Immunological profile of lactylation-related genes in Crohn's disease: a comprehensive analysis based on bulk and single-cell RNA sequencing data.

BACKGROUND: Crohn's disease (CD) is a disease characterized by intestinal immune dysfunction, often accompanied by metabolic abnormalities. Disturbances in lactate metabolism have been found in the intestine of patients with CD, but studies on the role of lactate and related Lactylation in the pathogenesis of CD are still unknown. METHODS: We identified the core genes associated with Lactylation by downloading and merging three CD-related datasets (GSE16879, GSE75214, and GSE112366) from the GEO database, and analyzed the functions associated with the hub genes and the correlation between their expression levels and immune infiltration through comprehensive analysis. We explored the Lactylation levels of different immune cells using single-cell data and further analyzed the differences in Lactylation levels between inflammatory and non-inflammatory sites. RESULTS: We identified six Lactylation-related hub genes that are highly associated with CD. Further analysis revealed that these six hub genes were highly correlated with the level of immune cell infiltration. To further clarify the effect of Lactylation on immune cells, we analyzed single-cell sequencing data of immune cells from inflammatory and non-inflammatory sites in CD patients and found that there were significant differences in the levels of Lactylation between different types of immune cells, and that the levels of Lactylation were significantly higher in immune cells from inflammatory sites. CONCLUSIONS: These results suggest that Lactylation-related genes and their functions are closely associated with changes in inflammatory cells in CD patients.

Retraction Notice to: Upregulation of lncRNA LINC00460 Facilitates GC Progression through Epigenetically Silencing CCNG2 by EZH2/LSD1 and Indicates Poor Outcomes.

[This retracts the article DOI: 10.1016/j.omtn.2019.12.041.].

RNA N6-Methyladenosine Methyltransferase METTL3 Facilitates Colorectal Cancer by Activating the m6A-GLUT1-mTORC1 Axis and Is a Therapeutic Target.

BACKGROUND & AIMS: RNA N6-methyladenosine (m6A) modification has recently emerged as a new regulatory mechanism in cancer progression. We aimed to explore the role of the m6A regulatory enzyme METTL3 in colorectal cancer (CRC) pathogenesis and its potential as a therapeutic target. METHODS: The expression and clinical implication of METTL3 were investigated in multiple human CRC cohorts. The underlying mechanisms of METTL3 in CRC were investigated by integrative m6A sequencing, RNA sequencing, and ribosome profiling analyses. The efficacy of targeting METTL3 in CRC treatment was elucidated in CRC cell lines, patient-derived CRC organoids, and Mettl3-knockout mouse models. RESULTS: Using targeted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 dropout screening, we identified METTL3 as the top essential m6A regulatory enzyme in CRC. METTL3 was overexpressed in 62.2% (79/127) and 88.0% (44/50) of primary CRCs from 2 independent cohorts. High METTL3 expression predicted poor survival in patients with CRC (n = 374, P < .01). Functionally, silencing METTL3 suppressed tumorigenesis in CRC cells, human-derived primary CRC organoids, and Mettl3-knockout mouse models. We discovered the novel functional m6A methyltransferase domain of METTL3 in CRC cells by domain-focused CRISPR screening and mutagenesis assays. Mechanistically, METTL3 directly induced the m6A-GLUT1-mTORC1 axis as identified by integrated m6A sequencing, RNA sequencing, ribosome sequencing, and functional validation. METTL3 induced GLUT1 translation in an m6A-dependent manner, which subsequently promoted glucose uptake and lactate production, leading to the activation of mTORC1 signaling and CRC development. Furthermore, inhibition of mTORC1 potentiated the anticancer effect of METTL3 silencing in CRC patient-derived organoids and METTL3 transgenic mouse models. CONCLUSIONS: METTL3 promotes CRC by activating the m6A-GLUT1-mTORC1 axis. METTL3 is a promising therapeutic target for the treatment of CRC.

Preservation of the fecal samples at ambient temperature for microbiota analysis with a cost-effective and reliable stabilizer EffcGut.

With the increasing researches on the role of gut microbiota in human health and disease, appropriate storage method of fecal samples at ambient temperature would conveniently guarantee the precise and reliable microbiota results. Nevertheless, less choice of stabilizer that is cost-efficient and feasible to be used in longer preservation period obstructed the large-scale metagenomics studies. Here, we evaluated the efficacy of a guanidine isothiocyanate-based reagent method EffcGut and compared it with the other already used storage method by means of 16S rRNA gene sequencing technology. We found that guanidine isothiocyanate-based reagent method at ambient temperature was not inferior to OMNIgene·GUT OM-200 and it could retain the similar bacterial community as that of -80 °C within 24 weeks. Furthermore, bacterial diversity and community structure difference were compared among different sample fraction (supernatant, suspension and precipitate) preserved in EffcGut and -80 °C. We found that supernatant under the preservation of EffcGut retained the similar community structure and composition as that of the low temperature preservation method.

Featured Gut Microbiomes Associated With the Progression of Chronic Hepatitis B Disease.

Dysbiosis of gut microbiota during the progression of HBV-related liver disease is not well understood, as there are very few reports that discuss the featured bacterial taxa in different stages. The aim of this study was to reveal the featured bacterial species whose abundances are directly associated with HBV disease progression, that is, progression from healthy subjects to, chronic HBV infection, chronic hepatitis B to liver cirrhosis. Approximately 400 fecal samples were collected, and 97 samples were subjected to 16S rRNA gene sequencing after age and BMI matching. Compared with the healthy individuals, significant gut microbiota alterations were associated with the progression of liver disease. LEfSe results showed that the HBV infected patients had higher Fusobacteria, Veillonella, and Haemophilus abundance while the healthy individuals had higher levels of Prevotella and Phascolarctobacterium. Indicator analysis revealed that 57 OTUs changed as the disease progressed, and their combination produced an AUC value of 90% (95% CI: 86-94%) between the LC and non-LC groups. In addition, the abundances of OTU51 (Dialister succinatiphilus) and OTU50 (Alistipes onderdonkii) decreased as the disease progressed, and these results were further verified by qPCR. The LC patients had the higher bacterial network complexity, which was accompanied with a lower abundance of potential beneficial bacterial taxa, such as Dialister and Alistipes, while they had a higher abundance of pathogenic species within Actinobacteria. The compositional and network changes in the gut microbiota in varied CHB stages, suggest the potential contributions of gut microbiota in CHB disease progression.

Upregulation of lncRNA LINC00460 Facilitates GC Progression through Epigenetically Silencing CCNG2 by EZH2/LSD1 and Indicates Poor Outcomes.

Non-protein-coding functional elements in the human genome in the postgenomic biology field have been drawing great attention in recent years. Thousands of long non-coding RNAs (lncRNAs) have been found to be expressed in various tumors. Yet only a small proportion of these lncRNAs have been well characterized. We have demonstrated that LINC00460 could affect cell proliferation through epigenetic regulation of KLF2 and CUL4A in human colorectal cancer. However, the clinical significance and biological role of LINC00460 in gastric cancer (GC) remain largely unknown. In this research, we discovered that LINC00460 is remarkably upregulated in GC tissues compared to the non-tumor tissues. Additionally, LINC00460 served as an independent prognostic marker in GC. Functionally, proliferation of GC cells could be regulated by LINC00460 both in vitro and in vivo. RNA sequencing (RNA-seq) analysis for the whole transcriptome indicated that LINC00460 may serve as a key regulatory factor in the tumorigenesis of GC. What's more, the biological function of LINC00460 was mediated, to certain extent, by the direct interaction with enhancer of zeste homolog 2 (EZH2) and lysine (K)-specific demethylase 1A (LSD1) proteins. Further analyses indicated that LINC00460 promoted GC proliferation at least partly through the downregulation of tumor suppressor-gene Cyclin G2 (CCNG2), which is mediated by EZH2 and LSD1. In conclusion, our results suggested that LINC00460 acted as an oncogene in GC to inhibit the expression of CCNG2 at least partly by binding with EZH2 and LSD1. Our study could provide additional insights into the development of novel target therapeutic methods for GC.

Long non-coding RNA ERICH3-AS1 is an unfavorable prognostic factor for gastric cancer.

Long non-coding RNAs (lncRNAs) play important roles in gastric cancer (GC), but the mechanism is not fully clear. ERICH3-AS1 (ERICH3 antisense RNA1) is affiliated with the non-coding RNA class which has proven to be involved in the prognostic of GC, but the function of ERICH3-AS1 is still unclear. In this study, we aim to explore the potential function of ERICH3-AS1 in the development of GC and analyze the prognostic role of ERICH3-AS1 in GC. We found that the lncRNA ERICH3-AS1 was significantly up-regulated in GC tissues in the analysis of The Cancer Genome Atlas (TCGA) data; the Kaplan-Meier analysis showed that the higher the expression of ERICH3-AS1 was, the earlier the recurrence and the poorer the prognosis would be in patients. Cox univariate and multivariate analyses revealed that ERICH3-AS1 was a risk factor of disease-free survival (DFS) (p < 0.05) and overall survival (OS) (p < 0.05) of patients. Through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, it demonstrated that the ERBB pathways, the mitogen-activated protein kinase (MAPK) pathways, the MTOR pathways, p53 pathways and Wnt pathways were differentially enriched in ERICH3-AS1 high expression phenotype. Furthermore, the correlation analysis showed that ERICH3-AS1 had significant correlations with apoptosis-related proteins such as BCL2L10 and CASP14; cell cycle-associated proteins CDK14 and invasion and migration-associated proteins such as MMP20, MMP26 and MMP27. In summary, we identified that increased ERICH3-AS1 might be a potential biomarker for diagnosis and independent prognostic factor of GC. Moreover, ERICH3-AS1 might participate in the oncogenesis and development of tumors via cell cycle and apoptosis pathway mediated by ERBB, MAPK, MTOR, p53 and Wnt pathways.

Macrophages-derived p38α promotes the experimental severe acute pancreatitis by regulating inflammation and autophagy.

BACKGROUND: Severe acute pancreatitis (SAP) is a common threat to human health. In the present study, we aimed to investigate the underlying mechanisms by which p38α in macrophages contributes to SAP. We used conditional knockout of p38α in macrophages and p38 MAPK inhibitors to understand the effects of p38α in macrophages on caerulein-induced inflammatory responses in SAP mice models. METHODS AND MATERIALS: Wild-type (WT) mice were randomly divided into three groups: a control group, SAP group, and SAP + p38MAPK inhibitor (SB203580) group, and mice with a conditional knockout (KO) of p38α in macrophages were included in a KO + SAP group. We evaluated pancreatic pathology and ultra-structure by hematoxylin and eosin staining and transmission electron microscopy. The pulmonary wet-to-dry weight ratio was calculated. The serum levels of TNF-α and IL-1ß were determined by ELISA. The mRNA and protein expression of inflammatory cytokines TNF-α, IL-1ß, IL-17, IL-18, MIF, and MCP-1 in pancreatic tissues were tested by qRT-PCR and immunohistochemistry analysis. The protein expression of p38, caspase-1, ULK1, LC3B and p62 in pancreatic tissues was examined by Western blotting. RESULTS: The results indicated that the severity of SAP as well as the expression of the cytokines TNF-α, IL-1ß, IL-17, IL-18 and MCP-1 were higher in the SAP group than those in the control group, but were lower in the SAP + SB203580 and KO + SAP groups as compared with the SAP group. The protein expression of p38, caspase-1, LC3B and p62 was increased in the SAP group than that in the control group, but this result was reversed in the SAP + SB203580 and KO + SAP groups as compared with the SAP group. In addition, the ULK1 level was significantly lower in the SAP group than that in the control group, but was increased in the SAP + SB203580 and KO + SAP groups as compared with the SAP group. CONCLUSIONS: Our findings demonstrated that, macrophage derived p38α promoted the experimental severe acute pancreatitis by regulating inflammation and autophagy.

Is dextran sulfate sodium a good inducer of acute experimental enteritis?

Animal models play critical roles in exploring the pathogenesis of human diseases and designing novel therapeutic schemes. Acute experimental colitis (AEC) models have been reported to be established in mice principally by oral administration of dextran sulfate sodium (DSS). However, little knowledge is known about whether DSS can be used to induce the acute experimental enteritis (AEE). In this study, different concentrations of DSS (0%, 2%, 3%, and 5%) were used to induce AEC and AEE models in two cohorts. After the establishment of these two models, the symptoms of the mice induced by DSS were noted, the length and average weight of each colon and small intestine were measured, and hematoxylin and eosin (HE) staining was conducted for assessing the inflammatory infiltration in these models. Generally, the comparison of the inflammatory scoring between AEC and AEE models was analyzed. As a consequence, we found that, the mice with 2%-5% DSS administration in a week could develop into AEC models in two cohorts and AEE models in one cohort, followed by the signs of diarrhea, gross rectal bleeding, weight loss of the body, and shortened colon and intestine length, as compared with the control group. HE staining showed that the inflammatory scoring was dramatically increased by 3%-5% DSS in AEC models in two cohorts but slightly elevated in AEE models in one cohort. Meanwhile, as compared with the severe AEC models, the extent of inflammatory infiltration induced by 3%-5% DSS in AEE models was much milder. In conclusion, oral administration of 3%-5% DSS is a good inducer of AEC models, but not AEE models.

Macrophage p38α promotes nutritional steatohepatitis through M1 polarization.

BACKGROUND & AIMS: p38 mitogen-activated protein kinases are important inflammatory factors. p38α alteration has been implicated in both human and mouse inflammatory disease models. Therefore, we aimed to characterize the cell type-specific role of p38α in non-alcoholic steatohepatitis (NASH). METHODS: Human liver tissues were obtained from 27 patients with non-alcoholic fatty liver disease (NAFLD) and 20 control individuals. NASH was established and compared between hepatocyte-specific p38α knockout (p38αΔHep), macrophage-specific p38α knockout (p38αΔMΦ) and wild-type (p38αfl/fl) mice fed with high-fat diet (HFD), high-fat/high-cholesterol diet (HFHC), or methionine-and choline-deficient diet (MCD). p38 inhibitors were administered to HFHC-fed wild-type mice for disease treatment. RESULTS: p38α was significantly upregulated in the liver tissues of patients with NAFLD. Compared to p38αfl/fl littermates, p38αΔHep mice developed significant nutritional steatohepatitis induced by HFD, HFHC or MCD. Meanwhile, p38αΔMΦ mice exhibited less severe steatohepatitis and insulin resistance than p38αfl/fl mice in response to a HFHC or MCD. The effect of macrophage p38α in promoting steatohepatitis was mediated by the induction of pro-inflammatory factors (CXCL2, IL-1ß, CXCL10 and IL-6) secreted by M1 macrophages and associated signaling pathways. p38αΔMΦ mice exhibited M2 anti-inflammatory polarization as demonstrated by increased CD45+F4/80+CD11b+CD206+ M2 macrophages and enhanced arginase activity in liver tissues. Primary hepatocytes from p38αΔMΦ mice showed decreased steatosis and inflammatory damage. In a co-culture system, p38α deleted macrophages attenuated steatohepatitic changes in hepatocytes through decreased secretion of pro-inflammatory cytokines (TNF-α, CXCL10 and IL-6), which mediate M1 macrophage polarization in p38αΔMΦ mice. Restoration of TNF-α, CXCL10 or IL-6 induced lipid accumulation and inflammatory responses in p38αfl/fl hepatocytes co-cultured with p38αΔMΦ macrophages. Moreover, pharmacological p38 inhibitors suppressed HFHC-induced steatohepatitis. CONCLUSIONS: Macrophage p38α promotes the progression of steatohepatitis by inducing pro-inflammatory cytokine secretion and M1 polarization. p38 inhibition protects against steatohepatitis. LAY SUMMARY: p38 mitogen-activated protein kinases are important inflammatory factors. In the present study, we demonstrated that p38α is upregulated in liver tissues of patients with non-alcoholic fatty liver diseases. Genetic deletion of p38α in macrophages led to ameliorated nutritional steatohepatitis in mice through decreased pro-inflammatory cytokine secretion and increased M2 macrophage polarization.