Detecting liver cancer (LC) remains a significant challenge in clinical practice. Small extracellular vesicle (sEV) miRNAs show promise as non-invasive biomarkers for LC detection, yet their diagnostic potential remains largely unexplored. This study aimed to identify specific sEV miRNA signatures for LC detection and develop a novel synchronized multi-miRNA detection platform to enhance diagnostic efficiency and sensitivity. High-throughput sequencing was conducted across four distinct cohorts: normal controls (NC), hepatitis B virus (HBV) patients, liver cirrhosis patients, and LC patients. This sequencing process identified miRNAs with differential expression, followed by RT-qPCR validation in serum sEV miRNAs from LC patients and NC. An innovative detection method, RCA-CRISPR, was introduced, combining rolling circle amplification (RCA) with CRISPR/Cas12a (RCA-CRISPR) for quick and sensitive miRNAs detection. Sequencing results showed a consistent elevation of hsa-miR-203b-5p, hsa-miR-4661-5p, and hsa-miR-219a-2-3p across all cohorts. RT-qPCR validations confirmed significant upregulation of these miRNAs in serum sEVs from LC patients, and the combined three-miRNA panel exhibited high diagnostic accuracy (p = 0.0003; AUC = 0.81). The RCA-CRISPR method demonstrated a detection limit of 3.12 pM for simultaneous multi-target miRNA detection, highlighting its exceptional sensitivity. Our study identifies hsa-miR-203b-5p, hsa-miR-4661-5p, and hsa-miR-219a-2-3p as promising sEV miRNA biomarkers for LC detection. The developed RCA-CRISPR sensor provides a robust tool for multi-miRNA analysis, potentially advancing non-invasive LC diagnostics. Future validation in larger, prospectively collected cohorts is essential to establish the clinical utility and performance of this biomarker panel and RCA-CRISPR sensor.

Cancer-associated fibroblasts (CAFs) are a major stromal component of the tumor microenvironment (TME) and actively participate in tumor progression. Although CAF heterogeneity in colorectal cancer (CRC) is increasingly recognized, how individual CAF subpopulations simultaneously coordinate extracellular matrix (ECM) remodeling, angiogenesis, and immune suppression within spatially organized niches remains poorly understood. CAF heterogeneity was systematically analyzed by integrating single-cell RNA sequencing, spatial transcriptomics, and bulk transcriptomic data. By combining cell clustering, pseudotime analysis, transcriptional regulatory network inference, spatial deconvolution, and ligand–receptor interaction analysis, we revealed the spatial distribution and cellular communication characteristics of CAF subpopulations. At the same time, machine learning-based prognostic models and transcriptional perturbation analysis were used to evaluate the clinical significance and potential therapeutic value of CAF-related transcriptional programs. We identified a prolyl 4-hydroxylase subunit alpha 1 (P4HA1)-positive collagen-modifying CAF subpopulation (P4HA1⁺ CAFs) that was enriched in tumor tissue and exhibited collagen remodeling characteristics. This subpopulation preferentially localized at the tumor-stroma interface, co-localizing with pro-angiogenic endothelial cells, immunosuppressive macrophages, and malignant epithelial cells to form a spatially organized niche with concurrent ECM remodeling, angiogenesis, and immune suppression. P4HA1⁺ CAFs were predicted to engage distinct ligand–receptor axes, including WNT5A–MCAM with endothelial cells, MDK–LRP1 with macrophages, and MDK–NCL with malignant epithelial cells. The MDK–NCL axis was associated with increased RANBP1 expression and enhanced tumor cell proliferation and invasion. Machine learning analysis further identified P4HA1, TIMP1, ARL4C, and SFRP4 as stable core genes associated with poor prognosis. Drug perturbation analysis showed that 2-methoxyestradiol and its combination with the histone deacetylase (HDAC) inhibitor JNJ-26,481,585 may partially reverse the malignant transcriptional program of P4HA1⁺ CAFs. This study characterizes a P4HA1⁺ collagen-modifying CAF subpopulation that resides within a spatially organized multicellular niche and engages distinct signaling axes to simultaneously promote angiogenesis, immune suppression, and tumor cell proliferation in CRC, highlighting its prognostic and potential therapeutic value.

BACKGROUND:The Capecitabine plus Oxaliplatin (XELOX) regimen is a first-line chemotherapy for colorectal cancer (CRC), but drug resistance remains a major therapeutic challenge. Our clinical observations suggest a positive correlation between fecal Akkermansia muciniphila bundance and chemotherapy responsiveness. Huang-Lian-Jie-Du Decoction (HLJDD), a traditional Chinese medicine with gut microbiota-modulating properties, may serve as a chemosensitizer, though its mechanisms are unclear. PURPOSE:This study aimed to evaluate whether HLJDD enhances XELOX efficacy in CRC and to elucidate the role of gut microbiota, particularly A. muciniphila, in this synergistic effect. METHODS:CRC patients receiving XELOX were analyzed for fecal A. muciniphila levels and treatment response. In MC38 and CT26 tumor-bearing mice, HLJDD was administered alone or combined with XELOX. Gut microbiota composition (16S rRNA sequencing), serum metabolites (untargeted metabolomics), and tumor immune infiltration (immunohistochemistry) were assessed. Co-housing experiments and direct A. muciniphila supplementation were conducted to validate microbiota-mediated mechanisms. RESULTS:Higher baseline A. muciniphila abundance correlated with improved XELOX response in CRC patients. HLJDD selectively promoted A. muciniphila proliferation in vitro and in vivo. In murine models, HLJDD synergized with XELOX, significantly suppressing tumor growth vs. XELOX alone. Multi-omics analyses revealed HLJDD-driven enrichment of tryptophan metabolism and increased 5-hydroxytryptophan levels, accompanied by enhanced CD8+ T cell infiltration and interferon-γ upregulation in tumors. Cohousing with HLJDD-treated mice or direct A. muciniphila gavage recapitulated the chemosensitizing effects, confirming gut microbiota's pivotal role. CONCLUSION:This study unveils a novel and multi-faceted therapeutic strategy: HLJDD overcomes XELOX resistance by specifically remodeling gut microbiota to expand A. muciniphila, which subsequently activates tryptophan metabolism and anti-tumor immunity. Our findings provide robust preclinical evidence for combining HLJDD with XELOX as a promising innovative approach to overcome CRC chemoresistance, with A. muciniphila serving as a predictive biomarker and potential therapeutic target.

Colorectal cancer (CRC) is the third most common cancer and leading cause of cancer-related deaths worldwide. However, current CRC screening methods are complex, invasive, and tend to exhibit low sensitivity. Recent evidence has highlighted gut microbiota dysbiosis, especially elevated Fusobacterium nucleatum levels, as a promising biomarker for CRC. In this study, a sensitive and specific detection platform was developed for F. nucleatum by combining a highly specific aptamer with rolling circle amplification (RCA) and the CRISPR/Cas12a technology. The aptamer enables specific target recognition, while RCA amplifies the target signal, and the Cas12a-mediated cleavage of a fluorescence-quenching substrate generates a quantifiable fluorescence or grayscale signal. Using a microplate reader, this assay achieved a limit of detection (LOD) of 3.68 CFU/mL; furthermore, by incorporating smartphone-assisted ImageJ grayscale analysis, it elevated the LOD to 4.30 CFU/mL, thereby enabling a dual-mode output along with on-site applicability. Additionally, the strong correlation between the two signals allowed for mutual validation. Upon application to clinical fecal samples, the developed method sensitively distinguished CRC patients from healthy controls, and its results correlated with the quantitative polymerase chain reaction results. This triple-synergistic platform, integrating aptamer specificity, RCA amplification, and CRISPR/Cas12a sensitivity, enables the noninvasive, ultrasensitive detection of F. nucleatum, supporting early CRC screening, prognosis monitoring, and microbiome-targeted therapy. Moreover, this approach overcomes the challenges of detecting low-abundance bacteria in early stage CRC and advances the precision of microbiome-based diagnostics for CRC.

The identification of specific miRNA biomarkers has considerably advanced colorectal cancer (CRC) diagnostics. Our prior research identified four key miRNA signatures-miR-10b-5p, miR-130a-3p, miR-561-5p, and miR-4684-5p-as potential diagnostic markers for CRC. To enhance the clinical utility of these biomarkers, we developed a novel multi-miRNA detection platform, RCA-SDA-CRISPR (RS-CRISPR), which integrates rolling circle amplification (RCA), strand displacement amplification (SDA), and CRISPR/Cas12a technologies. This platform employs RCA products as SDA templates, maximizes miRNA binding sites, and utilizes the spatial arrangement of miRNAs to bypass limitations of traditional enzymatic cleavage, enabling rapid, simultaneous detection of multiple miRNAs. RS-CRISPR achieved a detection sensitivity of 57.8fM for target miRNAs. In clinical validation, this method successfully distinguished tumor tissue from adjacent non-cancerous tissue in five patients with CRC. In addition, during the serum analysis of 12 normal controls (NC) and 12 CRC patients, we found that the levels of four specific miRNAs were significantly higher in CRC patients compared to NC (p = 0.00646), underscoring its diagnostic potential. These findings establish RS-CRISPR as a promising, precise, and efficient diagnostic tool for clinical CRC management through multi-miRNA detection.

Colorectal cancer (CRC) represents a significant global health issue, necessitating innovative approaches for early screening and diagnosis. Recent advances in molecular diagnostics have highlighted the potential of aptamers for use as highly specific and sensitive probes for detecting cancer biomarkers. In this study, we focus on the identification of aptamers that selectively bind to Solobacterium moorei (S. moorei), a bacterium associated with CRC. By integrating these aptamers into electrochemical sensor platforms, a reliable diagnostic tool is created for facile implementation in the clinical setting. More specifically, nucleic acid aptamers for S. moorei were obtained through whole-cell SELEX, and the affinity and specificity of these aptamers were validated. Subsequently, two types of electrochemical sensors were developed. Firstly, an electrochemical impedance spectroscopy sensor was developed to detect impedance changes on the electrode surface, which were caused by the binding of S. moorei to the aptamer. Secondly, a CRISPR/Cas12a-based electrochemical aptasensor was developed based on the ability of the Cas12a enzyme to be activated by specific single-stranded DNA, triggering its trans-cleavage activity. The limits of detection of these sensors for S. moorei were 30 and 6 CFU/mL, respectively. Clinical validation was performed using patient samples to assess the sensor efficacy in a real-world setting. The obtained results suggested that the abundance of S. moorei in the feces of CRC patients was significantly greater compared to that of healthy individuals. This integration of S. moorei aptamers into electrochemical sensors offers a noninvasive and cost-effective alternative to current screening methods available for CRC.

Theaflavin-3’-gallate was synthesised using polyphenol oxidase (PPO) from gallocatechin (EGC) and epicatechin gallonic acid (ECG). Using PPO, Box-Behnken design, and single factor test, the optimal reaction conditions were determined: ECG/EGC ratio of 3:7, magnetic stirring speed of 200 rpm, reaction temperature of 37°C, and enzyme concentration of 20 mg/100 mL. Under these conditions, the yield of TF-3'-G was 18.1%. These parameters represent the optimal conversion conditions for theaflavin-3’-monogallate.

miRNA is considered a novel biomarker for cancer diagnosis and due to its low level in vivo, the development of new detection methods for it has become a research hotspot in recent years. Here, we firstly found that miR-625-5p was significantly upregulated in colorectal cancer tissues by means of differential expression analysis of the dbDEMC database and clinical validation. Subsequently, it was found that miR-625-5p promoted cell proliferation and migration but inhibited apoptosis through phenotypic experiments; thus, we initially identified miR-625-5p as a potential biomarker for colorectal cancer. Moreover, in order to monitor slight changes in the miR-625-5p level, we developed a novel detection method for it based on strand displacement amplification (SDA). In this system, a hairpin was designed to recognize and pair with miR-625-5p, which was used as a primer to initiate SDA, and a large number of complementary DNAs were generated via cyclic amplification, followed by the addition of SYBR Gold to achieve quantitative analysis of miR-625-5p. Moreover, this method showed a good response to miR-625-5p with a detection limit of 8.6 pM and a dynamic range of 0.01 to 200 nM, and the specificity of it was verified using a set of other miRNAs as an interference. Finally, we set up different concentrations of biologic samples for detection to verify the practicability of the method. The results of this study indicate that this detection method has great potential in clinical diagnosis.

In terms of cancer diagnoses andcancer-related deaths worldwide,colorectal cancer (CRC) is now the third most common malignancy. Thedrawbacks of current screening methods are their exorbitant costs,difficult procedures, and lengthy implementation timelines. The benefitsof fecal screening for CRC are ease of operation, noninvasiveness,cost-effectiveness, and superior sensitivity. As a result of its enrichmentin the malignant tissues and feces of CRC patients, Fusobacteriumnucleatum (F. nucleatum) has emerged asa crucial biomarker for the incipient detection, identification, andprognostic prediction of CRC. Here, for the first time, the whole-bacteriumSELEX method was used to screen the highly specific and affinity aptamersagainst F. nucleatum by 13 cycles of selection. TheApt-S-5 linear correlation equation is y = 0.7363x (2.8315) (R (2) = 0.9864)with a limit of detection (LOD) of 851 CFU/mL. The results of theexperiment using fecal samples revealed a substantial disparity betweenthe microorganisms in the CRC patients' feces and those inthe feces of healthy individuals and were consistent with those ofqPCR. The aptamers may therefore offer a crucial approach to identifying F. nucleatum and hold tremendous promise for CRC diagnosisand prognostic prediction.

Colorectal cancer (CRC) becomes the second leading cause of cancer-related deaths in 2020. Emerging studies have indicated that microRNAs (miRNAs) play a key role in tumorigenesis and progression. The dysfunctions of miR-455-3p are observed in many cancers. However, its biological function in CRC remains to be confirmed. By sequencing serum sample, miR-455-3p was found to be up-regulated in CRC patients. RT-qPCR demonstrated that the miR-455-3p expression was both higher in the serum and tumor tissues of CRC patients. Furthermore, it indicated that miR-455-3p had the ability in promoting cell proliferation, suppressing cell apoptosis, and stimulating cell migration. In vivo experiments also showed that miR-455-3p promoted tumor growth. Additionally, H2AFZ was proved as the direct gene target of miR-455-3p by dual-luciferase assay. Taken together, miR-455-3p functioned as a tumor promoter in CRC development by regulating H2AFZ directly. Thus, it has enormous potential as a biomarker in the diagnosis of CRC.

Colorectal cancer (CRC) is one of the digestive tract malignancies whose early symptoms are not obvious. This study aimed to identify novel targets for CRC therapy, especially early-stage CRC, by reanalyzing the publicly available GEO and TCGA databases. Thyroid hormone receptor interactor 13 (TRIP13) correlated with tumor progression and prognosis of patients after several rounds of analysis, including weighted gene correlation network analysis (WGCNA), and further chosen for experimental validation in cancer cell lines and patient samples. We identified that mRNA and protein levels of TRIP13 increased in CRC cells and tumor tissues with tumor progression. miR-4693-5p was significantly downregulated in CRC tumor tissues and bound to the 3' untranslated region (3'UTR) of TRIP13, downregulating TRIP13 expression. DCZ0415, a small molecule inhibitor targeting TRIP13, induced anti-tumor activity in vitro and in vivo. DCZ0415 markedly suppressed CRC cell proliferation, migration, and tumor growth, promoted cell apoptosis, and resulted in the arrest of the cell cycle. Our research suggests that TRIP13 might play a crucial role in CRC progression and could be a potential target for CRC therapy.

Colorectal cancer (CRC) is a frequently occurring digestive system cancer and postoperative tumor metastasis and recurrence are the main reasons for the failure of CRC treatment. The aim of this study was to identifying and validating key genes associated with metastatic recurrence of CRC. RNA expression of three datasets (GSE17538, GSE32323, and GSE29623) was used for biomarker discovery. We identified integrin‐binding sialoprotein (IBSP) as a candidate biomarker which was validated in three clinical cohorts (GSE41258, GSE21510, and GSE39582) and our clinical specimens. The results suggested that IBSP expression significantly increased at mRNA and protein levels among CRC cases, which was associated with metastatic recurrence, metastasis, high risk of recurrence, and poor survival in CRC. Consistent results were obtained in CRC cells. The relative level of serum IBSP evidently increased among CRC patients relative to normal controls, and downregulated after operation. As suggested by gene set enrichment analysis (GSEA), the IBSP level was associated with cell‐matrix adhesion in CRC. Functional experiments in vitro showed that IBSP promoted the growth and aggressiveness of CRC, and the potential mechanism by which IBSP promoted carcinogenesis of CRC was the abnormal activation of Fyn/β‐catenin signaling pathway. To sum up, findings in the present work indicate that IBSP can serve as the candidate biomarker for the diagnosis, treatment, and prognosis of CRC.

Purpose We explored specific expression profiles of BGN and COL11A1 genes and studied their biological functions in CRC using bioinformatics tools. Patients and Methods A total of 68 pairs of cancer and non-cancerous tissues from CRC patients were enrolled in this study. Methods we used in this articles including: qRT-PCR, Western blot analysis, ELISA, GO and KEGG regulatory network analysis, tumor infiltration, luciferase reporter-based protein and etc. Results According to The Cancer Genome Atlas (TCGA) data, BGN and COL11A1 expression levels were significantly higher in CRC patient samples than in samples from healthy controls. Moreover, levels were much higher in late-stage CRC than in early-stage disease, warranting evaluation of these genes as CRC prognostic biomarkers. Subsequently, qRT-PCR, Western blot analysis, and ELISA results obtained from analyses of CRC cells, tissues, and patient sera aligned with TCGA results. GO and KEGG regulatory network analysis revealed BGN- and COL11A1-associated genes that were functionally related to extracellular matrix (ECM) receptor pathway activation, with transcription factor genes RELA and NFKB1 positively associated with BGN expression and CEBPZ and SIRT1 with COL11A1 expression. Meanwhile, BGN and COL11A1 expression were separately and significantly correlated to tumor infiltration by six immune cell types. Additionally, kinase genes PLK1 and LYN appeared to be downstream targets of differentially expressed BGN and COL11A1, respectively. In addition, the expression of PLK1 mRNA was down-regulated while BGN was down-regulated. Finally, BGN effects on CRC cell proliferation, cycle, apoptosis, invasion, and migration were studied using molecular biological methods, including luciferase reporter-based protein analysis, qRT-PCR, and Western blot results, which revealed that miR-6828-5p may regulate BGN expression. Conclusion We speculate that the use of BGN and COL11A1 as CRC biomarkers would improve CRC staging, while also providing several novel targets for use in the development of more effective CRC treatments.