Long Noncoding RNA PMS2L2 Downregulates miR-24 through Methylation to Suppress Cell Apoptosis in Ulcerative Colitis.

BACKGROUND: Ulcerative colitis (UC) is an inflammatory bowel disease characterized by chronic inflammation of the colon. It has been reported that PMS2L2 plays protective roles in inflammatory injury. This study aimed to investigate the role of the long noncoding RNA PMS2L2 in UC. METHODS: Sixty-two patients with UC as well as 62 age- and gender-matched healthy controls were enrolled. Expressions of PMS2L2 and miR-24 in plasma from UC patients and healthy controls were determined by RT-qPCR. The interaction between PMS2L2 and miR-24 was predicted by bioinformatics and confirmed by RNA immunoprecipitation and RNA pull-down. The role of PMS2L2 in the regulation of miR-24 gene methylation was analyzed by methylation-specific PCR. The effects of PMS2L2 and miR-24 on the expressions of apoptosis-related proteins were detected by Western blots. RESULTS: PMS2L2 was downregulated in the plasma of UC patients compared to that in age- and gender-matched healthy control. In human colonic epithelial cells (HCnEpCs), PMS2L2 overexpression inhibited miR-24 expression via promoting the methylation of miR-24 gene. In contrast, miR-24 overexpression failed to affect PMS2L2. In the detection of cell apoptosis, PMS2L2 overexpression could promote the expression of Bcl-2 and inhibit Bax, cleaved-caspase-3, and cleaved-caspase-9 expressions stimulated by LPS. Flow cytometer revealed that PMS2L2 elevation suppressed the apoptosis of HCnEpCs induced by LPS, but miR-24 aggravated the apoptosis. PMS2L2 overexpression rescued the detrimental effect of miR-24 on cell apoptosis. CONCLUSION: PMS2L2 may downregulate miR-24 via methylation to suppress cell apoptosis in UC.

Promotion of wound healing by acetate in murine colonic epithelial cell via c-Jun N-terminal kinase activation.

BACKGROUND AND AIM: Mucosal healing is an important clinical goal in patients with inflammatory bowel disease. Recently, short-chain fatty acids (SCFAs) have been reported to have multifaceted effects to host. However, the effects of SCFAs on wound healing in intestinal epithelial cells are unclear. In the present study, we investigated the effects of acetate, one of the major SCFAs, on the wound healing of murine colonic epithelial cells. METHODS: Young adult mouse colonic epithelial cells were used to determine the effect of acetate using wound healing assay. Mitogen-activated protein kinase and Rho kinase inhibitor were used to elucidate intracellular signal of wound healing treated with acetate. Meanwhile, Rho activation assays were utilized to measure Rho activation levels. To assess in vivo effects, C57B6 mice with dextran sodium sulfate for 7 days were treated with enema administration of acetate for 7 days. Body weight, disease activity index, colon length, and mucosal break ratio in histology were examined. RESULTS: Acetate enhanced wound healing and fluorescence intensity of actin stress fiber compared with control. These effects were canceled with pretreatment of c-Jun N-terminal kinase (JNK) inhibitor or Rho kinase inhibitor. Furthermore, JNK inhibitor reduced the activation of Rho induced by acetate. In the dextran sodium sulfate-induced colitis model, the mice with enema treatment of acetate significantly exhibited recovery. CONCLUSIONS: In this study, we demonstrated that acetate promoted murine colonic epithelial cell wound healing via activation of JNK and Rho signaling pathways. These findings suggested that acetate could have applications as a therapeutic agent for patients with intestinal mucosal damage, such as inflammatory bowel disease.

LncRNA MALAT1 Promotes Ulcerative Colitis by Upregulating lncRNA ANRIL.

BACKGROUND: LncRNA MALAT1 contributes to the inflammatory responses induced by lipopolysaccharides (LPS), which shares similar pathogenesis with ulcerative colitis (UC), indicating the potential involvement of MALAT1 in UC. METHODS: Expression of MALAT1 and lncRNA ANRIL in both UC patients and healthy controls was analyzed by RT-qPCR. ROC curve analysis was used to evaluate the diagnostic value of MALAT1 for UC. Cell transfections were performed to analyze the interactions between MALAT1 and ANRIL. Cell apoptosis was analyzed by cell apoptosis assay. RESULTS: In the present study, we found that MALAT1 was upregulated in colonic mucosa tissues of UC patients in comparison with healthy controls. Plasma levels of MALAT1 were also higher in UC patients than in healthy controls, and upregulation of plasma MALAT1 distinguished UC patients from healthy controls. ANRIL was also upregulated in colonic mucosa tissues of UC patients than in that of healthy controls. ANRIL and MALAT1 were significantly and positively correlated in UC patients but not in healthy controls. Normal colonic epithelial cells with ANRIL overexpression showed no significantly changed MALAT1 overexpression, while MALAT1 overexpression led to promoted ANRIL expression. MALAT1 and ANRIL overexpression led to promoted apoptosis of FHCs. CONCLUSION: MALAT1 promotes ulcerative colitis by upregulating ANRIL.

Interferon-γ induces a tryptophan-selective amino acid transporter in human colonic epithelial cells and mouse dendritic cells.

IDO1, which encodes the immunosuppressive and tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase-1 (IDO1), is a target for interferon-γ (IFN-γ). IDO1-mediated tryptophan catabolism in dendritic cells and macrophages arrests T cell proliferation, thereby providing a molecular basis for the immunosuppressive function of IDO1. Whether the entry of tryptophan into IDO1-expressing cells is also regulated by IFN-γ is not known. Here we used a human colonic epithelial cell line (CCD841) and a mouse dendritic cell line (DC2.4) to test the hypothesis that IFN-γ, which induces IDO1, also induces a tryptophan transporter to promote substrate availability to IDO1. Upon treatment with IFN-γ, there was a marked increase in IDO1 mRNA and a concomitant increase in tryptophan uptake in both cell lines. The induced uptake system was selective for tryptophan and saturable with a Michaelis constant of 36±3 µM in CCD841 cells and 0.5±0.1 µM in DC2.4 cells. The induction by IFN-γ and the tryptophan-selectivity of the induced transport system were demonstrable even in the presence of physiologic concentrations of all other amino acids. Since kynurenine, the catabolic end product of IDO1, is a signaling molecule as an agonist for the aryl hydrocarbon receptor (AhR), we examined if AhR signaling induces the tryptophan-selective transporter. Treatment of the cells with kynurenine and other AhR agonists increased tryptophan uptake. The present studies demonstrate that IFN-γ coordinately induces IDO1 and a tryptophan-selective transporter to maximize tryptophan depletion in IDO1-expressing cells and that the process involves a positive feedback mechanism via kynurenine-AhR signaling.

Evaluation of cellular retinoic acid binding protein 2 gene expression through the retinoic acid pathway by co-incubation of Blastocystis ST-1 with HT29 cells in vitro.

Blastocystis is a parasitic protist with a worldwide distribution that is commonly found in patients with colon and gastrointestinal pathological symptoms. Blastocystis infection has also commonly been reported in colorectal cancer and HIV/AIDS patients with gastrointestinal symptoms. To understand the pathway networks of gene regulation and the probable mechanisms influencing functions of HT-29 host cells in response to parasite infection, we examined the expression of 163 human oncogenes and kinases in human colon adenocarcinoma HT-29 cells co-incubated with Blastocystis by in-house cDNA microarray and PCR analysis. At least 10 genes were shown to be modified following Blastocystis co-incubation, including those with immunological, tumorigenesis, and antitumorigenesis functions. The expression of genes encoding cellular retinoic acid binding protein 2 (CRABP2) and proliferating cell nuclear antigen (PCNA) was markedly upregulated and downregulated, respectively. Reverse transcriptase-PCR validated the modified transcript expression of CRABP2 and other associated genes such as retinoic acid (RA)-related nuclear-receptor (RARα). Together, our data indicate that CRABP2, RARα, and PCNA expressions are involved in RA signaling regulatory networks that affect the growth, proliferation, and inflammation of HT-29 cells.

Bacteroides fragilis Toxin Induces Intestinal Epithelial Cell Secretion of Interleukin-8 by the E-Cadherin/ß-Catenin/NF-κB Dependent Pathway.

Enterotoxigenic Bacteroides fragilis (ETBF) has emerged as a gut microbiome pathogen that can promote colitis associated cancer in humans. ETBF secretes the metalloprotease, B. fragilis toxin (BFT), which can induce ectodomain cleavage of E-cadherin and IL-8 secretion through the ß-catenin, NF-κB, and MAPK pathways in intestinal epithelial cells. However, it is still unclear whether E-cadherin cleavage is required for BFT induced IL-8 secretion and the relative contribution of these signaling pathways to IL-8 secretion. Using siRNA knockdown and CRISPR knockout studies, we found that E-cadherin cleavage is required for BFT mediated IL-8 secretion. In addition, genetic ablation of ß-catenin indicates that ß-catenin is required for the BFT induced increase in transcriptional activity of NF-κB, p65 nuclear localization and early IL-8 secretion. These results suggest that BFT induced ß-catenin signaling is upstream of NF-κB activation. However, despite ß-catenin gene disruption, BFT still activated the MAPK pathway, suggesting that the BFT induced activation of the MAPK signaling pathway is independent from the E-cadherin/ß-catenin/NF-κB pathway. These findings show that E-cadherin and ß-catenin play a critical role in acute inflammation following ETBF infection through the inflammatory response to BFT in intestinal epithelial cells.

Atrial Natriuretic Peptide Attenuates Colitis via Inhibition of the cGAS-STING Pathway in Colonic Epithelial Cells.

Atrial Natriuretic Peptide (ANP) has known anti-inflammatory effects. However, the role of ANP in Ulcerative colitis (UC) remains unclear. This study aimed to explore the expression and function of ANP in UC, and its potential regulatory role in the stimulator of interferon genes (STING) pathway. Human colon biopsy and serum samples were collected between September 2018 and December 2019 at Wuhan Union Hospital. Levels of ANP and its receptors and STING pathway components were detected in people with UC and mice with dextran sulfate sodium (DSS)-induced colitis. These mice and HT-29 cells were treated with ANP and an agonist of the STING pathway. The level of inflammation, STING pathway, gut barrier, and endoplasmic reticulum (ER) stress-induced autophagy were measured. We found that the levels of ANP and its receptor decreased and the STING pathway activated statistically in people with UC and the mouse model of colitis. ANP treatment attenuated DSS-induced colitis and inhibited STING pathway phosphorylation in colonic tissue and epithelial cells. An interaction between cGAS and NPR-A was verified. ANP repaired the gut barrier and inhibited ER stress-induced autophagy via the STING pathway. ANP may thus alter colonic barrier function and regulate ER stress-induced autophagy as a promising therapy for UC.

Bisphenol A suppresses proliferation and induces apoptosis in colonic epithelial cells through mitochondrial and MAPK/AKT pathways.

Bisphenol A (BPA) can act as pathogenic agent on mammalian cells. However, whether BPA induces colonic epithelial cell damage remains unexplored. We used HCT116 cells as a model to investigate the negative effect of BPA on human colon cancer cells and explore the potential mechanism. Our results suggest that BPA decreased viability of HCT116 cells. BPA also caused serious oxidative damage to the colonic epithelium, as indicated by increased mitochondrial and intracellular reactive oxygen species (ROS) content and elevated malondialdehyde and H2O2 levels. Moreover, BPA depolarized the mitochondrial membrane potential and caused loss of mitochondrial integrity. Furthermore, BPA induced colonic epithelial cell apoptosis accompanied by upregulation of caspase3 and bax gene expression. Additionally, cell proliferation was inhibited significantly in HCT116 cells after the BPA treatment. We also studied the molecular mechanism involved in these effects and found that BPA inhibited proliferation of colonic epithelial cells through the mitogen activated protein kinase (MAPK) and AKT signaling pathways. Our data suggest that BPA triggers ROS generation as an initial step, followed by mediation of mitochondrial dysfunction, apoptosis, and proliferation of HCT116 cells. Moreover, MAPK/AKT signaling pathways were involved in BPA-induced toxicity of human colon cancer cells.

Bile acid: a potential inducer of colon cancer stem cells.

BACKGROUND: Although the unconjugated secondary bile acids, specifically deoxycholic acid (DCA) and lithocholic acid (LCA), are considered to be risk factors for colorectal cancer, the precise mechanism(s) by which they regulate carcinogenesis is poorly understood. We hypothesize that the cytotoxic bile acids may promote stemness in colonic epithelial cells leading to generation of cancer stem cells (CSCs) that play a role in the development and progression of colon cancer. METHODS: Normal human colonic epithelial cells (HCoEpiC) were used to study bile acid DCA/LCA-mediated induction of CSCs. The expression of CSC markers was measured by real-time qPCR. Flow cytometry was used to isolate CSCs. T-cell factor/lymphoid-enhancing factor (TCF/LEF) luciferase assay was employed to examine the transcriptional activity of ß-catenin. Downregulation of muscarinic 3 receptor (M3R) was achieved through transfection of corresponding siRNA. RESULTS: We found DCA/LCA to induce CSCs in normal human colonic epithelial cells, as evidenced by the increased proportion of CSCs, elevated levels of several CSC markers, as well as a number of epithelial-mesenchymal transition markers together with increased colonosphere formation, drug exclusion, ABCB1 and ABCG2 expression, and induction of M3R, p-EGFR, matrix metallopeptidases, and c-Myc. Inhibition of M3R signaling greatly suppressed DCA/LCA induction of the CSC marker ALDHA1 and also c-Myc mRNA expression as well as transcriptional activation of TCF/LEF. CONCLUSIONS: Our results suggest that bile acids, specifically DCA and LCA, induce cancer stemness in colonic epithelial cells by modulating M3R and Wnt/ß-catenin signaling and thus could be considered promoters of colon cancer.

GM-CSF facilitates the development of inflammation-associated colorectal carcinoma.

The granulocyte macrophage colony-stimulating factor (GM-CSF) is known to play a protective role in intestinal inflammation. However, the effects of GM-CSF on the progression of colitis to colorectal carcinoma have long remained unclear. We have recently demonstrated that GM-CSF produced by colonic epithelial cells promotes colorectal tumorigenesis by stimulating the secretion of vascular endothelial growth factor.