miR-3133 inhibits gastrointestinal cancer progression through activation of Hippo and p53 signalling pathways via multi-targets

Background

Malignant cell growth and chemoresistance, the main obstacles in treating gastrointestinal cancer (GIC), rely on the Hippo and p53 signalling pathways. However, the upstream regulatory mechanisms of these pathways remain complex and poorly understood.

Methods

Immunohistochemistry (IHC), western blot and RT-qPCR were used to analyse the expression of RNF146, miR-3133 and key components of Hippo and p53 pathway. CCK-8, colony formation, drug sensitivity assays and murine xenograft models were used to investigate the effect of RNF146 and miR-3133 in GIC. Further exploration of the upstream regulatory mechanism was performed using bioinformatics analysis, dual-luciferase reporter gene, immunoprecipitation assays and bisulfite sequencing PCR (BSP).

Results

Clinical samples, in vitro and in vivo experiments demonstrated that RNF146 exerts oncogenic effects in GIC by regulating the Hippo pathway. Bioinformatics analysis identified a novel miRNA, miR-3133, as an upstream regulatory factor of RNF146. fluorescence in situ hybridization and RT-qPCR assays revealed that miR-3133 was less expressed in gastrointestinal tumour tissues and was associated with adverse pathological features. Functional assays and animal models showed that miR-3133 promoted the proliferation and chemotherapy sensitivity of GIC cells. miR-3133 affected YAP1 protein expression by targeting RNF146, AGK and CUL4A, thus activating the Hippo pathway. miR-3133 inhibited p53 protein degradation and extended p53's half-life by targeting USP15, SPIN1. BSP experiments confirmed that miR-3133 promoter methylation is an important reason for its low expression.

Conclusion

miR-3133 inhibits GIC progression by activating the Hippo and p53 signalling pathways via multi-targets, including RNF146, thereby providing prognostic factors and valuable potential therapeutic targets for GIC.     

Risk of hospitalization for upper gastrointestinal bleeding in Helicobacter pylori eradicated patients newly started on warfarin or direct oral anticoagulants: A population-based cohort study

Background

To investigate risks of hospitalization for upper gastrointestinal bleeding (UGIB) in H. pylori-eradicated patients newly started on warfarin or direct oral anti-coagulants (DOACs).

Methods

We identified all patients who had previously received H. pylori eradication therapy or were found to have no H. pylori on endoscopy and were then newly started on warfarin or DOACs from a population-based electronic healthcare database. Primary analysis was the risk of UGIB between warfarin and DOACs users in H. pylori-eradicated patients. Secondary analysis included the UGIB risk between H. pylori-eradicated and H. pylori-negative patients who were newly started on warfarin or DOACs. The hazard ratio (HR) of UGIB was approximated by pooled logistic regression model incorporating the inverse propensity of treatment weightings with time-varying covariables.

Results

Among H. pylori-eradicated patients, DOACs had a significantly lower risk of UGIB (HR: 0.26, 95% CI 0.09–0.71) compared with warfarin. In particular, lower UGIB risks with DOACs were observed among older ( ≥ 65 years) patients, female, those without a history of UGIB or peptic ulcer, or ischemic heart disease, and non-users of acid-suppressive agents or aspirin. Secondary analysis showed no significant difference in UGIB risk between H. pylori-eradicated and H. pylori-negative patients newly started on warfarin (HR: 0.63,95% CI 0.33–1.19) or DOACs (HR: 1.37, 95% CI 0.45–4.22).

Conclusions

In H. pylori-eradicated patients, new users of DOACs had a significantly lower risk of UGIB than new warfarin users. Furthermore, the risk of UGIB in new warfarin or DOACs users was comparable between H. pylori-eradicated and H. pylori-negative patients.     

The 2.7 A crystal structure of the autoinhibited human c-Fms kinase domain

c-Fms, a member of the Platelet-derived Growth Factor (PDGF) receptor family of receptor tyrosine kinases (RTKs), is the receptor for macrophage colony stimulating factor (CSF-1) that regulates proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage. Abnormal expression of c-fms proto-oncogene is associated with a significant number of human pathologies, including a variety of cancers and rheumatoid arthritis. Accordingly, c-Fms represents an attractive therapeutic target. To further understand the regulation of c-Fms, we determined the 2.7 A resolution crystal structure of the cytosolic domain of c-Fms that comprised the kinase domain and the juxtamembrane domain. The structure reveals the crucial inhibitory role of the juxtamembrane domain (JM) that binds to a hydrophobic site immediately adjacent to the ATP binding pocket. This interaction prevents the activation loop from adopting an active conformation thereby locking the c-Fms kinase into an autoinhibited state. As observed for other members of the PDGF receptor family, namely c-Kit and Flt3, three JM-derived tyrosine residues primarily drive the mechanism for autoinhibition in c-Fms, therefore defining a common autoinhibitory mechanism within this family. Moreover the structure provides an understanding of c-Fms inhibition by Gleevec as well as providing a platform for the development of more selective inhibitors that target the inactive conformation of c-Fms kinase.     

Dynamics of competitive population abundance of Lactobacillus plantarum ivi gene mutants in faecal samples after passage through the gastrointestinal tract of mice

AIM: This study aims to evaluate the impact of mutation of previously identified in vivo-induced (ivi) genes on the persistence and survival of Lactobacillus plantarum WCFS1 in the gastrointestinal (GI) tract of mice. METHODS AND RESULTS: Nine Lact. plantarum ivi gene replacement mutants were constructed, focussing on ivi genes that encode proteins with a predicted role in cell envelope functionality, stress response and regulation. The in vitro growth characteristics of the mutants appeared identical to those observed for the wild-type strain, which agrees with the recombination-based in vivo expression technology suggestion that these genes are not transcribed in the laboratory. Quantitative PCR experiments demonstrated differences in the relative population dynamics of the Lact. plantarum ivi mutants in faecal samples after passage through the GI tract of mice. CONCLUSIONS: The in situ competition experiments revealed a 100- to 1000-fold reduction of the relative abundance of three of the ivi gene mutants, harbouring deletions of genes predicted to encode a copper transporter, an orphan IIC cellobiose PTS and a cell wall anchored extracellular protein. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments clearly establish that the proteins encoded by these three genes play a key role in Lact. plantarum performance during passage of the GI tract.     

Phylogenetic and gene-centric metagenomics of the canine intestinal microbiome reveals similarities with humans and mice

This study is the first to use a metagenomics approach to characterize the phylogeny and functional capacity of the canine gastrointestinal microbiome. Six healthy adult dogs were used in a crossover design and fed a low-fiber control diet (K9C) or one containing 7.5% beet pulp (K9BP). Pooled fecal DNA samples from each treatment were subjected to 454 pyrosequencing, generating 503 280 (K9C) and 505 061 (K9BP) sequences. Dominant bacterial phyla included the Bacteroidetes/Chlorobi group and Firmicutes, both of which comprised ∼35% of all sequences, followed by Proteobacteria (13–15%) and Fusobacteria (7–8%). K9C had a greater percentage of Bacteroidetes, Fusobacteria and Proteobacteria, whereas K9BP had greater proportions of the Bacteroidetes/Chlorobi group and Firmicutes. Archaea were not altered by diet and represented ∼1% of all sequences. All archaea were members of Crenarchaeota and Euryarchaeota, with methanogens being the most abundant and diverse. Three fungi phylotypes were present in K9C, but none in K9BP. Less than 0.4% of sequences were of viral origin, with >99% of them associated with bacteriophages. Primary functional categories were not significantly affected by diet and were associated with carbohydrates; protein metabolism; DNA metabolism; cofactors, vitamins, prosthetic groups and pigments; amino acids and derivatives; cell wall and capsule; and virulence. Hierarchical clustering of several gastrointestinal metagenomes demonstrated phylogenetic and metabolic similarity between dogs, humans and mice. More research is required to provide deeper coverage of the canine microbiome, evaluate effects of age, genetics or environment on its composition and activity, and identify its role in gastrointestinal disease.     

Impact of vitamin E or selenium deficiency on nematode-induced alterations in murine intestinal function

The effects of deficiencies in the antioxidant nutrients, vitamin E and selenium, on the host response to gastrointestinal nematode infection are unknown. The aim of the study was to determine the effect of antioxidant deficiencies on nematode-induced alterations in intestinal function in mice. BALB/c mice were fed control diets or diets deficient in selenium or vitamin E and the response to a secondary challenge inoculation with Heligmosomoides polygyrus was determined. Egg and worm counts were assessed to determine host resistance. Sections of jejunum were mounted in Ussing chambers to measure changes in permeability, absorption, and secretion, or suspended in organ baths to determine smooth muscle contraction. Both selenium and vitamin E deficient diets reduced resistance to helminth infection. Vitamin E, but not selenium, deficiency prevented nematode-induced decreases in glucose absorption and hyper-contractility of smooth muscle. Thus, vitamin E status is an important factor in the physiological response to intestinal nematode infection and may contribute to antioxidant-dependent protective mechanisms in the small intestine.     

Regeneration of the intestinal epithelia: Regulation of bone marrow-derived epithelial cell differentiation towards secretory lineage cells

The intestinal epithelia consists of four lineages of differentiated cells, all of which arise from stem cells residing in the intestinal crypt. For proper regeneration from epithelial damage, both expansion of the epithelial cell number and appropriate regulation of lineage differentiation from the remaining stem cells are thought to be required. In a series of studies, we have shown that bone-marrow derived cells could promote the regeneration of damaged epithelia in the human intestinal tract. Donor-derived epithelial cells substantially repopulated the gastrointestinal tract of bone-marrow transplant recipients during epithelial regeneration after graft-versus-host disease. Furthermore, precise analysis of epithelial cell lineages revealed that during epithelial regeneration, secretory lineage epithelial cells that originated from bone-marrow significantly increased in number. These findings may lead to a novel therapy to repair damaged intestinal epithelia using bone marrow cells, and provide an alternative therapy for refractory inflammatory bowel diseases.     

Analysis of serum copper and zinc concentrations in cancer patients

Several studies have shown that plasma copper concentrations are increased in various carcinomas. Zinc acts as a cellular growth protector, including growth of neoplastic cells, and its deficiency was demonstrated to be involved in several stages of malignant transformation. However, the usefulness of the serum zinc and copper determinations in cancer prevention, detection, monitoring treatment, and prognosis requires further investigations. The aim of the present study was to compare the serum copper and zinc levels in patients with cancer of the lung (PC), breast (BC), gastrointestinal tract (GIC), and gynecological (GYNC) malignancy with progress of the disease. The results of the study have shown a significant increase in the mean total serum Cu levels and the serum Cu/Zn ratio in all patient groups with cancer compared to a control group. Increased mean serum concentrations and Cu/Zn ratios were found in the whole group (ALLC), and for the GIC and GYNC groups with local as well as metastasized (Meta) disease in comparison with the control group. The mean serum concentrations of Zn were decreased only in metastasized ALLC and GYNC groups.     

Endocannabinoid‐related compounds in gastrointestinal diseases

The endocannabinoid system (ECS) is an endogenous signalling pathway involved in the control of several gastrointestinal (GI) functions at both peripheral and central levels. In recent years, it has become apparent that the ECS is pivotal in the regulation of GI motility, secretion and sensitivity, but endocannabinoids (ECs) are also involved in the regulation of intestinal inflammation and mucosal barrier permeability, suggesting their role in the pathophysiology of both functional and organic GI disorders. Genetic studies in patients with irritable bowel syndrome (IBS) or inflammatory bowel disease have indeed shown significant associations with polymorphisms or mutation in genes encoding for cannabinoid receptor or enzyme responsible for their catabolism, respectively. Furthermore, ongoing clinical trials are testing EC agonists/antagonists in the achievement of symptomatic relief from a number of GI symptoms. Despite this evidence, there is a lack of supportive RCTs and relevant data in human beings, and hence, the possible therapeutic application of these compounds is raising ethical, political and economic concerns. More recently, the identification of several EC‐like compounds able to modulate ECS function without the typical central side effects of cannabino‐mimetics has paved the way for emerging peripherally acting drugs. This review summarizes the possible mechanisms linking the ECS to GI disorders and describes the most recent advances in the manipulation of the ECS in the treatment of GI diseases.