Ruiyun Li

Small nucleolar RNA 42 acts as an oncogene in lung tumorigenesis

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death, reflecting the need for better understanding the oncogenesis, and developing new diagnostic and therapeutic targets for the malignancy. Emerging evidence suggests that small nucleolar RNAs (snoRNAs) have malfunctioning roles in tumorigenesis. Our recent study demonstrated that small nucleolar RNA 42 (SNORA42) was overexpressed in lung tumors. Here, we investigate the role of SNORA42 in tumorigenesis of NSCLC. We simultaneously assess genomic dosages and expression levels of SNORA42 and its host gene, KIAA0907, in 10 NSCLC cell lines and a human bronchial epithelial cell line. We then determine in vitro functional significance of SNORA42 in lung cancer cell lines through gain- and loss-of-function analyses. We also inoculate cancer cells with SNORA42-siRNA into mice through either tail vein or subcutaneous injection. We finally evaluate expression level of SNORA42 on frozen surgically resected lung tumor tissues of 64 patients with stage I NSCLC by using quantitative reverse transcriptase PCR assay. Genomic amplification and associated high expression of SNORA42 rather than KIAA0907 are frequently observed in lung cancer cells, suggesting that SNORA42 overexpression is activated by its genomic amplification. SNORA42 knockdown in NSCLC cells inhibits in vitro and in vivo tumorigenicity, whereas enforced SNORA42 expression in bronchial epitheliums increases cell growth and colony formation. Such pleiotropy of SNORA42 suppression could be achieved at least partially through increased apoptosis of NSCLC cells in a p53-dependent manner. SNORA42 expression in lung tumor tissue specimens is inversely correlated with survival of NSCLC patients. Therefore, SNORA42 activation could have an oncogenic role in lung tumorigenesis and provide potential diagnostic and therapeutic targets for the malignancy.

Overexpression of miR-214-3p in esophageal squamous cancer cells enhances sensitivity to cisplatin by targeting survivin directly and indirectly through CUG-BP1.

Based on its marked overexpression in multiple malignancies and its roles in promoting cell survival and proliferation, survivin is an attractive candidate for targeted therapy. Toward this end, a detailed understanding of the mechanisms regulating survivin expression in different cancer cells will be critical. We have previously shown that the RNA-binding protein (RBP) CUG-BP1 is overexpressed in esophageal cancer cells and post-transcriptionally regulates survivin in these cells. The objective of this study was to investigate the role of microRNAs (miRs) in regulating survivin expression in esophageal cancer cells. Using miR expression profiling analysis, we found that miR-214-3p is one of the most markedly downregulated miRs in two esophageal squamous cancer cell lines compared with esophageal epithelial cells. Interestingly, using miR target prediction programs, both survivin and CUG-BP1 mRNA were found to contain potential binding sites for miR-214-3p. Forced expression of miR-214-3p in esophageal cancer cells leads to a decrease in the mRNA and protein levels of both survivin and CUG-BP1. This effect is due to decreased mRNA stability of both targets. By contrast, silencing miR-214-3p in esophageal epithelial cells leads to an increase in both survivin and CUG-BP1 mRNA and protein. To determine whether the observed effect of miR-214-3p on survivin expression was direct, mediated through CUG-BP1, or both, binding studies utilizing biotin pull-down assays and heterologous luciferase reporter constructs were performed. These demonstrated that the mRNA of survivin and CUG-BP1 each contain two functional miR-214-3p-binding sites as confirmed by mutational analysis. Finally, forced expression of miR-214-3p enhances the sensitivity of esophageal cancer cells to cisplatin-induced apoptosis. This effect is abrogated with rescue expression of survivin or CUG-BP1. These findings suggest that miR-214-3p acts as a tumor suppressor and that its downregulation contributes to chemoresistance in esophageal cancer cells by targeting both survivin and CUG-BP1.

Sphingosine-1-Phosphate Regulates the Expression of Adherens Junction Protein E-Cadherin and Enhances Intestinal Epithelial Cell Barrier Function

BackgroundThe regulation of intestinal barrier permeability is important in the maintenance of normal intestinal physiology. Sphingosine-1-phosphate (S1P) has been shown to play a pivotal role in enhancing barrier function in several non-intestinal tissues. The current study determined whether S1P regulated function of the intestinal epithelial barrier by altering expression of E-cadherin, an important protein in adherens junctions.MethodsStudies were performed upon cultured differentiated IECs (IEC-Cdx2L1 line) using standard techniques.ResultsS1P treatment significantly increased levels of E-cadherin protein and mRNA in intestinal epithelial cells (IECs) and also led to E-cadherin localizing strongly to the cell–cell border. S1P also improved the barrier function as indicated by a decrease in 14C-mannitol paracellular permeability and an increase in transepithelial electrical resistance (TEER) in vitro.ConclusionsThese results indicate that S1P increases levels of E-cadherin, both in cellular amounts and at the cell–cell junctions, and leads to improved barrier integrity in cultured intestinal epithelial cells.

Sphingosine-1-Phosphate Protects Intestinal Epithelial Cells from Apoptosis Through the Akt Signaling Pathway

Objective The regulation of apoptosis of intestinal mucosal cells is important in maintenance of normal intestinal physiology. Summary Sphingosine-1-phosphate (S1P) has been shown to play a critical role in cellular protection to otherwise lethal stimuli in several nonintestinal tissues. Methods The current study determines whether S1P protected normal intestinal epithelial cells (IECs) from apoptosis and whether Akt activation was the central pathway for this effect. Results S1P demonstrated significantly reduced levels of apoptosis induced by tumor necrosis factor-alpha (TNF-α)/cycloheximide (CHX). S1P induced increased levels of phosphorylated Akt and increased Akt activity, but did not affect total amounts of Akt. This activation of Akt was associated with decreased levels of both caspase-3 protein levels and of caspase-3 activity. Inactivation of Akt by treatment with the PI3K chemical inhibitor LY294002 or by overexpression of the dominant negative mutant of Akt (DNMAkt) prevented the protective effect of S1P on apoptosis. Additionally, silencing of the S1P-1 receptor by specific siRNA demonstrated a lesser decrease in apoptosis to S1P exposure. Conclusion These results indicate that S1P protects intestinal epithelial cells from apoptosis via an Akt-dependent pathway.

Abstract B227: Overexpression of microRNA 214-3p in esophageal cancer cells enhances sensitivity to chemotherapy-induced apoptosis by targeting CUG-binding protein 1 (CUGBP1) and survivin.

Introduction: Survivin, a key regulator of apoptosis and cell division is overexpressed in esophageal cancer cells and has been correlated with decreased survival. Our previous studies indicate that the RNA-binding protein CUGBP1 is also overexpressed in esophageal cancer cells and contributes to survivin overexpression by stabilizing its mRNA. To date, little information exists on the regulation of CUGBP1 in esophageal cancer cells. Based on sequence analysis, miR 214-3p is predicted to bind both CUGBP1 and survivin mRNA with high binding affinity. The purposes of this study are to determine expression levels of miR 214-3p in esophageal cancer cells and to establish whether miR 214-3p directly interacts with CUGBP1 and survivin mRNA to regulate their expression and modulate sensitivity to chemotherapy-induced apoptosis. Methods: Studies were conducted in human esophageal epithelial (hESO) cells and in TE7 and TE10 human esophageal cancer cells. Global microRNA expression in these cell lines was determined by microarray analysis and confirmed by real-time PCR. MiR 214-3p function was tested by its overexpression and silencing. Levels of protein expression were measured by Western blot. Levels of mRNA were measured by real-time PCR. Binding of miR 214-3p to CUGBP1 and survivin mRNA was examined by biotinylated RNA pull-down assays. mRNA stability was determined by measuring its half-life after addition of Actinomycin D. The apoptotic phenotype was examined by caspase expression using Western blot and flow cytometry. Results: Levels of miR 214-3p in TE7 and TE10 esophageal cancer cells are reduced by approximately 3,000 log-fold in comparison to hESO cells. Biotinylated RNA-pull down assays confirm direct binding of miR 214-3p to CUGBP1 and survivin mRNA. CUGBP1 and survivin mRNA and protein levels are reduced in a time-dependent manner following miR 214-3p overexpression in TE7 and TE10 cells. Conversely, silencing miR 214-3p in hESO cells results in increased CUGBP1 and survivin mRNA and protein levels. The stability of both CUGBP1 and survivin mRNA is decreased following overexpression of miR 214-3p in TE7 cells. Caspase-3 protein levels are increased when TE7 cells are exposed to camptothecin following microRNA 214-3p overexpression. Conclusion: MiR 214-3p expression is markedly reduced in TE7 and TE10 esophageal cancer cells relative to hESO cells. MiR 214-3p binds and destabilizes both CUGBP1 and survivin mRNA. Overexpression of miR 214-3p in esophageal cancer cells results in decreased expression of CUGBP1 and survivin and leads to increased sensitivity to chemotherapy-induced apoptosis. These results strongly support the supposition that the loss of miR 214-3p in esophageal cancer cells contributes to enhanced resistance to chemotherapy-induced apoptosis by leading to increased CUGBP1 and survivin expression. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B227. Citation Format: Pornima Phatak, Daniel Mansour, Kimberly Byrnes, Shan Cao, Lan Liu, Ruiyun Li, Rao Jaladanki, Douglas J. Tuner, Jian Ying Wang, James M. Donahue. Overexpression of microRNA 214-3p in esophageal cancer cells enhances sensitivity to chemotherapy-induced apoptosis by targeting CUG-binding protein 1 (CUGBP1) and survivin. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B227.

Tu2060 Sphingosine-1-Phosphate Prevents LPS-Induced Loss of Permeability in Intestinal Epithelial Cells

Intestinal epithelial barrier dysfunction results from a wide variety of pathologic conditions; at the gastrointestinal mucosal layer cells must be capable of maintaining barrier integrity, and do this through the interplay of multiple active processes. Previous reports from our lab have shown that Sphingosine-1-phophate (S1P) promotes intestinal epithelial barrier function in part through regulation of barrier proteins, and S1P has also been found to be protective in various pathologic states. Lipopolysaccharide (LPS) has been shown to increase paracellular permeability, and recently, to also decrease intracellular S1P. In the current study we hypothesized that S1P would decrease paracellular permeability upon LPS exposure, and would act in part through regulation of caveolin-1 expression IEC-Cdx2L1 (Cdx) differentiated intestinal epithelial cells were utilized. Western blot analysis, real-time PCR, immunohistochemical staining, were utilized by standard techniques. Transwell permeability to C14-mannitol, FITC-dextran, and measurement of transepithelial electrical resistance (TEER) were utilized for permeability assessments. Sphingosine Kinase 1 (SphK-1) overexpression stable cell lines were selected in rat intestinal epithelial cells (IECs). SphK-1 activity and S1P production were measured by radioactive isotope assay. LPS-treated (50 mcM) Cdx cells show dramatically increased permeability at 4h, but pretreatment with S1P (0.5 mcM for one hour) was protective of this LPS-induced increase in permeability, and returned permeability to normal levels. S1P also prevented LPS-associated decreases in phosphorylated occludin, and in immunofluorescence studies S1P preserved cortical accumulation of occludin that was disrupted with LPS administration alone. S1P was found to increase levels of toll-like receptor (TLR) 2 in Cdx cells, with no change in levels of TLR4. Similarly, cells stably overexpressing SphK1 demonstrated increased levels of S1P and also increased levels of TLR2 and not TLR4. Cells overexpressing SphK1 and S1P showed dramatically increased plasma membrane levels of Stim1, TRPC1, and the scaffolding protein caveolin-1. Phosphorylated caveolin-1 was significantly decreased with exposure to LPS (5 mcM), however co-treatment with S1P preserved basal caveolin-1 levels. Finally, inhibition of caveolin-1 with siRNA prevented S1P rescue of LPS loss of permeability. Our findings demonstrate that S1P prevents LPS-associated loss of permeability, and this is in part through its ability to prevent LPS-associated loss of caveolin-1.

M1731 Sphingosine-1-Phosphate Regulates Barrier Function in Cultured Intestinal Epithelial Cells and Murine Intestinal Tissue

assessed by stable isotope methodology after administration of U-13C-glucose and 1-13Clactose. Concentrations of the EFA linoleic acid (LA), and the enzyme activity and mRNA expression of lactase, were measured in the mucosa of proximal, mid and distal small intestine. Results: Mice fed the EFA-deficient diet were markedly EFA-deficient (triene/ tetraene ratio 0.23±0.06 vs. 0.01±0.00 in controls, p<0.05) with a profound lower fat absorption of dietary fat (81±3 vs. 99±0% in controls, of ingested amount, p<0.05). EFA deficiency did not affect the histology or proliferative capacity of the small intestine as demonstrated by the histological and proliferative staining, and by the morphometrcal measurements of the small intestinal villi. Blood C6-glucose appearance and disappearance were similar in both groups, indicating unaffected monosaccharide absorption. In contrast, blood appearance of 13C-glucose, originating from 13C-lactose, was delayed in EFA-deficient mice. EFA deficiency profoundly reduced lactase activity (-58%, p<0.01) and mRNA expression (-55%, p<0.01) in mid small intestine. Both lactase activity and its mRNA expression strongly correlated with mucosal LA concentrations (r=0.89 and 0.79, resp., p< 0.01). Conclusions: EFA deficiency in mice inhibits the capacity to digest lactose, but does not affect small intestinal histology. These data underscore the observation that EFA deficiency functionally impairs the small intestine, possibly mediated by low LA levels in the enterocytes. This research was funded by the Dutch Digestive Foundation (MWO 04-38).