Identification of EGR4 as a prospective target for inhibiting tumor cell proliferation and a novel biomarker in colorectal cancer.

EGR4 (Early Growth Response 4) is a member of the EGR family, involving in tumorigenesis. However, the function and action mechanism of EGR4 in the pathogenesis of colorectal cancer (CRC) remain unclear. To address this, we assessed the prognosis of CRC based on EGR4 using the Kaplan-Meier plotter tool and tissue microarray. The abundance of immunoinfiltration was evaluated through ssGSEA, TISIDB, and TIMER. In vitro experiments involving knockdown or overexpression of EGR4 were performed, and RNA-sequencing was conducted to explore potential mechanisms. Furthermore, we used oxaliplatin and 5-fluorouracil to validate the impact of EGR4 on chemo-resistance. Pan-cancer analysis and tissue microarray showed that EGR4 was highly expressed in CRC and significantly correlated with an unfavorable prognosis. Moreover, EGR4 expression was associated with immunoinfiltration and cancer-associated fibroblasts in the CRC microenvironment. Functional enrichment demonstrated that high-expressional EGR4 were involved in chromatin and nucleosome assembly. Additionally, EGR4 promoted the proliferation of CRC cells. Mechanistically, EGR4 upregulated TNFα to activate the NF-κB signaling pathway, and its knockdown reduced p65 nuclear translocation. Importantly, combining shEGR4 with oxaliplatin and 5-fluorouracil significantly inhibited CRC proliferation. Taken together, these findings provide new insights into the potential prognosis and therapeutic targets of EGR4 in CRC.

ATP6AP1 as a potential prognostic biomarker in CRC by comprehensive analysis and verification.

The role of ATP6AP1 in colorectal cancer (CRC) remains elusive despite its observed upregulation in pan-cancer. Therefore, the current study aimed to assess the clinical significance of ATP6AP1 and its relationship with the immune infiltration in CRC. Transcriptome data of CRC were obtained from The Cancer Genome Atlas (TCGA) database and analyzed using the combination of R packages and tumor-related databases, including TIMER2, TISIDB, cBioPortal, and MethSurv. The tissue arrays and immunohistochemical staining were performed to verify the expression and clinical characteristics of ATP6AP1. The results revealed that ATP6AP1 expression was significantly elevated in CRC and associated with poor clinicopathological characteristics and prognosis. Furthermore, the analysis demonstrated ATP6AP1 expression was correlated with the infiltration of immune cells and cancer-associated fibroblasts in the microenvironment of CRC. Moreover, ATP6AP1 was found to be linked to various immune checkpoints and chemokines, with enrichment of cytoplasmic vesicle lumen, endopeptidase regulator activity, and endopeptidase inhibitor activity observed in the high ATP6AP1 expressional group. In conclusion, the findings of this study suggest that ATP6AP1 upregulation may serve as a biomarker for poor diagnosis in CRC and offer a potential target for immunotherapy in CRC.

The impact of SLC10A3 on prognosis and immune microenvironment in colorectal adenocarcinoma.

BACKGROUND: SLC10A3, a gene upregulated in pan-cancer, lacks full understanding regarding its prognostic implications and association with immune infiltration in colorectal cancer (CRC). This study comprehensively analyzed SLC10A3 in CRC, evaluating its prognostic significance and influence on the tumor's immune microenvironment. METHODS: Transcriptomic data from TCGA were obtained to compare SLC10A3 expression in both colorectal cancer (CRC) and normal tissues. Prognostic value was assessed for overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI). DNA methylation patterns of SLC10A3 and correlation with DNA mismatch repair (MMR) were explored. Genetic alterations in SLC10A3 were scrutinized. The study also delved into the influence of SLC10A3 on the immune microenvironment of CRC, including immune cell infiltration and chemokines. Involvement of cancer-associated fibroblasts (CAFs) was explored. Methylation status of specific CpG islands in the SLC10A3 gene correlated with CRC patient prognosis. CRC tissue microarray was performed to verify the expression of SLC10A3 and its relationship with prognosis. RESULTS: The research revealed that SLC10A3 is significantly upregulated in CRC and holds promise as a potential diagnostic marker. Elevated SLC10A3 expression was linked to poorer OS, DSS, and PFI. Methylation patterns of SLC10A3 displayed prognostic relevance, and genetic alterations in the gene were identified. SLC10A3 was shown to impact the immune microenvironment, with significant correlations observed between its expression and various immune cell types, chemokines, and markers associated with CAFs. Furthermore, an inverse relationship between SLC10A3 and MMR molecules was established. Methylation status of specific CpG islands within the SLC10A3 gene was associated with CRC patient prognosis. Tissue microarray showed that SLC10A3 was highly expressed in CRC and significantly correlated with poor prognosis. CONCLUSION: The study underscores the importance of elevated SLC10A3 in CRC, associating it with decreased survival and immune infiltration, proposing it as a diagnostic biomarker and appealing immunotherapy target, given its significant overexpression and influence on the immune microenvironment and prognosis through methylation patterns.

Establishment of a large-scale patient-derived high-risk colorectal adenoma organoid biobank for high-throughput and high-content drug screening.

BACKGROUND: Colorectal adenoma (CA), especially high-risk CA (HRCA), is a precancerous lesion with high prevalence and recurrence rate and accounts for about 90% incidence of sporadic colorectal cancer cases worldwide. Currently, recurrent CA can only be treated with repeated invasive polypectomies, while safe and promising pharmaceutical invention strategies are still missing due to the lack of reliable in vitro model for CA-related drug screening. METHODS: We have established a large-scale patient-derived high-risk colorectal adenoma organoid (HRCA-PDO) biobank containing 37 PDO lines derived from 33 patients and then conducted a series of high-throughput and high-content HRCA drug screening. RESULTS: We established the primary culture system with the non-WNT3a medium which highly improved the purity while maintained the viability of HRCA-PDOs. We also proved that the HRCA-PDOs replicated the histological features, cellular diversity, genetic mutations, and molecular characteristics of the primary adenomas. Especially, we identified the dysregulated stem genes including LGR5, c-Myc, and OLFM4 as the markers of adenoma, which are well preserved in HRCA-PDOs. Based on the HRCA-PDO biobank, a customized 139 compound library was applied for drug screening. Four drugs including metformin, BMS754807, panobinostat and AT9283 were screened out as potential hits with generally consistent inhibitory efficacy on HRCA-PDOs. As a representative, metformin was discovered to hinder HRCA-PDO growth in vitro and in vivo by restricting the stemness maintenance. CONCLUSIONS: This study established a promising HRCA-PDO biobank and conducted the first high-throughput and high-content HRCA drug screening in order to shed light on the prevention of colorectal cancer.

WTAP activates MAPK signaling through m6A methylation in VEGFA mRNA-mediated by YTHDC1 to promote colorectal cancer development.

N6-methyladenosine modification, especially Wilms tumor 1-associated protein (WTAP), is reportedly associated with a variety of cancers, including colorectal cancer (CRC). Angiogenesis also plays an important role in the occurrence and development of CRC. However, only a few studies have reported the biological mechanisms underlying this connection. Therefore, tissue microarray and public database were used to explore WTAP levels in CRC. Then, WTAP was down-regulated and over-expressed, respectively. CCK8, EdU, colony formation, and transwell experiments were performed to study the role of WTAP in CRC. Combined RNA sequencing and m6A RNA immunoprecipitation (MeRIP) sequencing, we found downstream molecules VEGFA. Moreover, a tube formation assay was executed for tumor angiogenesis. Finally, a subcutaneous tumorigenesis assay in nude mice was used to examine the tumor-promoting effect of WTAP in vivo. In the present study, WTAP was significantly upregulated in CRC cells and patients with CRC. Moreover, higher WTAP expression was observed in the TCGA and CPATC databases in CRC tissues. WTAP over-expression exacerbates cell proliferation, migration, invasion, and angiogenesis. Conversely, WTAP knockdown inhibited the malignant biological behavior of CRC cells. Mechanistically, WTAP positively regulated VEGFA, as identified using RNA sequencing and MeRIP sequencing. Moreover, we identified YTHDC1 as a downstream effector of the YTHDC1-VEGFA axis in CRC. Furthermore, increased WTAP expression activated the MAPK signaling pathway, which led to enhanced angiogenesis. In conclusion, our study revealed that the WTAP/YTHDC1/VEGFA axis promotes CRC development, especially angiogenesis, suggesting that it may act as a potential biomarker of CRC.

Identification and clinical validation of key genes as the potential biomarkers in colorectal adenoma.

BACKGROUND: Colorectal cancer (CRC), ranking third in cancer prevalence and second in mortality worldwide, is mainly derived from colorectal adenoma (CRA). CRA is a common benign disease in the intestine with rapidly increasing incidence and malignant potential. Therefore, this study aimed to recognize significant biomarkers and original pathogenesis in CRA. METHODS: Transcriptome data of GSE8671, GSE37364, and GSE15960 were downloaded from the Gene Expression Omnibus (GEO) datasets, and differentially expressed genes (DEGs) were screened. Functional pathways enrichment, protein-protein interaction (PPI) network, stem-correlation analysis, CIBERSORT, risk score and survival analyses were performed. RT-qPCR and immunohistochemical staining were applied to verify our results.  RESULTS: Screening for significant DEGs in each dataset, we identified 230 robust DEGs, including 127 upregulated and 103 downregulated genes. Functional pathways enrichment showed that these DEGs were distinctly enriched in various tumor-associated pathways, such as growth factor activity, extracellular structure organization, neutrophil activation, and inflammatory response. We filtered out two hub genes via STRING and Modules analysis, including CA2 and HSD11B2. Stem-correlation analysis displayed that hub genes were negatively associated with stem-related genes (Olfm4, CD44, CCND1 and MYC). The CIBERSORT algorithm indicated that Macrophage2, activated mast cells, and Neutrophils promoted CRA progression through inflammation. Survival analysis showed that CA2 and HSD11B2 were positively associated with survival outcomes in CRC. CONCLUSION: Our study has successfully identified the critical role of two core genes in the development and oncogenesis of CRA, which provides novel insight into the underlying pathogenesis, potential biomarkers and therapeutic targets.

Integrated analysis of mRNA and extrachromosomal circular DNA profiles to identify the potential mRNA biomarkers in breast cancer.

Extrachromosomal circular DNAs (eccDNAs) have been proved an inseparable relationship with cancer, based on the biological mechanisms of its biogenesis and impact on tumorigenesis, but still lacked of methods to analyze its function on the pathogenesis and progression of breast cancer (BC). The mRNA and eccDNA from BC cell samples (MDA-MB-453 and MCF-12A) were extracted with the removal of rRNA and linear DNA, respectively. High-throughput sequencing and bioinformatics analysis were performed to explore their expression level and molecular characterization of eccDNA. A total number of 161,062 eccDNA ranging from 33 bp to 54229 bp were detected with a median size of 1143 bp, distributed on all chromosomes and enriched on chromosome 20 the most. EccDNAs located in exons, upstream and downstream 2 kb regions were significantly increased compared with background. Analysis of eccDNA-related differentially expressed genes (eccDEGs) showed that FAT2 properly separated the two cells. CTNNB1, CACNA2D2 and CACNA1D were the hub genes with higher degrees in critical modules. All these four genes were significantly differentially expressed between breast invasive carcinoma (BRCA) tissues and normal ones. FAT2 and CTNNB1 correlated with significantly different overall survival (OS) when differentially expressed. The four genes showed a strong correlation with each other significantly and changed between tumor and normal samples. The results showed the potential of FAT2, CTNNB1, CACNA2D2 and CACNA1D as biomarkers with analysis of both DEGs and eccDEGs, which might assist in clinical medical treatment.

Effects of Immune Cells on Intestinal Stem Cells: Prospects for Therapeutic Targets.

The intestinal epithelium undergoes rapid cell turnover to maintain the integrity of the mucosal barrier, which is driven by the proliferation and differentiation of intestinal stem cells (ISCs). Due to their properties, ISCs are not only vulnerable targets during intestinal damage, but also act as the resources responsible for repair and regeneration. Moreover, the intestinal tract is the largest immune organ in the body, with the greatest number of immune cells including, but not limited to, macrophages, innate lymphoid cells and T cells. With the advance of intestinal organoid culture systems and single-cell RNA sequencing, the effects of immune cells on ISCs have been initially explored. As a component of the stem cell niche, these activated immune cells and their corresponding cytokines directly modulate apoptosis or survival of ISCs, leading to either destruction or protection of the intestinal epithelium in immune-mediated diseases, such as inflammatory bowel disease and graft-versus-host disease. In this review, we describe the effects of various immune cells on ISCs, as well as the mechanisms underlying these effects. We also highlight the remarkable role of ISCs in intestinal pathogenesis and raise the possibility of developing novel and effective therapeutic strategies for immune-mediated diseases based on ISCs.

CBX2 Expression in Colorectal Mucosa-adenoma-adenocarcinoma Sequence.

OBJECTIVE: To investigate the expression of chromobox 2 (CBX2) in colorectal adenoma (CRA) and colorectal cancer (CRC), and analyse its correlation with various clinicopathological parameters. STUDY DESIGN: Observational study. PLACE AND DURATION OF STUDY: Pathology Department, Huashan Hospital, Fudan University, from December 2019 to December 2020. METHODOLOGY: The mRNA level of CBX2 in colorectal mucosa, CRA and CRC was evaluated in gene expression profiling interactive analysis (GEPIA) and gene expression omnibus (GEO) dataset, then verified by quantitative real-time PCR (qRT-PCR). CBX2 expression by immunohistochemistry was determined in 122 samples, then its correlation was analysed with various clinicopathological parameters. Diagnostic value of CBX2 was estimated by the receiver operating characteristic curve (ROC). Prognostic value of CBX2 mRNA expression was evaluated via the Kaplan-Meier method in GEPIA. RESULTS: CBX2 expression rate in CRC (89.8%) was greater than adenoma (37.74%) and mucosa (20%). CBX2 protein levels were highest in adenocarcinoma and lowest in mucosa with intermediate level in adenoma. The area under curve (AUC) of CBX2 was 0.810 and 0.734, respectively, in distinguishing CRA from mucosa and CRC from CRA. CBX2 hyperexpression was not significantly correlated with clinicopathological variables, either in CRA or CRC. Kaplan-Meier survival analysis revealed that CBX2 mRNA hyperexpression was not associated with overall survival (OS) of CRC. Although the disease-free survival (DFS) of high CBX2 patients was shorter than those with low expression, but no obvious significance was found in colon adenocarcinoma (COAD, p=0.052) and rectal adenocarcinoma (READ, p=0.097). CONCLUSION: CBX2 expression progressively increased in the sequence of mucosa-adenoma-carcinoma, which may be used as a diagnostic biomarker and therapeutic target for CRA and CRC. Key Words: CBX2, Colorectal adenoma, Colorectal cancer, Biomarker.

Distinguishing colorectal adenoma from hyperplastic polyp by WNT2 expression.

BACKGROUND: Colorectal adenoma (CRA) is a classical premalignant lesion, with high incidence and mainly coexisting with hyperplastic polyp (HPP). Hence, this study aimed to distinguish CRA from HPP by molecular expression profiling and advance the prevention of CRA and its malignance. METHODS: CRA and paired HPP biopsies were collected by endoscopy. Through RNA-sequencing (RNA-seq), the differentially expressed genes (DEGs) were obtained. Functional enrichment analysis was performed based on the DEGs. The STRING database and Cytoscape were used to construct the protein-protein interaction (PPI) network and perform module analysis. Hub genes were validated by real-time quantitative PCR (RT-qPCR) and immunohistochemistry. The ROC curve was drawn to establish the specificity of the hub genes. RESULTS: 485 significant DEGs were identified including 133 up-regulated and 352 down-regulated. The top 10 up-regulated genes were DLX5, MMP10, TAC1, ACAN, TAS2R38, WNT2, PHYHIPL, DKK4, DUSP27, and ABCA12. The top 10 down-regulated genes were SFRP2, CHRDL1, KBTBD12, RERGL, DPP10, CLCA4, GREM2, TMIGD1, FEV, and OTOP3. Wnt signaling pathway and extracellular matrix (ECM) were up-regulated in CRA. Three hub genes including WNT2, WNT5A, and SFRP1 were filtered out via Cytoscape. Further RT-qPCR and immunohistochemistry confirmed that WNT2 was highly expressed in CRA. The area under the ROC curve (AUC) at 0.98 indicated the expression level of WNT2 as a candidate to differ CRA from HPP. CONCLUSION: Our study suggests Wnt signaling pathway and ECM are enriched in CRA, and WNT2 may be used as a novel biomarker for distinguishing CRA from HPP and preventing the malignance of CRA.

Acetylcholine ameliorates colitis by promoting IL-10 secretion of monocytic myeloid-derived suppressor cells through the nAChR/ERK pathway.

The alteration of the enteric nervous system (ENS) and its role in neuroimmune modulation remain obscure in the pathogenesis of inflammatory bowel diseases (IBDs). Here, by using the xCell tool and the latest immunolabeling-enabled three-dimensional (3D) imaging of solvent-cleared organs technique, we found severe pathological damage of the entire ENS and decreased expression of choline acetyltransferase (ChAT) in IBD patients. As a result, acetylcholine (ACh), a major neurotransmitter of the nervous system synthesized by ChAT, was greatly reduced in colon tissues of both IBD patients and colitis mice. Importantly, administration of ACh via enema remarkably ameliorated colitis, which was proved to be directly dependent on monocytic myeloid-derived suppressor cells (M-MDSCs). Furthermore, ACh was demonstrated to promote interleukin-10 secretion of M-MDSCs and suppress the inflammation through activating the nAChR/ERK pathway. The present data reveal that the cholinergic signaling pathway in the ENS is impaired during colitis and uncover an ACh-MDSCs neuroimmune regulatory pathway, which may offer promising therapeutic strategies for IBDs.

A novel endoscopic visible full-thickness cryoablation device on stomach.

Cryoablation has been used for the treatment of various sorts of solid visceral tumors, but few are reported on gastric tumor via endoscope, in terms of accurate control of ablation site, freezing depth and effective temperature. Thus, we developed a novel device, which could perform accurate cryoablation on the stomach via endoscope. This study aimed to evaluate the efficacy and safety of the device on porcine stomach. Results showed that the novel device could provide direct view of the operation space, allowing accurate and safe ablation of the stomach. Three minutes cryoablation caused a transmural, 1 cm radius gastric lesion. On serosal side, the temperature dropped to -64.2 °C, -34.1 °C, 26.1 °C at the center, 1 cm and 2 cm from center, respectively. Histopathology revealed acute ruptured cells with damaged glands in mucosa, partial disruption in muscularis propria and serosal slight exudation. Three months later, scar formed with complete recovery of gastric structure. No active bleeding or perforation of stomach, nor injury or adhesion of adjacent organs was observed. This endoscopic cryoablation device allowed safe, full-thickness cryoablation with effective temperature, which may provide an alternative treatment for gastric tumor.

H19/miR-148a/USP4 axis facilitates liver fibrosis by enhancing TGF-ß signaling in both hepatic stellate cells and hepatocytes.

Liver fibrosis is a wound-healing response represented by excessive extracellular matrix deposition. Activation of hepatic stellate cell (HSC) is the critical cellular basis for hepatic fibrogenesis, whereas hepatocyte undergoes epithelial-mesenchymal transition (EMT) which is also involved in chronic liver injury. Long noncoding RNA H19 has been found to be associated with cholestatic liver fibrosis lately. However, the role of H19 in liver fibrosis remains largely to be elucidated. In this study, we found that the expression of H19 was significantly upregulated in the liver tissue of CCl4 -induced mice, a toxicant-induced liver fibrogenesis model. Overexpression of H19 significantly aggravated activation of HSC and EMT of hepatocyte both by stimulating transforming growth factor-ß (TGF-ß) pathway. In terms of mechanism, H19 functioned as a competing endogenous RNA to sponge miR-148a and subsequently sustained the level of ubiquitin-specific protease 4 (USP4), which was an identified target of miR-148a and was able to stabilize TGF-ß receptor I. In conclusion, our findings revealed a novel H19/miR-148a/USP4 axis which promoted liver fibrosis via TGF-ß pathway in both HSC and hepatocyte, indicating that H19 could become a promising target for the treatment of liver fibrosis.

MicroRNA-212 activates hepatic stellate cells and promotes liver fibrosis via targeting SMAD7.

There has been an increasing number of researches about microRNAs (miRNAs) in the progression of liver fibrosis from the point of their comprehensive functions in regulating the activation of hepatic stellate cells (HSCs). Among them, it has been reported that miR-212 is up-regulated in activated rat primary HSCs. However, its mechanism has not been determined yet. Here, we confirmed that the level of miR-212-3p was up-regulated in livers of carbon tetrachloride (CCl4)-treated mice compared with the normal control, which is a classical model of chronically damaged fibrotic liver. In vitro, we demonstrated that TGF-ß, a master fibrogenic cytokine, could induce the level of miR-212. In turn, overexpression of miR-212 could induce the activation marker of HSC including α-smooth muscle actin (α-SMA) and collagens by activating TGF-ß signaling pathway. Furthermore, SMAD7, a dominant suppressor of TGF-ß pathway, was identified as a direct target of miR-212-3p. Our results indicate that miR-212-3p facilitates the activation of HSCs and TGF-ß pathway by targeting SMAD7, highlighting that it can be served as a novel biomarker or therapeutic target for liver fibrosis.

A five-gene based risk score with high prognostic value in colorectal cancer.

Colorectal cancer (CRC) is one of the most frequently occurring malignancies worldwide. The outcomes of patients with similar clinical symptoms or at similar pathological stages remain unpredictable. This inherent clinical diversity is most likely due to the genetic heterogeneity. The present study aimed to create a predicting tool to evaluate patient survival based on genetic profile. Firstly, three Gene Expression Omnibus (GEO) datasets (GSE9348, GSE44076 and GSE44861) were utilized to identify and validate differentially expressed genes (DEGs) in CRC. The GSE14333 dataset containing survival information was then introduced in order to screen and verify prognosis-associated genes. Of the 66 DEGs, the present study screened out 46 biomarkers closely associated to patient overall survival. By Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis, it was demonstrated that these genes participated in multiple biological processes which were highly associated with cancer proliferation, drug-resistance and metastasis, thus further affecting patient survival. The five most important genes, MET proto-oncogene, receptor tyrosine kinase, carboxypeptidase M, serine hydroxymethyltransferase 2, guanylate cyclase activator 2B and sodium voltage-gated channel a subunit 9 were selected by a random survival forests algorithm, and were further made up to a linear risk score formula by multivariable cox regression. Finally, the present study tested and verified this risk score within three independent GEO datasets (GSE14333, GSE17536 and GSE29621), and observed that patients with a high risk score had a lower overall survival (P<0.05). Furthermore, this risk score was the most significant compared with other predicting factors including age and American Joint Committee on Cancer stage, in the model, and was able to predict patient survival independently and directly. The findings suggest that this survival associated DEGs-based risk score is a powerful and accurate prognostic tool and is promisingly implemented in a clinical setting.