Humans ; Proto-Oncogene Proteins p21(ras)/genetics* ; Prognosis ; Biomarkers, Tumor/genetics* ; Mutation/genetics* ; Colorectal Neoplasms/genetics* ; Proto-Oncogene Proteins B-raf/metabolism*

BACKGROUND: Microsatellite instability (MSI) is a key biomarker for cancer immunotherapy and prognosis. Integration of MSI testing into a next-generation-sequencing (NGS) panel could save tissue sample, reduce turn-around time and cost, and provide MSI status and comprehensive genomic profiling in single test. We aimed to develop an MSI calling model to detect MSI status along with the NGS panel-based profiling test using tumor-only samples. METHODS: From January 2019 to December 2020, a total of 174 colorectal cancer (CRC) patients were enrolled, including 31 MSI-high (MSI-H) and 143 microsatellite stability (MSS) cases. Among them, 56 paired tumor and normal samples (10 MSI-H and 46 MSS) were used for modeling, and another 118 tumor-only samples were used for validation. MSI polymerase chain reaction (MSI-PCR) was performed as the gold standard. A baseline was built for the selected microsatellite loci using the NGS data of 56 normal blood samples. An MSI detection model was constructed by analyzing the NGS data of tissue samples. The performance of the model was compared with the results of MSI-PCR. RESULTS: We first intersected the target genomic regions of the NGS panels used in this study to select common microsatellite loci. A total of 42 loci including 23 mononucleotide repeat sites and 19 longer repeat sites were candidates for modeling. As mononucleotide repeat sites are more sensitive and specific for detecting MSI status than sites with longer length motif and the mononucleotide repeat sites performed even better than the total sites, a model containing 23 mononucleotide repeat sites was constructed and named Colorectal Cancer Microsatellite Instability test (CRC-MSI). The model achieved 100% sensitivity and 100% specificity when compared with MSI-PCR in both training and validation sets. Furthermore, the CRC-MSI model was robust with the tumor content as low as 6%. In addition, 8 out of 10 MSI-H samples showed alternations in the four mismatch repair genes ( MLH1 , MSH2 , MSH6 , and PMS2 ). CONCLUSION MSI status can be accurately determined along the targeted NGS panels using only tumor samples. The performance of mononucleotide repeat sites surpasses loci with longer repeat motif in MSI calling.
Humans ; Microsatellite Instability ; Colorectal Neoplasms/diagnosis* ; Microsatellite Repeats/genetics* ; DNA Mismatch Repair

BACKGROUND: Cholecystectomy is a standard surgery for patients suffering from gallbladder diseases, while the causal effects of cholecystectomy on colorectal cancer (CRC) and other complications are still unknown. METHODS: We obtained genetic variants associated with cholecystectomy at a genome-wide significant level ( P value <5 × 10 -8 ) as instrumental variables (IVs) and performed Mendelian randomization (MR) to identify the complications of cholecystectomy. Furthermore, the cholelithiasis was also treated as the exposure to compare its causal effects to those of cholecystectomy, and multivariable MR analysis was carried out to judge whether the effect of cholecystectomy was independent of cholelithiasis. The study was reported based on Strengthening the Reporting of Observational Studies in Epidemiology Using Mendelian Randomization guidelines. RESULTS: The selected IVs explained 1.76% variance of cholecystectomy. Our MR analysis suggested that cholecystectomy cannot elevate the risk of CRC (odds ratio [OR] =1.543, 95% confidence interval [CI]: 0.607-3.924). Also, it was not significant in either colon or rectum cancer. Intriguingly, cholecystectomy might decrease the risk of Crohn's disease (OR = 0.078, 95% CI: 0.016-0.368) and coronary heart disease (OR = 0.352, 95% CI: 0.164-0.756). However, it might increase the risk of irritable bowel syndrome (IBS) (OR = 7.573, 95% CI: 1.096-52.318). Cholelithiasis could increase the risk of CRC in the largest population (OR = 1.041, 95% CI: 1.010-1.073). The multivariable MR analysis suggested that genetic liability to cholelithiasis could increase the risk of CRC in the largest population (OR = 1.061, 95% CI: 1.002-1.125) after adjustment of cholecystectomy. CONCLUSIONS The study indicated that cholecystectomy might not increase the risk of CRC, but such a conclusion needs further proving by clinical equivalence. Additionally, it might increase the risk of IBS, which should be paid attention to in clinical practice.
Humans ; Mendelian Randomization Analysis ; Irritable Bowel Syndrome ; Colorectal Neoplasms/genetics* ; Cholelithiasis/complications* ; Cholecystectomy/adverse effects* ; Genome-Wide Association Study ; Polymorphism, Single Nucleotide

Humans ; Adsorption ; MicroRNAs/genetics* ; Cell Line, Tumor ; Colorectal Neoplasms/genetics* ; RNA, Long Noncoding ; Cell Proliferation ; Gene Expression Regulation, Neoplastic

Fusobacterium nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. The two-component system plays an important role in the regulation and expression of genes related to pathogenic resistance and pathogenicity. In this paper, we focused on the CarRS two-component system of F. nucleatum, and the histidine kinase protein CarS was recombinantly expressed and characterized. Several online software such as SMART, CCTOP and AlphaFold2 were used to predict the secondary and tertiary structure of the CarS protein. The results showed that CarS is a membrane protein with two transmembrane helices and contains 9 α-helices and 12 β-folds. CarS protein is composed of two domains, one is the N-terminal transmembrane domain (amino acids 1-170), the other is the C-terminal intracellular domain. The latter is composed of a signal receiving domain (histidine kinases, adenylyl cyclases, methyl-accepting proteins, prokaryotic signaling proteins, HAMP), a phosphate receptor domain (histidine kinase domain, HisKA), and a histidine kinase catalytic domain (histidine kinase-like ATPase catalytic domain, HATPase_c). Since the full-length CarS protein could not be expressed in host cells, a fusion expression vector pET-28a(+)-MBP-TEV-CarS_cyto was constructed based on the characteristics of secondary and tertiary structures, and overexpressed in Escherichia coli BL21-Codonplus(DE3)RIL. CarS_cyto-MBP protein was purified by affinity chromatography, ion-exchange chromatography, and gel filtration chromatography with a final concentration of 20 mg/ml. CarS_cyto-MBP protein showed both protein kinase and phosphotransferase activities, and the MBP tag had no effect on the function of CarS_cyto protein. The above results provide a basis for in-depth analysis of the biological function of the CarRS two-component system in F. nucleatum.
Humans ; Histidine Kinase/metabolism* ; Fusobacterium nucleatum/metabolism* ; Automobiles ; Protein Kinases/genetics* ; Escherichia coli/metabolism* ; Colorectal Neoplasms

Objective To study the pathological types,expression of mismatch repair protein,human epidermal growth factor receptor 2(HER2),and Pan-TRK,and Epstein-Barr virus(EBV)infection in patients with colorectal cancer resected in Tibet. Methods A total of 79 patients with colorectal cancer resected in Tibet Autonomous Region People's Hospital from December 2013 to July 2021 were enrolled in this study.The clinical and pathological data of the patients were collected.The expression of mismatch repair protein,HER2,and Pan-TRK was detected by immunohistochemical(IHC)staining,and detection of HER2 gene by fluorescence in situ hybridization(FISH)in the patients with HER2 IHC results of 2+ or above.EBV was detected by in situ hybridization with EBV-encoded small RNA. Results A total of 79 colorectal cancer patients were included in this study,with the male-to-female ratio of 1.26:1 and the mean age of(57.06±12.74)years(24-83 years).Among them,4 patients received preoperative neoadjuvant therapy.Colonic cancer and rectal cancer occurred in 57(57/79,72.15%,including 31 and 26 in the right colon and left colon,respectively)and 22(22/79,27.85%)patients,respectively.The maximum diameter of tumor varied within the range of 1-20 cm,with the mean of(6.61±3.33)cm.Among the 79 colorectal cancer patients,75(75/79,94.94%)patients showed adenocarcinoma.Lymph node metastasis occurred in 12(12/21,57.14%)out of the 21 patients with severe tumor budding,13(13/23,56.52%)out of the 23 patients with moderate tumor budding,and 2(2/31,6.45%)out of the 31 patients with mild tumor budding,respectively.The lymph node metastasis rate showed differences between the patients with severe/moderate tumor budding and the patients with mild tumor budding(all P<0.001).The IHC staining showed that mismatch repair protein was negative in 10(10/65,15.38%)patients,including 5 patients with both MSH2 and MSH6 negative,4 patients with both MLH1 and PMS2 negative,and 1 patient with MSH6 negative.Pan-TRK was negative in 65 patients.The IHC results of HER2 showed 0 or 1+ in 60 patients and 2+ in 5 patients.FISH showed no positive signal in the 5 patients with HER2 IHC results of 2+.The detection with EBV-encoded small RNA showed positive result in 1(1/65,1.54%)patient. Conclusions Non-specific adenocarcinoma of the right colon is the most common in the patients with colorectal cancer resected in Tibet,and 15% of the patients showed mismatch repair protein defects.EBV-associated colorectal carcer is rare,Pan-TRK expression and HER2 gene amplification are seldom.The colorectal cancer patients with moderate and severe tumor budding are more likely to have lymph node metastasis.
Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Adenocarcinoma ; Biomarkers, Tumor/genetics* ; Colorectal Neoplasms/pathology* ; DNA Mismatch Repair ; DNA-Binding Proteins/genetics* ; Epstein-Barr Virus Infections/diagnosis* ; Herpesvirus 4, Human/metabolism* ; In Situ Hybridization, Fluorescence ; Lymphatic Metastasis ; Tibet ; Young Adult ; Aged, 80 and over

We investigated the effects of decursin on the proliferation, apoptosis, and migration of colorectal cancer HT29 and HCT116 cells through the phosphatidylinositol 3-kinase(PI3K)/serine-threonine kinase(Akt) pathway. Decursin(10, 30, 60, and 90 μmol·L~(-1)) was used to treat HT29 and HCT116 cells. The survival, colony formation ability, proliferation, apoptosis, wound hea-ling area, and migration of the HT29 and HCT116 cells exposed to decursin were examined by cell counting kit-8(CCK8), cloning formation experiments, Ki67 immunofluorescence staining, flow cytometry, wound healing assay, and Transwell assay, respectively. Western blot was employed to determine the expression levels of epithelial cadherin(E-cadherin), neural cadherin(N-cadherin), vimentin, B-cell lymphoma/leukemia-2(Bcl-2), Bcl-2-associated X protein(Bax), tumor suppressor protein p53, PI3K, and Akt. Compared with the control group, decursin significantly inhibited the proliferation and colony number and promoted the apoptosis of HT29 and HCT116 cells, and it significantly down-regulated the expression of Bcl-2 and up-regulated the expression of Bax. Decursin inhibited the wound healing and migration of the cells, significantly down-regulated the expression of N-cadherin and vimentin, and up-regulated the expression of E-cadherin. In addition, it significantly down-regulated the expression of PI3K and Akt and up-regulated that of p53. In summary, decursin may regulate epithelial-mesenchymal transition(EMT) via the PI3K/Akt signaling pathway, thereby affecting the proliferation, apoptosis, and migration of colorectal cancer cells.
Humans ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; bcl-2-Associated X Protein ; Vimentin/metabolism* ; Cell Proliferation ; Signal Transduction ; Apoptosis ; Cell Line, Tumor ; Colorectal Neoplasms/genetics* ; Cadherins/genetics* ; Cell Movement

This study aimed to investigate the mechanism of resveratrol(RES) combined with irinotecan(IRI) in the treatment of colorectal cancer(CRC). The targets of RES, IRI, and CRC were obtained from databases, and the targets of RES combined with IRI in the treatment of CRC were acquired by Venn diagram. The protein functional cluster analysis, GO and KEGG enrichment analyses were performed. In addition, the protein-protein interaction(PPI) network was constructed. The core target genes were screened out and the target-signaling pathway network was set up. IGEMDOCK was used to dock the core target gene molecules. Besides, the relationship between the expression level of key target genes and the prognosis and immune infiltration of CRC was analyzed. Based on the in vitro cell experiment, the molecular mechanism of RES combined with IRI in the treatment of CRC was explored and analyzed. According to the results, 63 potential targets of RES combined with IRI were obtained for CRC treatment. Furthermore, cluster analysis revealed that protein functions included 23% transmembrane signal receptors, 22% protein modifying enzymes, and 14% metabolite converting enzymes. GO analysis indicated that BPs were mainly concentrated in protein autophosphorylation, CCs in receptor complex and plasma membrane, and MFs in transmembrane receptor protein tyrosine kinase activity. Moreover, KEGG signaling pathways were mainly enriched in central carbon metabolism in cancer. The key targets of RES combined with IRI in the treatment of CRC were PIK3CA, EGFR, and IGF1R, all of which were significantly positively correlated with the immune infiltration of CRC. As shown by the molecular docking results, PIK3CA had the most stable binding with RES and IRI. Compared with the results in the control group, the proliferation ability and EGFR protein expression of CRC cells in the RES-treated group, the IRI-treated group, and the RES+IRI treated group significantly decreased. Moreover, the cell proliferation ability and EGFR protein expression level of CRC cells in the RES+IRI treated group were remarkably lower than those in the IRI-treated group. In conclusion, PIK3CA, EGFR, and IGF1R are the key targets of RES combined with IRI in CRC treatment. In addition, RES can inhibit the proliferation of CRC cells and improve IRI chemoresistance by downregulating the EGFR signaling pathway.
Humans ; Irinotecan ; Colorectal Neoplasms/genetics* ; Resveratrol ; Molecular Docking Simulation ; ErbB Receptors/genetics*

Objective To investigate the effect of insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) on the proliferation, migration and tumor immune microenvironment of colorectal cancer cells and its possible molecular mechanism. Methods The Cancer Genome Atlas (TCGA) database was used to analyze the expression levels of IGF2BP2 and MYC in colorectal cancer and adjacent tissues. The expression of IGF2BP2 in HCT-116 and SW480 human colorectal cancer cells was silenced by RNA interference (RNAi), and the silencing effect was detected by quantitative real-time PCR. After knocking down IGF2BP2, colony formation assay, CCK-8 assay and 5-ethynyl-2'-deoxyuridine (EdU) assay were employed to detect cell colony formation and proliferation ability. Transwell^TM assay was used to detect cell migration ability. Quantitative real-time PCR was used to detect the mRNA expression of IGF2BP2, MYC, tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β) and interleukin-10 (IL-10). The protein expression of IGF2BP2 and MYC was detected by western blot. The binding ability of IGF2BP2 and MYC in HCT-116 cells was detected by quantitative real-time PCR after RNA immunoprecipitation. Results The results of TCGA database showed that the expression of IGF2BP2 and MYC in colorectal cancer tissues was significantly higher than that in adjacent tissues, and the survival time of colorectal cancer patients with high expression of IGF2BP2 was shorter. After silencing IGF2BP2, the viability, proliferation and migration of HCT-116 and SW480 cells were decreased. The mRNA expression of MYC, TGF-β and IL-10 in IGF2BP2 knockdown group was significantly decreased, while the expression of TNF-α mRNA was increased. The expression of MYC protein and the stability of MYC mRNA were significantly decreased. RIP-qPCR results showed that IGF2BP2 could bind to MYC mRNA. Conclusion Knockdown of IGF2BP2 inhibits colorectal cancer cell proliferation, migration and promotes tumor immunity by down-regulating MYC expression.
Humans ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Colorectal Neoplasms/metabolism* ; Gene Expression Regulation, Neoplastic ; Interleukin-10/metabolism* ; RNA, Messenger ; RNA-Binding Proteins/metabolism* ; Transforming Growth Factor beta/genetics* ; Tumor Microenvironment/immunology* ; Tumor Necrosis Factor-alpha/metabolism* ; Proto-Oncogene Proteins c-myc/metabolism*

Objective To investigate how the neutrophil extracellular traps (NETs) affect the proliferation and migration of mouse MC38 colorectal cancer cells and its mechanism. Methods Spleen neutrophils were extracted in mouse, followed by collection of NETs after ionomycin stimulation in vitro. The proliferation of MC38 cell was detected by CCK-8 assay, and migration ability were detected by Transwell^TM and cell scratch assay, after co-incubation with MC38 cells. The mRNA expression of cellular matrix metalloproteinase 2 (MMP2) and MMP9 were detected by real-time fluorescence quantitative PCR, and the expression of MMP2, MMP9 and focal adhesion kinase (FAK), phosphorylated FAK protein were detected by Western blot. After silencing MMP9 using small interfering RNA (siRNA), the effect of NETs on the proliferation and migration ability of MC38 cells and the altered expression of related molecules were examined by previous approach. Results NETs promoted the proliferation and migration of MC38 cells and up-regulated the MMP9 expression and FAK phosphorylation. Silencing MMP9 inhibited the promotion of MC38 proliferation and migration by NETs and suppressed FAK phosphorylation. Conclusion NETs up-regulates MMP9 expression in MC38 cells, activates FAK signaling pathway and promotes tumor cell proliferation and migration.
Animals ; Mice ; Focal Adhesion Protein-Tyrosine Kinases/metabolism* ; Matrix Metalloproteinase 2/metabolism* ; Matrix Metalloproteinase 9/metabolism* ; Extracellular Traps/metabolism* ; Cell Movement ; Cell Proliferation ; RNA, Small Interfering/genetics* ; Colorectal Neoplasms/genetics* ; Cell Line, Tumor