MiR-221 is involved in depression by regulating Wnt2/CREB/BDNF axis in hippocampal neurons.

OBJECTIVE: The aim of this study was to investigate the mechanism of miR-221 in depression. METHODS: The molecules expressions were measured by qRT-PCR and western blot. The sucrose preference test (SPT), forced swimming test (FST) and tail suspension test (TST) were used to detect depressive-like symptoms. MTT assay and flow cytometric was used to measure the proliferation and apoptosis of hippocampal neuronal. RESULTS: MiR-221 expression in the cerebrospinal fluid and serum of major depressive disorder patients and the hippocampus of chronic unpredictable mild stress (CUMS) mice were increased, while the expression of Wnt2, p-CREB and BDNF were decreased. Additionally, silence of miR-221 increased sucrose preference of CUMS mice and shortened the immobility time of CUMS mice in SPT and FST. MiR-221 could targeted regulate Wnt2, and knockdown of Wnt2 reversed the effect of miR-221 inhibitor on the proliferation and apoptosis of hippocampal neurons and countered the promoting effect of miR-221 inhibitor on the expression of Wnt2, p-CREB and BDNF. CONCLUSION: MiR-221 could promote the development of depression by regulating Wnt2/CREB/BDNF axis.

Knockdown of mediator subunit Med19 suppresses bladder cancer cell proliferation and migration by downregulating Wnt/ß-catenin signalling pathway.

Mediator complex subunit 19 (Med19), a RNA polymerase II-embedded coactivator, is reported to be involved in bladder cancer (BCa) progression, but its functional contribution to this process is poorly understood. Here, we investigate the effects of Med19 on malignant behaviours of BCa, as well as to elucidate the possible mechanisms. Med19 expression in 15 BCa tissues was significantly higher than adjacent paired normal tissues using real-time PCR and Western blot analysis. Immunohistochemical staining of 167 paraffin-embedded BCa tissues was performed, and the results showed that high Med19 protein level was positively correlated with clinical stages and histopathological grade. Med19 was knocked down in BCa cells using short-hairpin RNA. Functional assays showed that knocking-down of Med19 can suppress cell proliferation and migration in T24, UM-UC3 cells and 5637 in vitro, and inhibited BCa tumour growth in vivo. TOP/FOPflash reporter assay revealed that Med19 knockdown decreased the activity of Wnt/ß-catenin pathway, and the target genes of Wnt/ß-catenin pathway were down-regulated, including Wnt2, ß-catenin, Cyclin-D1 and MMP-9. However, protein levels of Gsk3ß and E-cadherin were elevated. Our data suggest that Med19 expression correlates with aggressive characteristics of BCa and Med19 knockdown suppresses the proliferation and migration of BCa cells through down-regulating the Wnt/ß-catenin pathway, thereby highlighting Med19 as a potential therapeutic target for BCa treatment.

Study of Wnt2 secreted by A-549 cells in paracrine activation of ß-catenin in co-cultured mesenchymal stem cells.

The canonical Wnt signal pathway is a key regulator of self-renewal and cell fate determination in various types of stem cells. The total pool of ß-catenin consists of two different forms: the signaling form of the protein transmits the Wnt signals from the cell membrane to the target genes, whereas the membrane ß-catenin is involved in formation of cell-to-cell contact at cadherin junctions. Earlier we developed an in vitro model of epithelial differentiation of mesenchymal stem cells (MSCs) co-cultured with epithelial A-549 cells. The purpose of the present work was to study the role of Wnt2 secreted by the A-549 cells in paracrine induction of ß-catenin in co-cultured MSCs. Using the somatic gene knockdown technique, we obtained A-549 cell cultures with down-regulated WNT2. The MSCs co-cultured with the control A-549 cells displayed an increase in the levels of total cellular and signaling ß-catenin and transactivation of a reporter construction containing the Lef/Tcf protein family binding sites. In contrast, ß-catenin was not induced in the MSCs co-cultured with the A-549 cells with down-regulated WNT2 expression, but the total protein level was increased. We suggest that Wnt2 secreted by A-549 cells induces in co-cultured MSCs the Wnt/ß-catenin signaling pathway, whereas the associated increase in total ß-catenin level should be due to another mechanism.

Aberrant changes of Wnt2/beta-catenin signaling pathway induced by sodium nitroprusside in human esophageal squamous cell carcinoma cell lines.

Inhibition of Wnt/beta-catenin pathway is an attractive method for therapy of various tumors including breast, colorectal, and cervical cancer, etc. However, little is known about the role of Wnt2/beta-catenin pathway in esophageal squamous cell carcinoma (ESCC). Here we identify that Wnt2/beta-catenin signaling pathway is activated in ESCC cells, and sodium nitroprusside (SNP) and siRNA against beta-catenin not only inhibit the expressions of beta-catenin and its major downstream effectors including c-myc and cyclin D1, but induce cell cycle arrest and apoptosis, suggesting that Wnt2/beta-catenin pathway may be a potential molecular target for ESCC therapy.

Wnt2 acts as a cell type-specific, autocrine growth factor in rat hepatic sinusoidal endothelial cells cross-stimulating the VEGF pathway.

UNLABELLED: The mechanisms regulating the growth and differentiation of hepatic sinusoidal endothelial cells (HSECs) are not well defined. Because Wnt signaling has become increasingly important in developmental processes such as vascular and hepatic differentiation, we analyzed HSEC-specific Wnt signaling in detail. Using highly pure HSECs isolated by a newly developed protocol selecting against nonsinusoidal hepatic endothelial cells, we comparatively screened the multiple components of the Wnt pathway for differential expression in HSECs and lung microvascular endothelial cells (LMECs) via reverse-transcription polymerase chain reaction (RT-PCR). As confirmed via quantitative RT-PCR and northern and western blotting experiments, Wnt2 (and less so Wnt transporter wls/evi) and Wnt coreceptor Ryk were overexpressed by HSECs, whereas Wnt inhibitory factor (WIF) was strongly overexpressed by LMECs. Exogenous Wnt2 superinduced proliferation of HSECs (P < 0.05). The Wnt inhibitor secreted frizzled-related protein 1 (sFRP1) (P < 0.005) and transfection of HSECs with Wnt2 small interfering RNA (siRNA) reduced proliferation of HSECs. These effects were rescued by exogenous Wnt2. Tube formation of HSECs on matrigel was strongly inhibited by Wnt inhibitors sFRP1 and WIF (P < 0.0005). Wnt signaling in HSECs activated the canonical pathway inducing nuclear translocation of beta-catenin. GST (glutathione transferase) pull-down and co-immunoprecipitation assays showed Fzd4 to be a novel Wnt2 receptor in HSECs. Gene profiling identified vascular endothelial growth factor receptor-2 (VEGFR-2) as a target of Wnt2 signaling in HSECs. Inhibition of Wnt signaling down-regulated VEGFR-2 messenger RNA and protein. Wnt2 siRNA knock-down confirmed Wnt2 specificity of VEGFR-2 regulation in HSECs. CONCLUSION: Wnt2 is an autocrine growth and differentiation factor specific for HSECs that synergizes with the VEGF signaling pathway to exert its effects.