Biomarker For Diagnosis Of Colorectal Cancer Research Articles

Image-based profiling and deep learning reveal morphological heterogeneity of colorectal cancer organoids

Patient-derived organoids have proven to be a highly relevant model for evaluating of disease mechanisms and drug efficacies, as they closely recapitulate in vivo physiology. Colorectal cancer organoids, specifically, exhibit a diverse range of morphologies, which have been analyzed with image-based profiling. However, the relationship between morphological subtypes and functional parameters of the organoids remains underexplored. Here, we identified two distinct morphological subtypes (“cystic” and “solid”) across 31360 bright field images using image-based profiling, which correlated differently with viability and apoptosis level of colorectal cancer organoids. Leveraging object detection neural networks, we were able to categorize single organoids achieving higher viability scores as “cystic” than “solid” subtype. Furthermore, a deep generative model was proposed to predict apoptosis intensity based on a apoptosis-featured dataset encompassing over 17000 bright field and matched fluorescent images. Notably, a significant correlation of 0.91 between the predicted value and ground truth was achived, underscoring the feasibility of this generative model as a potential means for assessing organoid functional parameters. The underlying cellular heterogeneity of the organoids, i.e., conserved colonic cell types and rare immune components, was also verified with scRNA sequencing, implying a compromised tumor microenvironment. Additionally, the “cystic” subtype was identified as a relapse phenotype featuring intestinal stem cell signatures, suggesting that this visually discernible relapse phenotype shows potential as a novel biomarker for colorectal cancer diagnosis and prognosis. In summary, our findings demonstrate that the morphological heterogeneity of colorectal cancer organoids explicitly recapitulate the association of phenotypic features and exogenous perturbations through the image-based profiling, providing new insights into disease mechanisms.

The multispecies microbial cluster of Fusobacterium, Parvimonas, Bacteroides and Faecalibacterium as a precision biomarker for colorectal cancer diagnosis.

The incidence of colorectal cancer (CRC) has increased worldwide, and early diagnosis is crucial to reduce mortality rates. Therefore, new noninvasive biomarkers for CRC are required. Recent studies have revealed an imbalance in the oral and gut microbiomes of patients with CRC, as well as impaired gut vascular barrier function. In the present study, the microbiomes of saliva, crevicular fluid, feces, and non-neoplastic and tumor intestinal tissue samples of 93 CRC patients and 30 healthy individuals without digestive disorders (non-CRC) were analyzed by 16S rRNA metabarcoding procedures. The data revealed that Parvimonas, Fusobacterium, and Bacteroides fragilis were significantly over-represented in stool samples of CRC patients, whereas Faecalibacterium and Blautia were significantly over-abundant in the non-CRC group. Moreover, the tumor samples were enriched in well-known periodontal anaerobes, including Fusobacterium, Parvimonas, Peptostreptococcus, Porphyromonas, and Prevotella. Co-occurrence patterns of these oral microorganisms were observed in the subgingival pocket and in the tumor tissues of CRC patients, where they also correlated with other gut microbes, such as Hungatella. This study provides new evidence that oral pathobionts, normally located in subgingival pockets, can migrate to the colon and probably aggregate with aerobic bacteria, forming synergistic consortia. Furthermore, we suggest that the group composed of Fusobacterium, Parvimonas, Bacteroides, and Faecalibacterium could be used to design an excellent noninvasive fecal test for the early diagnosis of CRC. The combination of these four genera would significantly improve the reliability of a discriminatory test with respect to others that use a single species as a unique CRC biomarker.

Exploring the Potential of Oral Microbiome Biomarkers for Colorectal Cancer Diagnosis and Prognosis: A Systematic Review.

There is growing evidence indicating that the oral microbiota, specifically certain periodontopathogens such as Fusobacterium nucleatum, may play a role in the development of colorectal cancer and that it could potentially be used as a biomarker for diagnosing colorectal cancer (CRC). The question beneath this systematic review is whether the development or progression of colorectal cancer can be attributed to the presence of certain oral bacteria, which could be used for discovering non-invasive biomarkers for CRC. This review aims to give an overview of the actual status of published studies regarding the oral pathogens related to colorectal cancer and assess the effectiveness of the oral microbiome derived biomarkers. A systematic literature search was performed using four databases, Web of Science, Scopus, PubMed, and Science Direct, on the 3rd and 4th of March 2023. The studies that did not have matching inclusion/exclusion criteria were winnowed out. A total of fourteen studies were included. The risk of bias was performed by using QUADAS-2. After assessing the studies, the general conclusion is that oral microbiota-based biomarkers can become a promising non-invasive tool for detecting CRC, but further research is needed in order to determine the mechanisms of oral dysbiosis in colorectal carcinogenesis.

Use of iTRAQ-based quantitative proteomic identification of CHGA and UCHL1 correlated with lymph node metastasis in colorectal carcinoma.

Metastatic dissemination of colorectal cancer (CRC), the third most common cancer type, is responsible for CRC deaths. Understanding the transition of lymph node metastasis (LNM) from Stage II to Stage III is beneficial in the prognosis and intervention of CRC. In this study, a quantitative proteomic survey was conducted to investigate the LNM-associated proteins and evaluate the clinicopathological characteristics of these target proteins in CRC. By using the LC-MS/MS iTRAQ technology, we analysed the proteomic changes between LMN II and LMN III. Fresh tumours from the CRC specimens consisting of 12 node-negative (Stage II) and 12 node-positive (Stage III) cases were analysed by LC-MS/MS iTRAQ proteome analysis. Subsequently, tissue microarray with immunohistochemistry staining was conducted to access the clinicopathological characteristics of these proteins in 116 paraffin-embedded CRC samples, each for non-LNM and LNM CRC. To study the effects of the differentially expressed proteins on the potential mechanism, Boyden chamber assay, flow cytometry and shRNA-based assessments were conducted to examine the role of the epithelial-mesenchymal transition (EMT) and the invasiveness of CRC cells and others in vivo xenograft mouse model experiments. Forty-eight proteins were found differentially expressed between non-LNM and LNM CRC tissues. Protein abundances of chromogranin-A (CHGA) and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) were observed in node-positive CRC (p < 0.05). Knockdown of CHGA and UCHL1 significantly regulate cancer behaviours of HCT-116, including inhibition of cell migration, invasiveness, cell cycle G1/S arrest and reactive oxygen species (ROS) generation. Mechanistically, the CHGA and UCHL1 inactivation displayed decreased levels of UCH-L1, chromogranin A, β-catenin, cyclin E, twist-1/2, vimentin, MMP-9, N-cadherin and PCNA through the activation of the Rho-GTPase/AKT/NFκB pathways. Histone modification of H3K4 trimethylation of CHGA and UCHL1 promoter were increased to activate their transcription through the signalling transduction such as Rho-GTPase, AKT and NFκB pathways. Our results indicated that UCHL1 and chromogranin A are novel regulators in CRC lymph node metastasis to potentially provide new insights into the mechanism of CRC progression and serve as biomarkers for CRC diagnosis at the metastatic stage.

Regularized survival learning and cross-database analysis enabled identification of colorectal cancer prognosis-related immune genes.

Colon adenocarcinoma is the most common type of colorectal cancer. The prognosis of advanced colorectal cancer patients who received treatment is still very poor. Therefore, identifying new biomarkers for prognosis prediction has important significance for improving treatment strategies. However, the power of biomarker analyses was limited by the used sample size of individual database. In this study, we combined Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) databases to expand the number of healthy tissue samples. We screened differentially expressed genes between the GTEx healthy samples and TCGA tumor samples. Subsequently, we applied least absolute shrinkage and selection operator (LASSO) regression and multivariate Cox analysis to identify nine prognosis-related immune genes: ANGPTL4, IDO1, NOX1, CXCL3, LTB4R, IL1RL2, CD72, NOS2, and NUDT6. We computed the risk scores of samples based on the expression levels of these genes and divided patients into high- and low-risk groups according to this risk score. Survival analysis results showed a significant difference in survival rate between the two risk groups. The high-risk group had a significantly lower overall survival rate and poorer prognosis. We found the receiver operating characteristic based on the risk score was showed to accurately predict patients' prognosis. These prognosis-related immune genes may be potential biomarkers for colorectal cancer diagnosis and treatment. Our open-source code is freely available from GitHub at https://github.com/gutmicrobes/Prognosis-model.git.

Exploring stool-based microrna-92a as a potential biomarker for colorectal cancer diagnosis

Exploring stool-based microrna-92a as a potential biomarker for colorectal cancer diagnosis

Development of fluorogenic substrates for colorectal tumor-related neuropeptidases for activity-based diagnosis.

The M3 metalloproteases, neurolysin and THOP1, are neuropeptidases that are expressed in various tissues and metabolize neuropeptides, such as neurotensin. The biological roles of these enzymes are not well characterized, partially because the chemical tools to analyse their activities are not well developed. Here, we developed a fluorogenic substrate probe for neurolysin and thimet oligopeptidase 1 (THOP1), which enabled the analysis of enzymatic activity changes in tissue and plasma samples. In particular, the probe was useful for studying enzyme activities in a single-molecule enzyme assay platform, which can detect enzyme activity with high sensitivity. We detected the activity of neurolysin in plasma samples and revealed higher enzyme activity in the blood samples of patients with colorectal tumor. The result indicated that single-molecule neurolysin activity is a promising candidate for a blood biomarker for colorectal cancer diagnosis.

Hypermethylation of MGMT Gene Promoter in Peripheral Blood Mononuclear Cells as a Noninvasive Biomarker for Colorectal Cancer Diagnosis.

Early colorectal cancer (CRC) diagnosis can drastically reduce CRC-related morbidity and mortality. In this regard, increasing attention is now being directed to DNA-based tests, especially the evaluation of methylation levels, to prioritize high-risk suspected persons for colonoscopy examination. Therefore, we aimed to assess the accuracy of MGMT gene promoter methylation levels in peripheral blood mononuclear cells (PBMCs) for distinguishing CRC patients from healthy people. For this study, a total of seventy individuals with CRC and 75 healthy individuals from Iran were included. The methylation level of MGMT in the DNA isolated from PBMCs was evaluated using the methylation quantification endonuclease-resistant DNA technique. To assess the diagnostic capability of the MGMT promoter methylation level, a receiver operating characteristic (ROC) curve was generated. The mean promoter methylation level of MGMT in the CRC and control groups was, respectively, 27.83 ± 22.80 vs. 12.36 ± 14.48. The average percentage of methylation of the MGMT promoter between the CRC and control groups was significantly different (P < 0.001). Also, the MGMT promoter was more hypermethylated in female patients than in males. ROC analyses indicated that the diagnostic power of the MGMT promoter methylation level for CRC was 0.754, with a sensitivity of 81.43% and a specificity of 75.71%, indicating a good biomarker for CRC diagnosis. Methylation evaluation of MGMT in PBMCs could be utilized as a diagnostic biomarker with high accuracy for prioritizing suspected CRC patients before colonoscopy.

Diet-mediated gut microbial community modulation and signature metabolites as potential biomarkers for early diagnosis, prognosis, prevention and stage-specific treatment of colorectal cancer

BackgroundOver the last decade, studies have shown an increased incidence of colorectal cancer (CRC), particularly early onset colorectal cancer (EOCRC). Researchers have demonstrated that dietary behavior, especially among young adults, influences alterations in the gut microbial community, leading to an increased accumulation of pathogenic gut microbiota and a decrease in beneficial ones. Unfortunately, CRC is likely to be diagnosed at a late stage, increasing CRC-related mortality. However, this alteration in the gut microbiota (gut dysbiosis) can be harnessed as a biomarker for non-invasive diagnosis, prognosis, prevention, and treatment of CRC in an effort to prevent late diagnosis and poor prognosis associated with CRC. Aim of reviewThis review discusses identification of potential biomarkers by targeting diet-mediated gut dysbiosis for the stage-specific diagnosis, prognosis, treatment, and prevention of CRC. Our findings provide a comprehensive insight into the potential of protumorigenic bacteria (e.g.pathogenic Escherichia coli,enterotoxigenic Bacteroides fragilis and Fusobacterium nucleatum) and their metabolites (e.g., colibactin and B. fragilis toxin) from gut dysbiosis as biomarkers for the diagnosis of CRC. Key scientific concepts of reviewCollectively, a detailed understanding of the available data from current studies suggests that, further research on quantification of metabolites and stage-specific pathogenic microbial abundance is required for the diagnosis and treatment of CRC based on microbial dysbiosis. Specifically, future studies on faecal samples, from patient with CRC, should be conducted for F. nucleatum among different opportunistic bacteria, given its repeated occurrence in faecal samples and CRC biopsies in numerous studies. Finally, we discuss the potential of faecal microbial transplantation (FMT) as an intervention to restore damaged gut microbiota during CRC treatment and management.

Abstract A030: Gut microbiome as a promising biomarker for colorectal cancer diagnosis and immunotherapy response prediction

Abstract Background and objective: The association between human intestinal microbes and disease is an emerging topic in microbiome research, with potential implications in cancer diagnosis. Meanwhile, immune checkpoint inhibitor (ICI) therapy has been extensively applied to cancer patients but with relatively low overall response rate owing to pathological complexity. Thus, accurate prediction of cancer onset as well as patients’ response to ICI treatment through non-invasive methods, such as gut microbiome profiling, could greatly facilitate cancer diagnosis and treatment practice, which essentially improves patients’ quality of life. With the rapid accumulation of microbiome sequencing data and advanced machine learning algorithms, the use of gut microbiome as predictive biomarkers presents a feasible approach to address these problems. Methods and results: The first model we built is a predictive model for classifying healthy people from patients with colon cancer (CRC). The data (originated from Curated Metagenomics Data of Human Microbiome) consists of 166 fecal samples from healthy population and equal number of CRC patients from two studies (PRJEB24748, PRJEB6070). The data were divided into training and testing sets with a 3:1 ratio. Using the training dataset, we developed a Gradient Boosting Decision Tree model. To demonstrate the model's generalizability, we evaluated it using the testing dataset and the model achieved an AUC score of 0.93 (accuracy: 89%). Moreover, using an external independent cohort (which included 52 CRC patients from PRJEB12449 and 52 healthy individuals), we obtained an AUC score of 0.94 (accuracy: 88%). We also detected bacteria highly related to CRC pathology including Solobacterium_moorei, which could be potential biomarkers for CRC diagnosis. In addition, to predict CRC patients’ response to ICI, we built a random forest model based on patients’ fecal microbiome data before the ICI treatment. In total, stool metagenomics data from 63 CRC patients were used, including 30 patients responding to ICI treatment and 33 non-responders. With five-fold cross-validation, we achieved training and testing AUC score of 0.89 and 0.72, respectively. With an external data with 11 CRC patients’ samples, the classifier achieved an AUC of 0.78. Several microbial taxa were listed as important features contributing to the classification, e.g. Eubacterium hallii – a butyrate producer, which previously report to have a positive association with ICI clinical benefits. Conclusion: Using the non-invasive human gut microbiome data and advanced machine learning techniques, we present here two models with great performance to predict the occurrence of CRC and clinical response to ICI treatment in CRC patients. Similar strategy could be readily transferred to other cancer types to improve early diagnosis and patient stratification. Moreover, the microbial taxa identified to be essential features in the model could be developed as potential therapeutic targets. Citation Format: Die Dai, Xiaomin Xu, Wei Shen, Zhengnong Zhu, Fang Li, Xiaochen Yin, Yan Kou. Gut microbiome as a promising biomarker for colorectal cancer diagnosis and immunotherapy response prediction [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A030.

5-Methylcytosine (m5C) modification in peripheral blood immune cells is a novel non-invasive biomarker for colorectal cancer diagnosis.

Current non-invasive tumor biomarkers failed to accurately identify patients with colorectal cancer (CRC), delaying CRC diagnosis and thus leading to poor prognosis. Dysregulation of 5-Methylcytosine (m5C) RNA has gradually been reported in various cancers, but their role in tumor diagnosis is rarely mentioned. Our study aimed to determine the role of m5C methylation modification in blood immune cells for the diagnosis of CRC. Peripheral blood samples were obtained from a total of 83 healthy controls and 196 CRC patients. We observed that m5C RNA contents in blood immune cells of CRC patients were markedly enhanced in both training set and validation set. Moreover, levels of m5C increased with CRC progression and metastasis but reduced after treatment. Compared with common blood tumor biomarkers, m5C levels in peripheral blood immune cells had superior discrimination and reclassification performance in diagnosing CRC. Besides, bioinformatics and qRT-PCR analysis identified increased expression of m5C-modified regulators NSUN5 and YBX1 in CRC patients’ blood. A series of animal models and cell co-culture models further demonstrated that CRC tumor cells could increase immune cells’ m5C levels and m5C-modified regulators. Monocyte was the predominant m5C-modified immune cell type in CRC patients’ blood by Gene set variation analysis (GSVA). Taken together, m5C methylation modification in peripheral blood immune cells was a promising biomarker for non-invasive diagnosis of CRC.

2D-DIGE-MS Proteomics Approaches for Identification of Gelsolin and Peroxiredoxin 4 with Lymph Node Metastasis in Colorectal Cancer.

Simple SummarySchematic presentation of the histone modification of gelsolin (GSN) and peroxiredoxin-4 (PRDX4) promoters by endogenous EGFR/RhoA/PKC/ERK signaling pathways in DLD-1 cells. Identifying lymph node metastasis (LNM)-associated proteins by two-dimensional difference gel electrophoresis MS/MS in colorectal cells (CRC) indicates the reliability of candidate markers of GSN and PRDX4 and therapeutic targets for CRC.Background/Aims: A combination of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry approach was used to search for potential markers for prognosis and intervention of colorectal cancer (CRC) at different stages of lymph node metastasis (LMN). This quantitative proteomic survey aimed to investigate the LNM-associated proteins and evaluate the clinicopathological characteristics of these target proteins in CRC from stage I to stage IV. Methods: Sixteen CRC cases were categorized into paired non-LNM and LNM groups, and two-dimensional difference gel electrophoresis and MS proteome analysis were performed. Differential protein expression between non-LNM and LNM CRC was further validated in a tissue microarray, including 40 paraffin-embedded samples by immunohistochemistry staining. Moreover, a Boyden chamber assay, flow cytometry, and shRNA were used to examine the epithelial–mesenchymal transition and mechanism invasiveness of the differentially expressed proteins in DLD-1 cells and in vivo xenograft mouse model. Results: Eighteen differentially expressed proteins were found between non-LNM and LNM CRC tissues. Among them, protein levels of Gelsolin (GSN) and peroxiredoxin 4 (PRDX4) were abundant in node-positive CRC. Downregulation of GSN and PRDX4 markedly suppressed migration and invasiveness and also induced cell cycle G1/S arrest in DLD-1. Mechanistically, the EGFR/RhoA/PKCα/ERK pathways are critical for transcriptional activation of histone modification of H3 lysine 4 trimethylation (H3K4me3) of GSN and PRDX4 promoters, resulting in upregulation of GSN, PRDX4, Twist-1/2, cyclinD1, proliferating cell-nuclear antigen, β-catenin, N-cadherin, and matrix metalloprotein-9. Conclusions: GSN and PRDX4 are novel regulators in CRC lymph node metastasis to potentially provide new insights into the mechanism of CRC progression and serve as a biomarker for CRC diagnosis at the metastatic stage.

LncRNA XIST sponges microRNA-448 to promote malignant behaviors of colorectal cancer cells via regulating GRHL2.

Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are essential regulators in human cancers, while the role of lncRNA X-inactive-specific transcript (XIST) in colorectal cancer (CRC) via regulating miR-448 remains largely unknown. Herein, we aimed to elucidate the effect of the XIST/miR-448/grainyhead-like 2 (GRHL2) axis on CRC development. XIST, miR-448, and GRHL2 expression in CRC tissues from patients and in human CRC cell lines was assessed. Loss- and gain-function assays were implemented to unveil the roles of XIST, miR-448, and GRHL2 in screened CRC cells. The tumor growth in vivo was observed in nude mice. Binding relations among XIST, miR-448, and GRHL2 were evaluated. XIST and GRHL2 expressed highly whereas miR-448 expressed lowly in CRC tissues and cells. XIST or GRHL2 downregulation, or miR-448 elevation suppressed the malignant behaviors of CRC cells in vitro, and downregulated XIST or upregulated miR-448 also inhibited the tumor growth in vivo. miR-448 upregulation reversed the role of XIST elevation in CRC cells. XIST particularly bound to miR-448, and miR-448 targeted GRHL2. Knockdown of XIST upregulates miR-448 to impede malignant behaviors of CRC cells via inhibiting GRHL2. This study may provide novel biomarkers for CRC diagnosis and treatment.

Abstract 1484: Emerging microRNAs as biomarkers for colorectal cancer diagnosis: A meta-analysis

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer deaths worldwide. The current method of clinical screening is mainly based on colonoscopy. However, many CRC patients are unwilling to undergo colonoscopy because of the high procedure cost and the discomfort during examination. Therefore, it appears necessary to find novel and non-invasive biomarkers that would improve CRC early detection. MicroRNAs (miRNAs) are a class of non-coding RNAs with critical regulatory roles in fundamental cellular processes, such as proliferation, migration, and apoptosis. They can act as potential oncogenes or tumor suppressors in cancer development. Numerous studies have shown significant differences in miRNAs expression in serum or plasma of CRC patients and healthy subjects. Thus, the potential use of circulating miRNAs as diagnostic biomarkers in CRC seems to be a fast and relevant alternative to traditional and invasive methods. Here, we reviewed selected studies which identified dysregulated circulating miRNAs in CRC and evaluated their diagnostic value. Methods: This meta-analysis study was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The articles were searched in online databases such as PubMed, Google Scholar to identify all eligible studies that evaluated the diagnostic value of miRNAs for CRC. Then we explored the diagnostic accuracy of each miRNA using the random-effects model to estimate the pooled sensitivity (Se), specificity (Sp), Positive likelihood ratio (PLR) and Negative likelihood ratio (NLR) and the area under the curve (AUC), along with their corresponding 95% confidence intervals (CIs). The statistical analyses were performed using the Review Manager (Revman) 5.4 software and the Meta Disc software. Results: Thirteen studies involving five microRNAs were included in our analysis. Our results suggested that serum miR-29a levels showed great potential as a high diagnostic biomarker for CRC. The overall pooled sensitivity, specificity, area under the curve (AUC), PLR and NLR of circulating miR-29a were 69.75%, 88.25%, 0.8351, 5.93 and 0.342 respectively. Conclusion: miRNAs have become increasingly recognized molecular tools in CRC diagnosis. According to our results, circulating miR-29a level has potential value for early detection and might be used as a novel diagnostic biomarker for CRC in combination with other biomarkers. Citation Format: Sofia Fathi, Fadila Guessous, Mehdi Karkouri. Emerging microRNAs as biomarkers for colorectal cancer diagnosis: A meta-analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1484.

Small Extracellular Vesicles: Key Forces Mediating the Development and Metastasis of Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cancer worldwide, and its incidence and mortality rates have been increasing annually in recent years. A variety of different small extracellular vesicles (sEVs) are important mediators of intercellular communication and have an important role in tumor metastasis and progression. The development and metastasis of CRC are closely linked to tumor-cell-derived sEVs, non-tumor-cell-derived sEVs, and intestinal-microbiota-derived sEVs. Numerous studies have shown that the tumor microenvironment (TME) is a key component in the regulation of CRC proliferation, development, and metastasis. These sEVs can create a TME conducive to CRC growth and metastasis by forming an immunosuppressive microenvironment, remodeling the extracellular matrix, and promoting tumor cell metabolism. Therefore, in this paper, we review the role of different types of sEVs in colorectal cancer development and metastasis. Furthermore, based on the properties of sEVs, we further discuss the use of sEVs as early biomarkers for colorectal cancer diagnosis and the potential for their use in the treatment of CRC.

Identification of circ_0000375 and circ_0011536 as novel diagnostic biomarkers of colorectal cancer.

BACKGROUNDColorectal cancer (CRC) imposes a tremendous burden on human health, with high morbidity and mortality. Circular ribonucleic acids (circRNAs), a new type of noncoding RNA, are considered to participate in cancer pathogenesis as microRNA (miRNA) sponges. However, the dysregulation and biological functions of circRNAs in CRC remain to be explored.AIMTo identify potential circRNA biomarkers of CRC and explore their functions in CRC carcinogenesis.METHODSCircRNAs and miRNAs differentially expressed in CRC tissues were identified by analyzing expression profiles from the Gene Expression Omnibus (GEO) database. Circ_0000375 and circ_0011536 were selected as CRC biomarker candidates. Quantitative real-time polymerase chain reaction was utilized to evaluate the expression of these 2 circRNAs in CRC tissues, serums and cell lines. Receiver operating characteristic curves were generated to assess the diagnostic performances of these 2 circRNAs. Then, functional experiments, including cell counting kit-8, wound healing and Transwell invasion assays, were performed after the overexpression of circ_0000375 and circ_0011536 in CRC cell lines. Furthermore, candidate target miRNAs of circ_0000375 and circ_0011536 were predicted via bioinformatics analysis. The expression levels of these miRNAs were explored in CRC cell lines and tissues from GEO datasets. A luciferase reporter assay was developed to examine the interactions between circRNAs and miRNAs. Based on the target miRNAs and downstream genes, functional enrichment analyses were applied to reveal the critical signaling pathways involved in CRC carcinogenesis.RESULTSDownregulated circ_0000375 and circ_0011536 expression was observed in CRC tissues in GSE126095, clinical CRC tissue and serum samples and CRC cell lines. The areas under the curve for circ_0000375 and circ_0011536 were 0.911 and 0.885 in CRC tissue and 0.976 and 0.982 in CRC serum, respectively. Moreover, the serum levels of these 2 circRNAs were higher in patients at 30 d postsurgery than in patients before surgery, suggesting that the serum expression of circ_0000375 and circ_0011536 is related to CRC tumorigenesis. Circ_0000375 and circ_0011536 overexpression inhibited the proliferation, migration and invasion of CRC cells. Furthermore, miR-1182 and miR-1246, which were overexpressed in CRC tissues in GSE41655, GSE49246 and GSE115513, were verified as target miRNAs of circ_0000375 and circ_0011536, respectively, by luciferase reporter assays. The downstream genes of miR-1182 and miR-1246 were enriched in some CRC-associated pathways, such as the Wnt signaling pathway.CONCLUSIONCirc_0000375 and circ_0011536 may function as tumor suppressors in CRC progression, serving as novel biomarkers for CRC diagnosis and as promising candidates for therapeutic exploration.

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

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.

Systematic study on expression and prognosis of E2Fs in human colorectal cancer.

E2Fs are important components of transcription factors and play key roles in occurrence or advancement of various cancers, but the expression and exact roles of each E2F in colorectal cancer (CRC) are rarely known. To address this issue, we investigated the roles and prognostic values of E2Fs expressions in CRC patients by searching ONCOMINE, cBioPortal, GEPIA, Matascape and UALCAN. E2F1, 3-8 were upregulated at the mRNA level and E2F2 was less expressed in CRC tissues than in normal tissues. The eight E2Fs were correlated with tumor stages of CRC. Survival analysis using GEPIA revealed that high expressions of E2F3, 4 were related with short overall survival in all CRC patients. The mutation rate of E2Fs (60%) was high and genetic alteration in E2Fs was linked with longer overall survival in CRC patients. Functional analysis implied that E2Fs and their 50 nearby genes were concentrated in tumor-related pathways. E2Fs may be candidate biomarkers for CRC diagnosis and E2F3, 4 are potential prognosis biomarkers of CRC. Nevertheless, our findings must be validated in the future to popularize the clinical application of E2Fs in CRC.

Expression of Th17 axis as a biomarker panel in diagnosis and prognosis of colorectal cancer

Little is known about how the immunologic microenvironment alters throughout tumor progression. Currently, murine models have revealed the T-helper 17 (Th17) pathway to play a valuable role in promoting colorectal cancer (CRC). The aim of this study was to evaluate integrative analysis of Th17 axis genes expression in CRC. The expression level of Th17 axis genes including IL17A, IL17F, IL17RA, IL21, IL21R, IL22 and IL22RA1, were evaluated according to TCGA database. Subsequently, the transcription levels of mentioned genes were assessed using qRT-PCR technique. ROC curve was used to evaluate the specificity and sensitivity of genes expression in CRC. Statistically significant logRank p-values was used in order to determine the relation between gene expression and patient survival. The results from both conditions indicated that the expression of IL-17A, IL-17F and IL-22 genes significantly increased, while the expression levels of IL-17RA, IL-21, IL- 21R and IL-22RA1 genes showed a significant decrease in CRC tissues. Additionally, based on analysis with Kaplan Meyer survival curves, no significant relation between gene expression levels and survival rate of CRC patients was found. However, analysis by ROC curves, demonstrated that these genes could be used as specific biomarkers for the diagnosis of CRC. Th17 axis genes might be useful biomarkers for CRC diagnosis and prognosis.

Expression profiles of miR-196, miR-132, miR-146a, and miR-134 in human colorectal cancer tissues in accordance with their clinical significance : Comparison regarding KRAS mutation.

Colorectal cancer (CRC) is among the most widespread malignancies in the world. MicroRNA (miRNA) has been identified as an important modulator of the biological processes of the cells. This group of noncoding RNAs also has apivotal function in the growth and development of human cancers, including CRC. Among these miRNAs, miR-196, miR-132, miR-146a, and miR-134 have fundamental impacts on the regulation of cancers. The current study aimed to investigate the involvement of these miRNAs in CRC patients. In this study, 50pairs of tumor and tumor margin samples of CRC patients were investigated to assess the expression levels of miR-196, miR-132, miR-146a, and miR-134 in this cancer. For this purpose, firstly, quantitative real-time PCR (qRT-PCR) was applied. Also, KRAS mutation and clinicopathological characteristics of the CRC patients were analyzed in the study groups. The findings demonstrated the overexpression of miR-196 (P-value = 0.0045) and miR-146a (P-value = 0.0033) in tumor tissues compared to controls. Conversely, the expression levels of miR-132 (P-value = 0.00032) and miR-134 (P-value < 0.0001) were downregulated in tumor tissues. Also, miR-146a was the only miRNA with significant expression change in the case of the KRAS gene mutation. Interestingly, the expression ratio of these miRNAs was significantly associated with some of the clinicopathological features of the patients, such as lymph node and distant metastases. Our data demonstrated that these miRNAs appear to be promising novel biomarkers for early diagnosis of CRC and may pave the way for the future establishment of novel therapeutic options for CRC.