BackgroundColorectal carcinoma (CRC) is one of the leading causes of cancer-related mortality worldwide. MicroRNAs (miRNAs, miRs) play important roles in carcinogenesis. MiR-126 has been shown to be down-regulated in CRC. In this study, we identified the potential effects of miR-126 on some important biological properties of CRC cells and clarified the regulation of insulin receptor substrate 1 (IRS-1) and its possible signaling pathway by miR-126.MethodsThe effect of miR-126 on IRS-1, AKT, and ERK1/2 expression was assessed in the CRC cell lines HT-29 and HCT-116 with a miR-126 mimic or inhibitor to increase or decrease miR-126 expression. Furthermore, the roles of miR-126 in regulation of the biological properties of CRC cells were analyzed with miR-126 mimic or inhibitor-transfected cells. The 3′-untranslated region (3′-UTR) of IRS-1 regulated by miR-126 was analyzed by using a dual-luciferase reporter assay.ResultsWe found that IRS-1 is the functional downstream target of miR-126 by directly targeting the 3′-UTR of IRS-1. Endogenous miR-126 and exogenous miR-126 mimic inhibited IRS-1 expression. Furthermore, gain-of-function or loss-of-function studies showed that over-expression of miR-126 down-regulated IRS-1, suppressed AKT and ERK1/2 activation, CRC cells proliferation, migration, invasion, and caused cell cycle arrest, but had no effect on cell apoptosis. Knockdown of miR-126 promoted these processes in HCT-116 cells and promoted AKT and ERK1/2 activation by up-regulating the expression of the IRS-1 protein.ConclusionsMiR-126 may play roles in regulation of the biological behavior of CRC cells, at least in part, by targeting IRS-1 via AKT and ERK1/2 signaling pathways.

The role of the adenosine A3 receptor (A3AR) in experimental colitis is controversial. The A3AR agonist N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) has been shown to have a clinical benefit, although studies in A3AR-deficient mice suggest a pro-inflammatory role. However, there are no studies on the effect of 2-Cl-IB-MECA and the molecular mechanism of action of A3AR in murine colitis models in vivo. Is it the same as that observed in vitro? The interaction between 2-CL-IB-MECA and A3AR in a murine colitis model and the signaling pathways associated with this interaction remain unclear. Here we demonstrate a role for the NF-κB signaling pathway and its effect on modifying the activity of proinflammatory factors in A3AR-mediated biological processes. Our results demonstrated that A3AR activation possessed marked effects on experimental colitis through the NF-κB signaling pathway.

To investigate the expression of adenosine A3 receptor (A3AR) in human colonic epithelial cells and the effects of A3AR activation on tumor necrosis factor alpha (TNF-α-) induced inflammation in order to determine its mechanism of action in human colonic epithelial cells, human colonic epithelial cells (HT-29 cells) were treated with different concentrations of 2-Cl-IB-MECA prior to TNF-α stimulation, followed by analysis of NF-κB signaling pathway activation and downstream IL-8 and IL-1β production. A3AR mRNA and protein were expressed in HT-29 cells and not altered by changes in TNF-α or 2-Cl-IB-MECA. Pretreatment with 2-Cl-IB-MECA prior to stimulation with TNF-α attenuated NF-κB p65 nuclear translocation as p65 protein decreased in the nucleus of cells and increased in the cytoplasm, inhibited the degradation of IκB-α, and reduced phosphorylated-IκB-α level significantly, compared to TNF-α-only-treated groups. Furthermore, 2-Cl-IB-MECA significantly decreased TNF-α-stimulated IL-8 and IL-1β mRNA expression and secretion, compared to the TNF-α-only treated group. These results confirm that A3AR is expressed in human colonic epithelial cells and demonstrate that its activation has an anti-inflammatory effect, through the inhibition of NF-κB signaling pathway, which leads to inhibition of downstream IL-8 and IL-1β expression. Therefore, A3AR activation may be a potential treatment for gut inflammatory diseases such as inflammatory bowel disease.

Abstractβ‐Catenin signalling plays an important role in regulating tubular epithelial‐to‐mesenchymal transition (EMT), an indispensable programme for driving renal fibrosis. As an adapter protein, four and a half LIM domain protein 2 (FHL2) acts as a coregulator of β‐catenin in several other cell types. To determine whether FHL2 affects β‐catenin signalling and thus is involved in tubular EMT, we examined its expression and function in the process of TGF‐β1‐induced EMT. FHL2 mRNA and protein were induced by TGF‐β1 in rat tubular epithelial cells (NRK‐52E), an effect that intracellular Smad signalling was required. Ectopic expression of FHL2 inhibited E‐cadherin and enhanced α‐smooth muscle actin (α‐SMA) and fibronectin expression, whereas knockdown of FHL2 partially restored E‐cadherin and reduced α‐SMA and fibronectin induction stimulated by TGF‐β1. Overexpression of FHL2 increased β‐catenin dephosphorylation (Ser37/Thr41), nuclear translocation and β‐catenin‐mediated transcription and up‐regulated expression of β‐catenin target, EMT‐related genes, such as Snail, Twist, vimentin, plasminogen activator inhibitor‐1 and matrix metalloproteinase‐7. Conversely, knockdown of FHL2 increased β‐catenin phosphorylation (Ser33/37/Thr41), decreased its nuclear translocation and inhibited β‐catenin‐mediated transcription and target genes expression. TGF‐β1 induced a FHL2/β‐catenin interaction in NRK‐52E cells, especially in the nuclei. In a mouse model of obstructive nephropathy, FHL2 mRNA and protein were induced in a time‐dependent fashion, and the extent and pattern of renal β‐catenin activation were positively correlated with FHL2 induction. Collectively, this study suggests that FHL2, via modulating β‐catenin signalling, may implicate in regulation of TGF‐β1‐mediated tubular EMT and could be a potential therapeutic target for fibrotic kidney disease.

MicroRNAs (miRNAs, miRs) are suspected to play important roles in carcinogenesis. MiR-32 has altered expression in colorectal cancer (CRC); however, the clinical significance of miR-32 expression in the process of carcinogenesis is poorly understood. In this study, we determined the levels of, the correlation between, and the clinical significance of the expression of miR-32 and phosphatase and tensin homologue (PTEN), a tumor suppressor targeted by miR-32, in CRC. The levels of miR-32 and PTEN gene expression in 35 colorectal carcinoma samples, 35 corresponding cancer-adjacent tissue samples, 27 colorectal adenoma samples, and 16 normal tissue samples were quantified using real-time quantitative reverse transcriptase-polymerase chain reaction. PTEN protein expression was determined using western blot and immunohistochemistry (IHC). The relationship between the miR-32 and PTEN protein expression and clinicopathological factors was analyzed. Significant upregulation of miR-32 expression and reduction of PTEN were identified in CRC tissues. High miR-32 levels were significantly associated with lymph node and distant metastasis, and Kaplan-Meier analysis indicated that patients with high miR-32 expression had a poor overall survival. Low PTEN protein expression was also significantly correlated with distant metastasis. An inverse relationship between miR-32 and PTEN protein expression was identified. In addition, IHC analysis revealed weak or indiscernible PTEN staining in tumor tissue. MiR-32 overexpression was correlated with specific CRC clinicopathological features and may be a marker of poor prognosis in CRC patients. MiR-32 and PTEN expression were inversely correlated, and miR-32 may be associated with the development of CRC.