17]. Moreover, NKD1 is highly expressed in colon carcinomas and enhances colon cancer growth according to both bioinformatics analyses and experimental validations [18].…”

Analysis of the key prognostic genes and potential traditional Chinese medicine therapeutic targets in glioblastoma based on bioinformatics and network pharmacology methods

“…Washah et al ( 6 ) analyzed the principle and prospects of bioinformatics tools and databases in detail and concluded that they offer significant advantages and have broad prospects in the treatment of cancer. Relevant predictions indicate that CCNB1, CCNA2, COL1A1, and COL1A2 (extracted by bioinformatics screening) play a key role in malignant progression and the prognosis of hepatocellular carcinoma and colon cancer ( 7 , 8 ).…”