Isolation and target identification of antihepatoma polyprenylphenols from the edible mushroom Suillus granulatus.

Eight polyprenylphenol derivatives were isolated from the wild edible mushroom Suillus granulatus, including seven novel compounds, named suillin F-L (2-8), and one previously identified compound (1). The structures of the new compounds were elucidated using HR-ESI-MS and 1D and 2D NMR data. The absolute configuration of compound 8 was assigned based on the comparison of the experimental and calculated ECD data. All isolated compounds were evaluated for their cytotoxicity against HepG2 cancer cell lines. Compounds 1 and 3-6 demonstrated significant antitumor activity compared to the positive control (cisplatin), with IC50 values ranging from 8.19 to 13.97 µM. Furthermore, DARTS assay and LC-MS/MS analysis were used to identify HSP90AA1 as the direct target of compound 5, and the interaction between compound 5 and HSP90AA1 was verified by molecular docking.

Anti-Hyperglycemic Effect of the Brown Slime Cap Mushroom Chroogomphus rutilus (Agaricomycetes) Crude Polysaccharide In Vitro and In Vivo.

The prevalence of diabetes is increasing worldwide, and it is very important to study new hypoglycemic active substances. In this study, we investigated the hypoglycemic effect of Chroogomphus rutilus crude polysaccharide (CRCP) in HepG2 cells and streptozotocin-induced diabetic mice. A glucose consumption experiment conducted in HepG2 cells demonstrated the in vitro hypoglycemic activity of CRCP. Furthermore, CRCP exhibited significant hypoglycemic effects and effectively ameliorated insulin resistance in insulin resistant HepG2 cells. In high-fat diet and streptozotocin-induced diabetic mice, after 4 weeks of CRCP administration, fasting blood glucose, fasting serum insulin, triglyceride, total cholesterol, low-density lipoprotein cholesterol, glutamate transaminase, alanine transaminase, and insulin resistance index significantly decreased, while high-density lipoprotein cholesterol and insulin sensitivity index (ISI) were markedly increased. Moreover, hematoxylin-eosin (HE) staining and immunofluorescence labeling of tissue sections indicated that CRCP attenuated the pathological damage of liver and pancreas in diabetic mice. These results indicate that CRCP is a potential hypoglycemic agent.

ALOX15+ M2 macrophages contribute to epithelial remodeling in eosinophilic chronic rhinosinusitis with nasal polyps.

BACKGROUND: Epithelial remodeling is a prominent feature of eosinophilic chronic rhinosinusitis with nasal polyps (eCRSwNP), and infiltration of M2 macrophages plays a pivotal role in the pathogenesis of eCRSwNP, but the underlying mechanisms remain undefined. OBJECTIVE: We sought to investigate the role of ALOX15+ M2 macrophages in the epithelial remodeling of eCRSwNP. METHODS: Digital spatial transcriptomics and single-cell sequencing analyses were used to characterize the epithelial remodeling and cellular infiltrate in eCRSwNP. Hematoxylin and eosin staining, immunohistochemical staining, and immunofluorescence staining were used to explore the relationship between ALOX15+ M2 (CD68+CD163+) macrophages and epithelial remodeling. A coculture system of primary human nasal epithelial cells (hNECs) and the macrophage cell line THP-1 was used to determine the underlying mechanisms. RESULTS: Spatial transcriptomics analysis showed the upregulation of epithelial remodeling-related genes, such as Vimentin and matrix metalloproteinase 10, and enrichment of epithelial-mesenchymal transition (EMT)-related pathways, in the epithelial areas in eCRSwNP, with more abundance of epithelial basal, goblet, and glandular cells. Single-cell analysis identified that ALOX15+, rather than ALOX15-, M2 macrophages were specifically highly expressed in eCRSwNP. CRSwNP with high ALOX15+ M2THP-1-IL-4+IL-13 macrophages had more obvious epithelial remodeling features and increased genes associated with epithelial remodeling and integrity of epithelial morphology versus that with low ALOX15+ M2THP-1-IL-4+IL-13 macrophages. IL-4/IL-13-polarized M2THP-1-IL-4+IL-13 macrophages upregulated expressions of EMT-related genes in hNECs, including Vimentin, TWIST1, Snail, and ZEB1. ALOX15 inhibition in M2THP-1-IL-4+IL-13 macrophages resulted in reduction of the EMT-related transcripts in hNECs. Blocking chemokine (C-C motif) ligand 13 signaling inhibited M2THP-1-IL-4+IL-13 macrophage-induced EMT alteration in hNECs. CONCLUSIONS: ALOX15+ M2 macrophages are specifically increased in eCRSwNP and may contribute to the pathogenesis of epithelial remodeling via production of chemokine (C-C motif) ligand 13.

Six new polyphenolic metabolites isolated from the Suillus granulatus and their cytotoxicity against HepG2 cells.

Edible mushrooms are an important source of nutraceuticals and for the discovery of bioactive metabolites as pharmaceuticals. In this work, six new polyphenolic metabolites suillusol A-D (1-4), suillusinoic acid (5), ethyl suillusinoate (6), were isolated from the Suillus granulatus. The structures of new compounds were elucidated using high-resolution electrospray ionization mass spectroscopy, nuclear magnetic resonance data, and single-crystal X-ray diffraction analysis. As far as we know, compound 1 represents an unprecedented type of natural product and compound 3 represents a new type of polyphenol fungal pigment, which may be biosynthetically related to thelephoric acid. The cytotoxicity against HepG2 cells of the new compounds were also evaluated. Compound 2 demonstrate significant inhibitory activity against HepG2 cells with IC50 values of 10.85 µM, surpassing that of positive control cisplatin. Moreover, compound 1 and 3 also exhibited moderate cytotoxic activity with their IC50 values measured at 35.60 and 32.62 µM, respectively. Our results indicate that S. granulatus is a rich source of chemical constituents that may provide new lead compounds for the development of anticancer agents.

Chemical Compounds, Bioactivities, and Potential Applications of the Mushroom Species of Genus Suillus (Agaricomycetes): A Review.

The genus Suillus, also known as "Song mo," falls under the order Boletales and consists of various higher fungi. It establishes mycorrhizae primarily with pine trees and has a good taste and medicinal values. Herein, we reviewed the chemical compounds present in the genus Suillus, including polysaccharides, steroids, phenols, polyprenyl phenol derivatives, fatty acids, organic acids, and amino acids, and their reported bioactivities and potential applications. This review aims to promote the utilization of the resources belonging to the genus Suillus and serves as a theoretical basis for their future studies and clinical applications.

Vitamin D promotes epithelial tissue repair and host defense responses against influenza H1N1 virus and Staphylococcus aureus infections.

BACKGROUND: Early studies indicated that vitamin D (VD) exerted pleiotropic extra-skeletal effects in the airway, but the definite linkage between VD deficiency and airway host responses remains unclear. METHODS: 142 cases of clinical data from Department of Otolaryngology, the Seventh Affiliated Hospital of Sun Yat-sen University, were collected to characterize the relationship between VD deficiency and chronic rhinosinusitis (CRS). Based on the clinical observations, 2.5-D airway epithelial organoids cultured at the air-liquid interface (ALI) were used to simulate the effects of VD treatment in the development of airway epithelium and the modulation of the host responses against influenza H1N1 virus (representing viral infections) and Staphylococcus aureus (representing bacterial infections) infections in the airway. The intrinsic mechanisms of VD deficiency underlying epithelial remodeling were mapped by transcriptomic as well as proteomic analyses. RESULTS: In this study we observed prevailing VD deficiency among inpatients suffering from CRS, a common disease predominantly characterized by epithelial impairment and remodeling. Relative to control organoids cultured without VD, long-term incubation with VD accelerated basal cell proliferation during nasal epithelial development. Under infectious conditions, VD treatment protected the organoids against influenza H1N1 virus and Staphylococcus aureus invasions by reinforcing the respiratory host defenses, including upregulation of LL37, suppression (or inhibition) of proinflammatory cytokines, strengthening of epithelial integrity, and mucociliary clearance. In silico analysis of transcriptomics and proteomics suggested that VD modulated the epithelial development and remodeling, involving epithelial cell proliferation/differentiation, epithelial-mesenchymal transition (EMT), and cytokine signaling in the immune system, as well as responses to microbe, cell junction organization, and extracellular matrix organization via PTEN signaling, independent of TGF-ß signaling. CONCLUSIONS: Our findings emphasize the importance of managing VD deficiency in clinical settings for the sake of alleviating pathological epithelial remodeling. Vitamin D promotes epithelial tissue repair and host defense responses against influenza H1N1 and Staphylococcus aureus infections.

[Analysis of clinical features and PAK1 gene variant in a child with epilepsy and global developmental delay].

OBJECTIVE: To investigate the clinical phenotype and genetic basis of a child with epilepsy and global developmental delay. METHODS: A child with epilepsy and global developmental delay who had visited West China Second University Hospital, Sichuan University on April 1, 2021 was selected as the study subject. Clinical data of the child were reviewed. Genomic DNA was extracted from peripheral blood samples of the child and his parents. Whole exome sequencing (WES) was carried out for the child, and candidate variant was verified by Sanger sequencing and bioinformatic analysis. A literature review was also carried out by searching databases such as Wanfang data knowledge service platform, China National Knowledge Infrastructure, PubMed, ClinVar and Embase to summarize the clinical phenotypes and genotypes of the affected children. RESULTS: The child was a 2-year-and-2-month-old male with epilepsy, global developmental delay and macrocephaly. Results of WES showed that the child has harbored a c.1427T>C variant of the PAK1 gene. Sanger sequencing confirmed that neither of his parents has carried the same variant. Only one similar case had been recorded by the dbSNP, OMIM, HGMD, and ClinVar databases. No frequency for this variant among Asian population was available in the ExAC, 1000 Genomes, and gnomAD databases. Prediction with IFT, PolyPhen-2, LRT, Mutation Taster, and FATHMM online software suggested that this variant is deleterious to the function of encoded protein. Based on the Standards and Guidelines for the Interpretation of Sequence Variants: A Joint Consensus Recommendation of the American College of Medical Genetics and Genomics (ACMG), the PAK1 gene c.1427T>C variant was determined to be likely pathogenic. CONCLUSION: The PAK1 gene c.1427T>C variant probably underlay the epilepsy and global developmental delay in this child, which has provided a reference for the clinical diagnosis and genetic counseling in children with similar disorders.

Arterial spin labeling for presurgical localization of refractory frontal lobe epilepsy in children.

BACKGROUND: Epilepsy is one of the most common chronic neurological diseases. Despite the great variety and prevalence of antiepileptic drug treatments, one-third of epilepsies remain drug resistant. The frontal lobe is extensive, and frontal lobe seizures are difficult to locate, which increases the difficulty of the preoperative localization of the epileptogenic zone. CASE PRESENTATION: Two previously healthy girls with refractory frontal lobe epilepsy showed significant perfusion abnormalities in the right frontal lobe using the cerebral blood perfusion (CBF) quantitative analysis system. They became seizure-free after lesionectomy of the frontal lobe by ASL combined with electroencephalography (EEG) rapid localization. The histopathological diagnosis was focal cortical dysplasia (FCD) type IIa and IIb. CONCLUSIONS: The positive outcome suggests that the combined use of ASL with EEG could be a beneficial option for the presurgical evaluation of pediatric epilepsy.

Novel hypoglycemic compounds from wild mushroom Paxillus involutus.

Wild mushrooms are an important source of secondary metabolites possessing a broad range of biological activities. In this study, eight new compounds, named furanopaxin A-F (1-6), deoxybisinvolutone (7), and coumarinvol (8) along with two known compounds were isolated from fruiting bodies of the wild mushroom Paxillus involutus (Batsch) Fr. Their structures were established based on HR-ESI-MS and 1D and 2D NMR spectroscopic data. The results of hypoglycemic assays indicated that compounds 5-8 possessed significant α-glucosidase inhibitory activities, with IC50 values ranging from 14.65 ± 1.68 to 47.55 ± 1.47 µM, and each compound could enhance glucose consumption in insulin-resistance HepG2 cells. Further analysis by molecular docking implied that compounds 5-8 could interact with the amino acid residues of α-glucosidase, supporting the hypoglycemic activity of the compounds.

Modified adenoid grading system for evaluating adenoid size in children: a prospective validation study.

Nasal endoscopy is the best choice for evaluation of adenoid size, but very few studies published on the endoscopic quantitative assessment. This study aimed to newly propose and validate a modified adenoid grading system (MAGS) with the existing endoscopic scoring methods of adenoid size. A prospective study on children with chronic mouth breathing and having endoscopic nasal examination was conducted. Digital images obtained during endoscopic examination were evaluated with the traditional method and the MGAS. Adenoid size was also evaluated by intraoperative nasal endoscopy among those underwent adenoidectomy. One hundred and thirty patients were enrolled. The MAGS showed high inter-rater reliability with a Kappa score of 0.869. Sixty of 130 patients underwent adenoidectomy and assessed with intraoperative nasal endoscopy. The MAGS significantly correlated to the percentage of nasopharyngeal obstruction of intraoperative endoscopy (Spearman's r = 0.796, gamma coefficient = 0.94), and the percentage of choanal obstruction of preoperative endoscopy (Spearman's r = 0.816, gamma coefficient = 0.859). Our findings suggest that the MAGS has high reliability and validity for assessment of adenoid size. It may be a more suitable and reliable grading system for endoscopic evaluation of adenoid size.

Novel 2,5-Diarylcyclopentenone Derivatives from the Wild Edible Mushroom Paxillus involutus and Their Antioxidant Activities.

Wild edible mushrooms are important as a source of nutraceuticals and for the discovery of bioactive metabolites as pharmaceuticals. In this work, 10 rare 2,5-diarylcyclopentenone derivatives were isolated from the wild edible mushroom Paxillus involutus (Batsch) Fr., including eight novel compounds termed involutenone A-H (1-8) and two previously identified compounds (9-10). Their structures were established using high-resolution electrospray ionization mass spectroscopy and 1D and 2D nuclear magnetic resonance data. The absolute configurations of compounds 1-3 and 6-8 were assigned based on the comparison of the experimental and calculated electronic circular dichroism data. The antioxidant activities of 1-8 were tested through DPPH free radical scavenging, hydroxyl radical scavenging, and superoxide anion radical scavenging assays. Compounds 3, 5, 6, and 7 demonstrated significant antioxidant activity compared to the positive control (tert-butylhydroquinone). These compounds could be effective natural antioxidants with considerable pharmaceutical value.

Exploration of the relationship between tumor mutation burden and immune infiltrates in colon adenocarcinoma.

Background: Tumor mutation burden (TMB) was correlated with the immunotherapeutic response in various malignancies. We aimed to evaluate the TMB immune signature in colon adenocarcinoma (COAD). Methods: Gene expression profile, mutation and clinical data of COAD patients were obtained from The Cancer Genome Atlas (TCGA) database. The samples were divided into high and low TMB level groups to identify differentially expressed genes (DEGs). Functional enrichments analyzes were performed to identify the biological functions of the DEGs. Then, immune cell infiltration signatures were calculated by the CIBERSORT algorithm. Finally, Cox proportional hazard model was constructed to estimate the prognostic value of the identified immune-related genes. Results: Gene set enrichment analysis in the high-TMB level group showed that DEGS were enriched in immune-related pathways, such as antigen processing and presentation, Toll-like receptor signaling and natural killer cell-mediated cytotoxicity. A higher infiltration level of CD8+ T cells, CD4+ T cells, activated NK cells , M1 Macrophages and T follicular helper cells was observed in the high-TMB level group. Furthermore, a Cox regression model combined with survival analysis based on the expression level of four identified prognostic genes was constructed, validated anf revealed that higher risk-score levels conferred poor survival outcomes in COAD patients. Conclusions: Our data demonstrate that the high TMB levels are associated with an immune signature in COAD and deepen the molecular understanding of TMB function in tumor immunotherapy.

An arbuscular mycorrhizal fungus increased the macroaggregate proportion and reduced cadmium leaching from polluted soil.

NOVELTY STATEMENT: AMF significantly increased the GRSP content and the macroaggregate proportion in soil, which contributed to reducing the Cd concentration in pore water and its leaching loss from soil.

[Fine modeling of nasal cavity and its application in precision surgical design].

In recent years, the research hotspot of rhinology is looking forward to effective method for changing the anatomical structure of nasal cavity and paranasal sinus. And it will build up the optimal relationship between airflow field and biological effects in rhinology surgency. Now in precise medical treatment, we are eager to find a more reasonable and optimized surgical method before operation, with the help of information technology and big data science. It will not only achieve the greatest biological effect, but also maximize the protection of the lateral wall of the nasal cavity, in order to eliminate the potential recurrence of sinusitis. This paper mainly includes three parts: status and trends of nasal cavity modeling, biomechanics research progress and progress in evaluation of nasal surgery. It is very helpful for promoting the applying of up-to-date model in precise surgical design.

Association of tricellulin expression with poor colorectal cancer prognosis and metastasis.

Tricellulin is a tight­junction transmembrane protein that regulates cell­cell interactions. Altered tricellulin expression could promote tumor cell invasions and metastasis in human cancers. The present study assessed tricellulin expression in colorectal cancer tissues for any association with clinicopathological features of colorectal cancer patients and then investigated the underlying molecular events using quantitative proteomic analysis and in vitro experiments. Tissue samples from 98 colorectal cancer patients and 15 volunteers were collected for immunohistochemistry. Colorectal cell lines were used to overexpress or knockdown tricellulin expression in various assays. The data revealed that upregulated tricellulin expression was associated with lymph node and distant metastases and poor prognosis, while tricellulin overexpression promoted colorectal cancer cell migration and invasion in vitro. In contrast, tricellulin knockdown had positive effects on the tumor cells. Furthermore, TMT­LC­MS/MS and bioinformatics analyses revealed that tricellulin was involved in EMT and reduction of apoptosis through the NF­κB signaling pathway. These findings highlight for the first time the significance of tricellulin in colorectal cancer development and progression. Further study may validate tricellulin as a novel biomarker and target for colorectal cancer.

Protective effect of coumarin-pi against t-BHP-induced hepatotoxicity by upregulating antioxidant enzymes via enhanced Nrf2 signaling.

Coumarin-pi, a new coumarin derivative isolated from the mushroom Paxillus involutus, has antioxidative activity, but the underlying mechanism against intracellular oxidative stress is still unclear. This study investigated its cytoprotective effects and the antioxidative mechanism in tert-butyl hydroperoxide (t-BHP)-induced HepG2 cells. The results demonstrated that coumarin-pi suppressed t-BHP-stimulated cytotoxicity, cell apoptosis, and generation of reactive oxygen species (ROS). Additionally, coumarin-pi promoted nuclear factor erythroid 2-related factor 2 (Nrf2) expression and upregulated the protein expression of antioxidantenzymes, including heme oxygenase-1 (HO-1), NAD(P)H: quinone oxidase (NQO1), glutamyl cysteine ligase catalytic subunit (GCLC), and glutamate-cysteine ligase regulatory subunit (GCLM). After coumarin-pi treatment, transcriptome sequencing and bioinformatic analysis revealed that 256 genes were differentially expressed; interestingly, only 20 genes were downregulated, and the rest of the genes were upregulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation were used to identify changes in metabolic pathways. Collectively, the results presented in this study indicate that coumarin-pi has a protective effect against t-BHP-induced cellular damage and oxidative stress.

Evi-1 Influence on Clinical Progress of Colorectal Cancer Patients.

OBJECTIVE: Aim of this study is to assess effects of Evi-1 on regulation of c-Jun N-terminal kinase (JNK)/c-Jun pathway on colorectal cancer and the relationship of their expression with clinicopathological characteristics and prognosis. METHODS: The clinical data of 394 CRC patients and the mRNA expression of Evi-1 were downloaded from The Cancer Genome Atlas (TCGA). Immunohistochemical (IHC) method was used to detect the protein expression of Evi-1, JNK and c-Jun in CRC tissues and adjacent normal tissues. RESULTS: The TCGA dataset demonstrated that Evi-1 mRNA was upregulated in CRC. Evi-1 mRNA expression was significantly correlated with lymphatic metastasis, T stage, distant metastasis and TNM stage. IHC staining showed that Remmele immunoreactive Score (IRS) of Evi-1, JNK and c-Jun were highly expressed in CRC tissues compared with normal tissues adjacent to cancer. Evi-1 in CRC tissues was negatively correlated with JNK and c-Jun. Overexpression of Evi-1, JNK and c-Jun was significantly correlated with the degree of differentiation, T staging, lymphatic metastasis, distant metastasis and TNM classification. CONCLUSION: The present study shows that Evi-1 expression is negatively correlated with JNK and c-Jun expression, and closely related to some clinical data. Further, Evi-1 combined with JNK and c-Jun can be used for adverse prognosis of CRC.

Cinnamaldehyde improves the growth performance and digestion and absorption capacity in grass carp (Ctenopharyngodon idella).

The present study evaluated the effect of cinnamaldehyde (CIN) on the growth performance and digestion and absorption capacity of grass carp (Ctenopharyngodon idella). Fish were fed five diets including graded levels of CIN for 60 days. The results indicated that (1) appropriate CIN supplementation increased the growth performance and promoted the intestine growth of grass carp; (2) dietary appropriate CIN supplementation increased the digestion and absorption capacity by increasing the activities of intestinal and hepatopancreas digestive enzymes (lipase, chymotrypsin, trypsin, and amylase) and intestinal brush border enzymes (creatine kinase (CK), Na+/K+-ATPase, γ-glutamyl transpeptidase (γ-GT), and alkaline phosphatase (AKP)); (3) dietary CIN increased the absorption capacity which may be associated with the upregulated messenger RNA (mRNA) abundances of their amino acid transporters (AATs) in the intestine, which might be associated with activating the target of rapamycin (TOR) signaling pathway. The best CIN supplementation in the diets of grass carp was estimated to be 76.40 mg kg-1 diet based on the best percent weight gain (PWG). In general, CIN increased the digestion and absorption capacity of grass carp and raised the mRNA abundances of AATs which may be partly related to activation of the TOR signaling pathway.

High expression of CDCA7 predicts tumor progression and poor prognosis in human colorectal cancer.

Colorectal cancer (CRC) is one of the most fatal types of cancer worldwide. This study aimed to determine the predictive and prognostic values of cell division cycle associated protein 7 (CDCA7) in CRC. Firstly, the relationship between CDCA7 and CRC was assessed through bioinformatics analysis. Subsequently, CDCA7 expression levels were detected in various CRC cell lines, as well as 15 fresh human CRC tissues and their paired adjacent normal colorectal tissues using reverse transcription­quantitative PCR and western blotting. Additionally, immunohistochemical staining was used to determine the levels of CDCA7 in 104 CRC tissues and their paired adjacent normal colorectal tissues. The present study revealed that CDCA7 expression was upregulated in CRC tissues and cell lines. The positive expression rates of CDCA7 in normal and CRC tissues were 26.92 and 75.96%, respectively. The intensities of CDCA7 immunostaining were significantly associated with CRC invasion depth, lymph node metastasis, tumor­node­metastasis stage and distant metastasis. However, no significant differences in sex, age, tumor size and CRC differentiation were found between high and low CDCA7 expression groups. Furthermore, patients with low CDCA7 expression exhibited a greater overall survival rate of CRC compared to those with high CDCA7 expression. The findings of this study indicated that CDCA7 may serve a significant role in CRC prognosis and progression, and may be considered a novel biomarker for the prediction of patient survival after colectomy.

Upregulation of nectin-4 is associated with ITGB1 and vasculogenic mimicry and may serve as a predictor of poor prognosis in colorectal cancer.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Unlike endothelium-dependent vasculature, vasculogenic mimicry (VM) is an alternative type of blood supply in tumors that is frequently associated with poor patient outcome. Nectin-4 serves a vital role in the formation and maintenance of adherens junctions; integrin ß-1 (ITGB1) promotes tumor invasion, metastasis and VM formation. In the present study, the analysis of nectin-4 mRNA expression in a database of The Cancer Genome Atlas (TCGA) was combined with that of another non-overlapping cohort of 68 patients with CRC. TCGA data were used to examine nectin-4 mRNA expression in CRC and its correlation with the clinicopathological features of patients. Data from the non-overlapping cohort of patients was used to determine nectin-4 and ITGB1 protein expression in CRC by immunohistochemical (IHC) staining. Cluster of differentiation 34/periodic acid-Schiff double staining was performed to validate the presence of VM formation. The association with, and significance of combining nectin-4, ITGB1 protein expression and VM formation for predicting patient prognosis was evaluated. The TCGA dataset demonstrated that nectin-4 mRNA was upregulated in CRC, which was significantly relate to lymph node metastasis (P=0.0017), distant metastasis (P=0.0045), and tumor-node-metastasis (TNM) stage (P=0.0015). Of the 68 patients analyzed by IHC staining, 48 (70.6%) were positive for nectin-4, 46 (67.6%) for ITGB1 and 17 (25%) for VM formation. Nectin-4 protein expression was associated with ITGB1 protein expression (P<0.01) and VM formation (P<0.05). Nectin-4, ITGB1 expression and VM formation were associated with distant metastasis stage (P<0.05) and TNM stage (P<0.05). Based on these findings it was concluded that nectin-4 was upregulated in CRC tissues compared with normal mucosal tissues, and was associated with ITGB1 expression and VM formation. Furthermore, nectin-4 and ITGB1 protein expression, together with VM formation may be used to predict poor prognosis in CRC.