ObjectivesToll-like receptors (TLRs) are important initiators in native immune responses to microbial infections. TLR4 is up-regulated in response to H.pylori infection in gastric epithelial cells. However, the regulatory mechanisms for the expression of TLR4 in H.pylori infection have not been clearly defined. The aims of this study are to present the evidence that microRNA let-7b directly regulates TLR4 expression in human gastric epithelial cells, and subsequently influences the activation of NF-κB and the expression of the downstream genes in H.pylori infection.MethodsThe expression of let-7b was determined in gastric mucosa specimens and in two gastric epithelial cell lines using quantitative RT-PCR. The expression of TLR4 was determined by immunohistochemistry staining and RT-PCR. The potential target of let-7b was identified by luciferase reporter assay and Western blot. Let-7b mimics and inhibitors were used to examine the effects of let-7b on NF-κB activity. The expression of the downstream genes of NF-κB was also determined in cells infected with H.pylori 26695.ResultsLet-7b was significantly decreased in gastric mucosa specimens and in gastric epithelial cell lines (AGS, GES-1) infected with H.pylori 26695 (cagA+). Let-7b was complementary to the 3′-UTR of TLR4 mRNA and regulated TLR4 expression via post-transcriptional suppression in gastric epithelium. Infection of H.pylori induced the expression of TLR4 and activated NF-κB in AGS and GES-1 cells. Overexpression of let-7b by mimics downregulated TLR4, and subsequently attenuated NF-κB, MyD88, NF-κB1/p50, RelA/p65. The expression of IL-8, COX-2 and CyclinD1 was inhibited in H.pylori infected cells with let-7b overexpression. Both TAK-242 (TLR4 inhibitor) and SN50 (NF-κB inhibitor) significantly inhibited the H.pylori induced downregulation of let-7b.ConclusionsLet-7b targets at TLR4 mRNA, and regulates the activation of NF-κB and the expression of the downstream genes related to the inflammation and immune responses in H.pylori infection.

Dysregulated Notch signaling has a critical role in the tumorigenesis. Jagged1, a Notch ligand, is overexpressed in various human cancers. Recent studies revealed the involvement of Jagged1 in colorectal cancer (CRC) development. These basic studies provide a promising potential for inhibition of the Notch pathway for the treatment of CRC. Herein, we aimed to investigate the consequences of targeting Jagged1 using shRNA on CRC both in vitro and in vivo to test their potential to inhibit this key element for CRC treatment. We found that downregulation of Jagged1 with lentiviral Jagged1-shRNA resulted in decreased colon cancer cell viability in vitro, most likely mediated through reduced cell proliferation. Importantly, Jagged1 knockdown induced G0/G1 phase cell cycle arrest, with reduced Cyclin D1, Cyclin E and c-Myc expression. Silencing of Jagged1 reduced the migration and invasive capacity of the colon cancer cells in vitro. Furthermore, colon cancer cells with knockdown of Jagged1 had much slower growth rate than control cells in a xenograft mouse model in vivo, with a marked downregulation of cell proliferation markers (PCNA, Ki-67, and c-Myc) and metastasis markers (MMP-2 and MMP-9). These findings rationalize a mechanistic approach to CRC treatment based on Jagged1-targeted therapeutic development.

The interleukin-6 (IL-6)/STAT3 signaling regulates survival and proliferation of intestinal epithelial cells and plays an important role in the pathogenesis of inflammatory bowel disease and colorectal cancer. Embelin is a small molecule inhibitor of X-linked inhibitor of apoptosis protein (XIAP), with antioxidant, anti-inflammatory, and antitumor activities. We previously showed that embelin inhibits the growth of colon cancer cells in vitro, and effectively suppresses 1,2-dimethylhydrazine dihydrochloride-induced colon carcinogenesis in mice. Here, we explored the antitumor effects and mechanisms of embelin on colitis-associated cancer (CAC) using the azoxymethane/dextran sulfate sodium (AOM/DSS) model, with a particular focus on whether embelin exerts its effect through the IL-6/STAT3 pathway. We found that embelin significantly reduced incidence and tumor size in CAC-bearing mice. In addition to inhibiting proliferation of tumor epithelial cells, embelin suppressed colonic IL-6 expression and secretion, and subsequently STAT3 activation in vivo. Importantly, in vitro studies have revealed that in colon cancer cells, embelin diminished both the constitutive and IL-6-induced STAT3 activation by stimulating Src homology domain 2-containing protein tyrosine phosphatase (SHP2) activity. Moreover, embelin protected mice from AOM/DSS-induced colitis before tumor development. Embelin decreased IL-1b, IL-17a, and IL-23a expression as well as the number of CD4 þ T cells and macrophages infiltrating the colonic tissues. Thus, our findings demonstrated that embelin suppresses CAC tumorigenesis, and its antitumor effect is partly mediated by limiting IL-6/STAT3 activation and Th17 immune response. Embelin may be a potential agent in the prevention and treatment of CAC. Mol Cancer Ther; 13(5); 1206-16. Ó2014 AACR.