Abstract Background Exosomes are extensively reported to be strongly associated with many immunologic diseases, including Crohn disease (CD). Meanwhile, the dysfunction of macrophage activation has been proposed to be critical for the pathogenesis of CD. However, it is an unsettled issue whether serum exosomes from CD could activate macrophages and participate in its pathogenesis. Our study intended to clarify the role of CD-derived exosomes on macrophages to elucidate a novel mechanism and possible diagnostic and therapeutic strategies. Methods Serum exosomes were isolated and identified. Functional assays in vitro were performed on Raw264.7 macrophages, followed by exosomal microRNA (miRNA) profiling and bioinformatics analyses via high-throughput sequencing. In animal experiments, exosomes were intraperitoneally injected into dextran sulfate sodium–induced colitis. Results In vitro CD-derived exosomes induced proinflammatory cytokine expression and increased macrophage counts. Meanwhile, the intervention of exosomes from CD with epithelial cells led to increased permeability of the intestinal epithelial barrier. In vivo, CD-derived exosomes could circulate into the intestinal mucosa and significantly aggravate colitis. Furthermore, CD changed the miRNA profile of exosomes and further analysis revealed a differential expression of let-7b-5p. Mechanistically, the let-7b-5p/TLR4 pathway was recognized as a potential contributor to macrophage activation and inflammatory response. Furthermore, serum exosome–mediated let-7b-5p mimic delivery alleviated colitis significantly. Conclusions Our study indicated that serum exosomes can circulate into the intestinal mucosa to aggravate colitis by regulating macrophage activation and epithelial barrier function. In addition, CD showed altered exosomal miRNA profiles. Furthermore, serum exosome–mediated let-7b-5p-mimic delivery may significantly alleviate colitis, providing potential novel insight into an exosome-based strategy for the diagnosis and treatment of CD.
Accumulated evidence supports that long non-coding RNAs (lncRNAs) are involved significantly in the development of human cancers. Enhancer RNAs (eRNAs), a subtype of lncRNAs, have recently attracted much attention about their roles in carcinogenesis. Colon adenocarcinoma is one of the most commonly diagnosed tumors with unfavorable prognosis. It highlights the great significance of screening and identifying novel biomarkers. More importantly, it remains to be elucidated with respect to the function of eRNAs in colon adenocarcinoma, as is in pan-cancers. The expression of LINC02257 was determined based on the data obtained from The Cancer Genome Atlas (TCGA). Further evaluation was performed on the basis of the following analyses: clinicopathology and survival analysis, gene ontology (GO) terms, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, as well as multi-omics immunotherapy-related analysis and co-expression analysis. The statistical analysis was conducted in R software, and immune cell infiltration of LINC02257 expression in cancers was investigated by using the CIBERSORT algorithm. By large-scale data mining, our study highlighted that a total of 39 eRNA genes were associated with colon adenocarcinoma prognosis, among which 25 eRNAs showed significant associations with their predicted target genes. LINC02257 was identified as the most significant survival-associated eRNA, with DUSP10 as its target gene. Besides, the high expression of LINC02257 in colon adenocarcinoma was more vulnerable to unfavorable prognosis and correlated with various clinical characteristics. GO and KEGG analyses revealed that LINC02257 was closely correlated with extracellular matrix organization via the PI3K-Akt signaling pathway. Besides, LINC02257 expression correlated with a multi-omics analysis of 33 cancer types, such as survival analysis [overall survival (OS), disease-specific survival (DSS), disease-free interval (DFI), and progression-free interval (PFI)] and immunotherapy-related analysis [tumor microenvironment (TME), tumor mutational burden (TMB), and microsatellite instability (MSI)]. Finally, we investigated the co-expression genes of LINC02257 and its potential signaling pathways across different cancer types. LINC02257 is screened and can function as an independent prognostic biomarker through the PI3K-Akt signaling pathway for colon adenocarcinoma. Simultaneously, LINC02257 may be a multifaceted and significant immunotherapy-related eRNA in different cancers.
The ubiquitously expressed multifunctional protein, CUEDC2 (CUE domain-containing 2), is involved in many physiological and pathological processes, including the cell cycle regulation and inflammation. Although it is known that CUEDC2 is expressed disparately in breast cancer, ovarian carcinoma, hepatocellular carcinoma, cholangiocarcinoma, glioma, lung adenocarcinoma, colon cancers, and is involved in the Warburg's effect, its role in oncogenesis remains to be further explored. In this review, we examine the expression of CUEDC2 in various tumors, and discuss several fundamental signaling pathways that are impacted by CUEDC2.
Abstract Background Esophageal squamous cell cancer (ESCC) poses serious threats to human life. Hence, the search for effective bio-markers to predict the occurrence and development of ESCC is of emerging significance. Methods We used immunohistochemistry to semi-quantitatively detect CUEDC2 expression in 50 ESCC cases and 20 adjacent tissues, analyzing the relationship with clinicopathological parameters and prognosis outcomes. Additionally, investigating the differences between CUEDC2 and CD68 in ESCC. Result CUEDC2 expression was higher in 9 ESCC tissues and lower in 41 ESCC tissues. Whereas, CUEDC2 expression was higher in 11 adjacent tissues and lower of the rest 9 cases, and the differences were statistically significant (P<0.05).CUEDC2 and ESCC clinicopathological characteristics exerted no significant difference (P>0.05). Via the Kaplan-Meier method, CUEDC2 and tumor grade demonstrated an impact on ESCC prognosis ( P<0.05). By double-immunofluorescence, there was an expression difference between CD68 and CUEDC2, and the difference was statistically significant (P<0.05). There showed co-localization of CUEDC2 and CD68 fluorescence. Conclusion CUEDC2 was relatively lower expressed in ESCC and higher in adjacent tissues. There was no significant difference between CUEDC2 and ESCC clinicopathological characteristics.CUEDC2 and tumor grade presented an impact on ESCC prognosis. There might be an interaction between CD68 and CUEDC2.
Background: There has been growing evidence that the aberrantly expressed Homeobox-C 4 (HOXC4) plays crucial roles in the development of some cancer types. However, it remains unclear as far as its expression patterns and prognostic significance are concerned, as is tumor immunity. Methods: To investigate the expression levels and prognostic implications of HOXC4, multiple data sources were used in conjunction with quantitative real-time polymerase chain reaction (qRT-PCR) verification. Afterward, diverse immunological-related analyses, along with anti-cancer drug sensitivity, were performed in a number of cancer types. A further exploration of the underlying mechanisms of HOXC4 in tumorigenesis and immunity was carried out using the Gene Set Enrichment Analysis (GSEA) and the Gene Set Variation Analysis (GSVA). Results: Based on extensive database mining, HOXC4 was ubiquitously expressed across 21 tumor cell lines and significantly higher than that of normal tissues in 21 tumor types. The outcome of survival analysis including overall survival (OS), disease-free interval (DFI), disease-specific survival (DSS) and progression-free interval (PFI) revealed that upregulation of HOXC4 expression in several cancers was associated with worse prognosis. Additionally, HOXC4 was observed to correlate closely with colon adenocarcinoma (COAD), head and neck squamous cell carcinoma (HNSC), lower grade glioma (LGG), liver hepatocellular carcinoma (LIHC), rectum adenocarcinoma (READ), and thyroid carcinoma (THCA) in terms of tumor immune cells infiltration. As a result of our comprehensive pan-cancer study, we have identified a significant link between the expression of HOXC4 and the efficacy of immunotherapy-related treatments, together with anti-cancer drug sensitivity. As a final note, HOXC4 was found to modulate multiple signaling pathways involved in tumorigenesis and immunity. Conclusion: HOXC4 has been implicated in our study for the first time as an oncogene in cancers with a poor prognosis, potentially laying the groundwork for promising clinical biomarkers and immunotherapy approaches.
Background: Necroptosis contributes significantly to colon adenocarcinoma (COAD). We aim to assess the relationship between immunoinfiltration and stemness in COAD patients through the development of a risk score profile using necroptosis-related long noncoding RNAs (NRLs). Methods: Our study was based on gene expression data and relevant clinical information from The Cancer Genome Atlas (TCGA). Necroptosis-related genes (NRGs) were obtained from the Kyoto Encyclopedia of Genes and Genome (KEGG) database. Pearson correlation analysis, Cox regression, and least absolute shrinkage and selection operator (LASSO) regression were used to determine the NRL prognositic signature (NRLPS). NRLs expression was examined using qRT-PCR method. Several algorithms were used to identify relationships between immune cell infiltration and NRLPS risk scores. Further analysis of somatic mutations, tumor stemness index (TSI), and drug sensitivity were also explored. Results: To construct NRLPS, 15 lncRNAs were investigated. Furthermore, NRLPS patients with high-risk subgroups had lower survival rates than that of patients with low-risk subgroups. Using GSEA analysis, NRL was found to be enriched in Notch, Hedgehog and Smoothened pathways. Immune infiltration analysis showed significant differences in CD8+ T cells, dendritic cell DCs, and CD4+ T cells between the two risk groups. In addition, our NRLPS showed a relevance with the regulation of tumor microenvironment, tumor mutation burden (TMB) and stemness. Finally, NRLPS demonstrated potential applications in predicting the efficacy of immunotherapy and chemotherapy in patients with COAD. Conclusion: Based on NRLs, a prognostic model was developed for COAD patients that allows a personalized tailoring immunotherapy and chemotherapy to be tailored.
Background/Aims: Primary angiosarcoma of the small intestine is a rare neoplasia, and there are limited data from systematic analyses. The aim of this study is to describe the clinical and pathological characteristics in addition to the prognostic factors for this rare neoplasia. Methods: We retrospectively collected the clinical records and prognostic information of 66 patients with small intestine angiosarcoma reported between 1970 and 2017. We used the Chi-square test, the log-rank test, and Cox regression analyses to evaluate the data. Results: There were 66 patients diagnosed with small intestine angiosarcoma. The onset age ranged from 24–92 years old. There were 24 patients diagnosed before the year 2000, and 42 patients were diagnosed after 2000. The data indicated that 49 cases were diagnosed as primary disease, and the remaining 15 cases were secondary disease. The main clinical symptoms were nonspecific and included gastrointestinal (GI) bleeding and abdominal pain. Additionally, we found multi-center foci were one of the characteristics of this disease. Radiation-induced small intestine angiosarcoma (RSIA) is a special type of disease with a similar prognosis. This type was more frequent in females and decreased after the year 2000. We also found that GI bleeding was less common in RSIA cases. The log-rank test results revealed that old-age, poor differentiation, and GI bleeding were associated with worse prognosis. Surgical treatment showed a trend toward a prolonged survival time. However, the result was not statistically significant. Our results show treatment with adjuvant therapy improved prognosis. The multivariate Cox analysis demonstrated adjuvant therapy was an independent indicator of a favorable outcome in small intestine angiosarcoma patients. Conclusion: Pay attention to the unexplained gastrointestinal bleeding could lead to a faster diagnosis and control of small intestine angiosarcoma. Furthermore, treatments including adjuvant therapy can effectively improve the prognosis.
Background Lactate metabolism is critically involved in the tumor microenvironment (TME), as well as cancer progression. It is important to note, however, that lactate metabolism-related long non-coding RNAs (laRlncRNAs) remain incredibly understudied in colon adenocarcinoma (COAD). Methods A gene expression profile was obtained from the Cancer Genome Atlas (TCGA) database to identify laRlncRNA expression in COAD patients. A risk signature with prognostic value was identified from TCGA and Gene Expression Omnibus (GEO) cohort based on laRlncRNA pairs by the least absolute shrinkage and selection operator (LASSO) and Cox regression analyses. Quantitative real-time polymerase chain reaction (qRT-PCR) and functional experiments were carried out to verify the expression of laRlncRNAs in COAD. The relationship of laRlncRNA pairs with immune landscape as well as the sensitivity of different therapies was explored. Results In total, 2378 laRlncRNAs were identified, 1,120 pairs of which were studied to determine their prognostic validity, followed by a risk signature established based on the screened 5 laRlncRNA pairs. The laRlncRNA pairs-based signature provided a better overall survival (OS) prediction than other published signatures and functioned as a prognostic marker for COAD patients. According to the calculated optimal cut-off point, patients were divided into high- and low-risk groups. The OS of COAD patients in the high-risk group were significantly shorter than that of those in the low-risk group (P=4.252e-14 in the TCGA cohort and P=2.865-02 in the GEO cohort). Furthermore, it remained an effective predictor of survival in strata of gender, age, TNM stage, and its significance persisted after univariate and multivariate Cox regressions. Additionally, the risk signature was significantly correlated with immune cells infiltration, tumor mutation burden (TMB), microsatellite instability (MSI) as well as immunotherapeutic efficacy and chemotherapy sensitivity. Finally, one of the laRlncRNA, LINC01315, promotes proliferation and migration capacities of colon cancer cells. Conclusion The newly identified laRlncRNAs pairs-based signature exhibits potential effects in predicting prognosis, deciphering patients’ immune landscape, and mediating sensitivity to immunotherapy and chemotherapy. Findings in our study may provide evidence for the role of laRlncRNAs pairs as novel prognostic biomarkers and potentially individualized therapy targets for COAD patients.
To the Editor: The enhanced activation of immune cells is a decisive factor for the pathological progress of inflammatory bowel disease (IBD), which can release generous inflammatory cytokines and bioactive molecules suggesting their roles as potential therapeutic targets for IBD. Purinergic receptors are a family of membranous proteins found extensively in many mammalian tissues and organs. As a subtype of purinergic receptors, the expressions of P2X7 receptor (P2X7R) can be detected abundantly in epithelial cells and most immune cells. During inflammation, adenosine triphosphate (ATP) is released extracellularly by various stimuli. In general, through the activation of P2X7R, extracellular ATP (eATP) can stimulate inflammation, while the degradation of ATP to adenosine usually exerts anti-inflammation roles. Consequently, ATP/P2X7R signaling can stimulate immune cells to produce various biological activities. Currently, mouse models of colitis have clarified the role of P2X7R in inflammation, which was supported by the increase in extracellular purines in the process of inflammation and the deregulated expression of purine receptor genes and proteins.[1,2] In addition, nuclear factor kappa B (NF-κB) is a pleiotropic transcription factor, which can drive the expression of pro-survival genes in intestinal epithelial cells and coordinate the expression of pro-inflammatory genes in innate and adaptive immune system cells. NF-κB has been proven to be a key regulator in the progression of IBD.[3] It is well known that the tight junctions at the top of intestinal epithelial cells play a significant role in regulating epithelial barrier permeability. Both IBD patients and modeled animals had increased epithelial barrier permeability, even before the onset of the disease. In addition, submucosal inflammation of IBD may damage the integrity of intestinal mucosal barrier structure, leading to increased permeability of intestinal mucosa, hence allowing paracellular permeation of luminal antigens and aggravation of intestinal inflammation. Based on the findings above, we hypothesized that P2X7R may be over-activated in macrophages of IBD and promote the production of inflammatory cytokines through inactivating the NF-κB pathway, which may further induce dysfunction of the intestinal mucosal barrier and thus exacerbate the progression of colitis. The experimental animals were 40 C57BL/6 wild-type 7–8-week-old male mice weighing 21 ± 2 g (procured from the Animal Center of Central South University). Drinking water was administrated in mice in the control group while 2.5% dextran sulfate sodium (DSS) in drinking water for the other four groups, namely DSS,3'-O-(4-benzoyl) benzoyl adenosine 5'-triphosphate(BzATP, Sigma, B6396-5mg, America), brilliant blue G (BBG, sc-203733, Santa Cruz Biotechnology, CA, America), and apyrase, for 8 consecutive days. Additionally, BzATP (5 mg/kg, a P2X7R agonist), BBG (40 mg/kg, the P2X7R antagonist), and apyrase (Sigma, 40 μg/kg, an ATP scavenger), were injected intraperitoneally into mice in the BzATP, BBG, and apyrase groups, respectively, while the same volume of normal saline was injected intraperitoneally in the control and DSS groups. All mice were euthanized on the eighth day after DSS treatment. After weighing and measuring the length of the colon, the whole colon was fixed in 10% formalin to prepare paraffin-embedded sections for hematoxylin & eosin (H&E) staining. A TCS-SP5 AOBS (Leica, Wetzlar, Germany) confocal laser scanning microscope (Leica, Heidelberg, Germany) was used to visualize protein expression and localization of P2X7R and F4/80, with the selection of representative images for further use. Blood samples were collected from 33 Crohn's disease (CD) patients and 34 healthy subjects from the Department of Gastroenterology, Xiangya Hospital, Central South University from January 2020 and December 2020. Venous blood samples in the EDTA anticoagulant tubes were subject to centrifugation. PBMCs were separated with a lymphocyte isolation agent by ficoll-hypaque density gradient centrifugation. PBMCs washed twice in PBS were suspended at 5 × 105 cells/mL in RPMI-1640 (R0883-100mL, Sigma-Aldrich®, St. Louis, Missouri, America) medium and cultured in six-well plates. Macrophages were obtained when the suspended cells were removed after overnight incubation at 37°C containing 5% CO2. The obtained macrophages were cultured continuously in RPMI-1640 medium containing 10% FBS based on different protocols: (1) without any additives for 5 h; (2) lipopolysaccharide (LPS) for 5 h; (3) LPS for 4 h + BzATP for 1 h; (4) LPS for 4 h + BBG for 1 h; (5) LPS for 4 h + apyrase for 1 h; (6) LPS for 4 h + JSH-23 (NF-κB inhibitor; Santa Cruz Biotechnology) for 1 h; and (7) LPS for 4 h + BBG + JSH-23 for 1 h. Among them, the concentrations of LPS, BzATP, BBG, apyrase, and JSH-23 were 1 μg/mL, 300 μmol/L, 1 μmol/L, 4 U/mL, and 20 μmol/L, respectively. The β-catenin mRNA levels were assessed via quantitative polymerase chain reaction. The total and nuclear β-catenin expressions were assessed via western blotting. IL-1β and IL-6 concentrations (picograms per meter) in the supernatants of cultured PBMCs were measured using enzyme-linked immunosorbent assay (ELISA) using corresponding kits (CUSABIO, CSB-E08054M and CSB-E04639M, America) in duplicate. The weight of mice decreased significantly in the DSS and BzATP groups from the fourth day of DSS administration compared with that in the control group (both P <0.01), with a more serious weight loss in the BzATP than in the DSS group (both P <0.01). However, compared with the DSS group, there was a significant alleviation in weight loss of the mice in both the BBG and apyrase groups (P <0.01) [Supplementary Figure A, https://links.lww.com/CM9/B565]. Furthermore, disease activity index (DAI) scores were increased in the DSS and BzATP groups, which, however, were partially prevented in the BBG and apyrase groups (all P <0.01) [Supplementary Figure A, https://links.lww.com/CM9/B565]. In addition, H&E staining [Supplementary Figure B, https://links.lww.com/CM9/B565] revealed that DSS treatment induced massive destruction of colonic epithelial structures, loss of goblet cells, diffuse loss of crypts, and robust inflammatory cell infiltration in the mucosa. Significantly, the BBG and apyrase groups showed partial improvement in the pathological changes of the colonic sections in mice. Previous studies have shown that P2X7R was widely expressed in the epithelium and the crypt bottom of the colon from DSS-induced mice. To determine major immune cells that co-expressed P2X7R, anti-P2X7R was incubated with F4/80 in tissue sections. As presented in Supplementary Figure C, https://links.lww.com/CM9/B565, concerning the co-localization of P2X7R (green) with macrophages (F4/80) (red), in the epithelium of DSS and BzATP treated mice, P2X7R was co-localized with macrophages. However, the expression of P2X7R decreased obviously in the DSS + BBG group (P <0.01) and the DSS + apyrase group (P <0.01) in comparison with that in the DSS group [Supplementary Figure C,D, https://links.lww.com/CM9/B565]. The role of NF-κB in IBD has been recognized and the p65 protein is the representative member, and phosphorylation is the starting point in its activation. In our experiment, NF-κB p65-Ser536 phosphorylation level was elevated in the DSS group compared with that in the control group (P <0.001). In contrast, the DSS + BBG and DSS + apyrase groups showed a decreased NF-κB p65-Ser536 phosphorylation level when compared with the DSS group [Supplementary Figure E, https://links.lww.com/CM9/B565]; similar changes of NF-κB p65-Ser536 were also observed in the western blotting and quantitative polymerase chain reaction [Supplementary Figure F, https://links.lww.com/CM9/B565]. To understand the underlying role of ATP/P2X7R signaling on macrophages, we determined the effect of the NF-κB (P65) pathway on IL-1β and IL-6 levels in an in vitro experimental model. Consequently, the LPS-stimulated IL-1β release from macrophages in CD patients was significantly higher compared with macrophages in CD patients cultured in RPMI alone (P <0.05; Supplementary Figure G, https://links.lww.com/CM9/B565). These changes could also be found in cultured macrophages from healthy controls. However, the LPS-stimulated effect in cultured macrophages from healthy controls was less obvious than that from CD patients. However, the production of IL-1β in the LPS + BBG and LPS + apyrase groups was significantly decreased compared with that in the CD + LPS group (both P <0.05). In addition, similar changes were also observed in IL-6 levels in the macrophages' culture medium [Supplementary Figure G, https://links.lww.com/CM9/B565]. The additional JSH-23 treatment also decreased IL-1β and IL-6 levels in the CD + LPS group, while no difference was found in their release between the LPS + BBG and LPS + JSH-23 groups. Interestingly, there was no synergistic effect when BBG and JSH-23 were used to intervene in macrophages simultaneously. In general, ATP is derived from symbiotic bacteria harbored in the intestine or from damaged cells in the inflammatory tissue. It can continuously activate P2X7R to increase cell permeability and induce cell death.[4,5] In addition, DSS and BzATP treatment resulted in an elevated count of apoptotic cells in the crypts. The abnormally increased apoptosis of intestinal epithelial cells could further damage the epithelial barrier, lead to overexposure to lumen bacteria, and ultimately destroy the intestinal epithelium's integrity and intestinal homeostasis. Based on these findings, our study established a connection of P2X7R-regulated ATP levels with intestinal inflammation. Interestingly, apyrase can significantly attenuate DSS-induced colitis following the downregulation of ATP levels via promoting ATP hydrolysis. It suggests that the dynamic changes of ATP are a key factor in DSS-induced colitis. Collectively, the findings in our study may provide complementary information to demonstrate that ATP/P2X7R signaling was a potentially important pathway involved in affecting epithelial cell proliferation, differentiation, and cell death. Our study discovered that macrophages played a pivotal role in mediating the severity of colitis by interacting with ATP and P2X7R. These interactions could both induce macrophage-mediated inflammation and aggravate the progression by promoting macrophage infiltration. Collectively, our study indicates that macrophages have important roles in DSS-induced colitis and human intestinal inflammation via mediating the release of various inflammatory cytokines and mediators. Funding This work was supported by the National Natural Science Foundation of China (Nos. 82230019, 81770584, and 82000502), China Postdoctoral Science Foundation (No. 2021M693572), Natural Science Foundation of Hunan Province (Nos. 2020SK2068 and 2020JJ5941), Free exploration and innovation project of Central South University (No. 506021711), and Fundamental Research Funds for the Central Universities of Central South University (No. 2020zzts268). Conflicts of interest None.
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