AIM To explore the mechanism by which microRNA-155 (miR-155) regulates the pathogenesis of experimental colitis. METHODS A luciferase assay was performed to confirm the binding of miR-155 to the SHIP-1 3'-UTR. MiR...AIM To explore the mechanism by which microRNA-155 (miR-155) regulates the pathogenesis of experimental colitis. METHODS A luciferase assay was performed to confirm the binding of miR-155 to the SHIP-1 3'-UTR. MiR-155 mimics, negative controls and SHIP-1 expression/knockdown vectors were established and then utilized in gain-and loss-of-function studies performed in raw264.7 cells and primary bone marrow-derived macrophages (BMDMs). Thereafter, dextran sulfate sodium (DSS)-induced colitis mouse model with or without antagomiR-155 treatment was established, and the levels of miR-155 and SHIP-1, as well as the pro-inflammatory capabilities, were measured by western blot, quantitative polymerase chain reaction, and immunohistochemistry. RESULTS MiR-155 directly bound to the 3'-UTR of SHIP-1 mRNA and induced a significant decrease in SHIP-1 expression in both raw264.7 cells and primary BMDMs. MiR-155 markedly promoted cell proliferation and proinflammatory secretions including IL-6, TNF-alpha, IL-1 beta, and IFN-gamma, whereas these effects could be reversed by the restoration of SHIP-1 expression. In vivo studies showed that antagomiR-155 administration could alleviate DSS-induced intestinal inflammation in Balb/c mice. Moreover, significantly increased SHIP-1 expression, as well as decreased Akt activation and in-flammatory response, were observed in the antagomiR-155-treated mice. CONCLUSION MiR-155 promotes experimental colitis by repressing SHIP-1 expression. Thus, the inhibition of miR-155 might be a promising strategy for therapy.展开更多
SHIP-1 is an SH2 domain containing inositol-5-phosphatase that appears to be a negative regulator of hematopoiesis. To the potential effects of SHIP-1 on MMP2 secretion and migration of cancer cells, three murine SHIP...SHIP-1 is an SH2 domain containing inositol-5-phosphatase that appears to be a negative regulator of hematopoiesis. To the potential effects of SHIP-1 on MMP2 secretion and migration of cancer cells, three murine SHIP-1 mutants were made： △SH2-SHIP-1, △Ptase-SHIP-1, △Cter-SHIP-1. These mutant forms were subcloned as well as the wild type （WT） of murine SHIP-1 cDNA were subcloned into pcDNA3 expression vector, then transfected into and overexpressed SHIP-1 and its mutants in a Src-transformed 3Y1 cellline （SR3Y1）. The results showed that overexpression of wild type of SHIP-1 does not affect the MMP2 secretion in both SR3Y1 and 3Y1 cells, but can induce MMP9 secretion, while either WT SHIP-1, the SH2 domain, phosphatase domain, or C terminus deletion mutants could significantly block the MMP2 and MMP9 secretion in SR3Y1 cells and suppress cell invasion ability. The results confirmed SHIP-1 as a negative regulator for cell migration and invasion in transformed cells, and implied that it may function through each of its three domains.展开更多
文摘AIM To explore the mechanism by which microRNA-155 (miR-155) regulates the pathogenesis of experimental colitis. METHODS A luciferase assay was performed to confirm the binding of miR-155 to the SHIP-1 3'-UTR. MiR-155 mimics, negative controls and SHIP-1 expression/knockdown vectors were established and then utilized in gain-and loss-of-function studies performed in raw264.7 cells and primary bone marrow-derived macrophages (BMDMs). Thereafter, dextran sulfate sodium (DSS)-induced colitis mouse model with or without antagomiR-155 treatment was established, and the levels of miR-155 and SHIP-1, as well as the pro-inflammatory capabilities, were measured by western blot, quantitative polymerase chain reaction, and immunohistochemistry. RESULTS MiR-155 directly bound to the 3'-UTR of SHIP-1 mRNA and induced a significant decrease in SHIP-1 expression in both raw264.7 cells and primary BMDMs. MiR-155 markedly promoted cell proliferation and proinflammatory secretions including IL-6, TNF-alpha, IL-1 beta, and IFN-gamma, whereas these effects could be reversed by the restoration of SHIP-1 expression. In vivo studies showed that antagomiR-155 administration could alleviate DSS-induced intestinal inflammation in Balb/c mice. Moreover, significantly increased SHIP-1 expression, as well as decreased Akt activation and in-flammatory response, were observed in the antagomiR-155-treated mice. CONCLUSION MiR-155 promotes experimental colitis by repressing SHIP-1 expression. Thus, the inhibition of miR-155 might be a promising strategy for therapy.
基金the Sasagawa Medical Fellowship from Japan-Sino Medical Association with funds from the Nippon Foundation.
文摘SHIP-1 is an SH2 domain containing inositol-5-phosphatase that appears to be a negative regulator of hematopoiesis. To the potential effects of SHIP-1 on MMP2 secretion and migration of cancer cells, three murine SHIP-1 mutants were made： △SH2-SHIP-1, △Ptase-SHIP-1, △Cter-SHIP-1. These mutant forms were subcloned as well as the wild type （WT） of murine SHIP-1 cDNA were subcloned into pcDNA3 expression vector, then transfected into and overexpressed SHIP-1 and its mutants in a Src-transformed 3Y1 cellline （SR3Y1）. The results showed that overexpression of wild type of SHIP-1 does not affect the MMP2 secretion in both SR3Y1 and 3Y1 cells, but can induce MMP9 secretion, while either WT SHIP-1, the SH2 domain, phosphatase domain, or C terminus deletion mutants could significantly block the MMP2 and MMP9 secretion in SR3Y1 cells and suppress cell invasion ability. The results confirmed SHIP-1 as a negative regulator for cell migration and invasion in transformed cells, and implied that it may function through each of its three domains.
