Expression Of Wnt10b Research Articles

1983-P: WNT Pathway and Bone Fragility in Postmenopausal Women with Type 2 Diabetes

Fragility fractures are major complications in type 2 diabetes (T2D), but mechanisms underlying bone fragility are still poorly clarified. Bone mineral density (BMD) underestimates T2D fracture incidence, since a normal or even higher BMD is associated with a greater fracture risk than healthy population. We hypothesized that bone fragility in T2D results from poor bone microarchitecture and bone quality caused by WNT signaling impairment and lower bone formation. We aimed to evaluate the association between bone gene expression of WNT pathway and features of bone microarchitecture and strength. We enrolled T2D (n=13) and nondiabetic (controls, n=75) postmenopausal women undergoing standard hip arthroplasty. Serum and proximal femur samples from recruited subjects were collected at the moment of surgical procedure. Gene expression of sclerostin (SOST), WNT5a, WNT10b, SFRP5, GSK3β, osteocalcin and RUNX2 was analyzed by real-time PCR. Total-body, lumbar and femoral BMD were assessed by bone densitometry. A trabecular core from bone samples was analyzed by Micro X-ray computed tomography and by compression tests. Age (75.23 ± 8.47 vs. 73.24 ± 5.77) and BMI (29.92 ± 5.38 vs. 27.68 ± 5.62 Kg/m2) did not differ between T2D and controls respectively. Glycated hemoglobin was 6.48 ± 1.7 mmol/mol. We found that T2D subjects had no differences in BMD and in bone microarchitecture parameters compared to controls. Neither bone turnover nor WNT serum markers differed among groups. SOST gene expression was increased (p=0.003) whereas RUNX2 expression was decreased in T2D vs. controls (p=0.017), independently by BMI and age. Osteocalcin and other WNT related genes expression did not differ among groups. Compression tests did not show impairment in bone strength in T2D vs. controls. In conclusion, although a good glycemic control and no alterations in bone density, microarchitecture and strength, we found an increase in SOST and a decrease in RUNX2 gene expression, implying a lower bone formation in T2D. Disclosure A. Piccoli: None. F. Cannata: None. F. Russo: None. V.L. Greto: None. C. Isgrò: None. G. Leanza: None. R. Strollo: None. C. Massaroni: None. C. Pedone: None. S. Silvestri: None. G. Vadalà: None. V. Denaro: None. T. Bisogno: None. R. Papalia: None. P. Pozzilli: None. M. Maccarrone: None. N. Napoli: None. Funding Campus Bio-Medico University

The role of Wnt7B in the mediation of dentinogenesis via the ERK1/2 pathway

ObjectivesThis study investigates the role of Wnt7b in mouse dentin formation. DesignC57BL/6 mouse tooth germs at different developmental stages were collected to measure the expression of Wnt7b by immunohistochemical staining. The morphology of mandibles of Dmp1-cre;ROSA26-Wnt7b transgenic mice and ROSA26-Wnt7b littermates was analyzed by Micro-CT and HE staining. The ultramicrostructure of dentin was scanned with an electron microscope. Primary mouse dental papillae cells (MDPCs) and odontoblastic cell line (A11) were cultured and infected with adenovirus to overexpress Wnt7b. Cell proliferation and cell apoptosis were evaluated using CCK-8 and flow cytometry. Osteogenic differentiation of MDPCs and A11 was assessed by Alizarin red staining, and qPCR detection of osteogenic gene expression. The activation of signaling pathways was measured by the use of western blot analysis. The ERK1/2 inhibitor was used to test the effect of Wnt7b regulated cell differentiation. ResultsWnt7b was expressed principally in the mouse odontoblast layer after the early bell stage. In transgenic mice, Wnt7b was over-expressed in tooth mesenchyme, with a thinner predentin layer and thicker intertubular dentin. Both the micro-hardness value and the Ca/Pi ratio of dentin of transgenic mice were higher. Wnt7b promoted proliferation and mineralization of MDPCs and A11. The protein level of p-ERK1/2 was found to be higher in A11 infected with Ad-Wnt7b. The ERK signaling pathway inhibitor partly rescued the Wnt7b-induced differentiation of A11. ConclusionsWnt7b enhances dentinogenesis by increasing the proliferation and differentiation of dental mesenchymal cells partly through ERK1/2 pathway.

Narrowband UVB treatment induces expression of WNT7B, WNT10B and TCF7L2 in psoriasis skin

WNT/β-catenin signaling pathways play a pivotal role in the human immune defense against infections and in chronic inflammatory conditions as psoriasis. Wnt gene alterations are linked to known comorbidities of psoriasis as obesity, diabetes and Crohn’s disease. The objective of this study was to investigate WNT7B, WNT10B, WNT16 and TCF7L2 gene and protein expression in lesional and non-lesional skin and in the peripheral blood of patients with chronic plaque psoriasis compared with healthy individuals. To investigate the effect of narrowband UVB radiation, expression of these genes were analyzed before and after narrowband UVB treatment. Associations between single nucleotide polymorphisms for WNT7B, WNT10B, WNT16 and TCF7L2 genes and psoriasis were tested. Our results show significantly decreased WNT7B, WNT10B and TCF7L2 gene expression in lesional skin compared with non-lesional skin and healthy controls. Narrowband UVB treatment significantly increased expression of these genes in lesional skin. Immunohistochemistry shows increased WNT16 expression in lesional skin. No significant differences in allele or genotype frequencies for Wnt or TCF7L2 gene polymorphisms were found between patient and control group. This study shows for the first time significant UVB induced upregulation of WNT7B, WNT10B and TCF7L2 in patients with psoriasis and suggests a potential role of these genes in psoriasis pathogenesis.

Regulation of Wnt Singaling Pathway by Poly (ADP-Ribose) Glycohydrolase (PARG) Silencing Suppresses Lung Cancer in Mice Induced by Benzo(a)pyrene Inhalation Exposure.

Benzo(a)pyrene (BaP) is a polycyclic aromatic hydrocarbon that specifically causes cancer and is widely distributed in the environment. Poly (ADP-ribosylation), as a key post-translational modification in BaP-induced carcinogenesis, is mainly catalyzed by poly (ADP-ribose) glycohydrolase (PARG) in eukaryotic organisms. Previously, it is found that PARG silencing can counteract BaP-induced carcinogenesis in vitro, but the mechanism remained unclear. In this study, we further examined this process in vivo by using heterozygous PARG knockout mice (PARG+/−). Wild-type and PARG+/− mice were individually treated with 0 or 10 μg/m3 BaP for 90 or 180 days by dynamic inhalation exposure. Pathological analysis of lung tissues showed that, with extended exposure time, carcinogenesis and injury in the lungs of WT mice was progressively worse; however, the injury was minimal and carcinogenesis was not detected in the lungs of PARG+/− mice. These results indicate that PARG gene silencing protects mice against lung cancer induced by BaP inhalation exposure. Furthermore, as the exposure time was extended, the protein phosphorylation level was down-regulated in WT mice, but up-regulated in PARG+/− mice. The relative expression of Wnt2b and Wnt5b mRNA in WT mice were significantly higher than those in the control group, but there was no significant difference in PARG+/− mice. Meanwhile, the relative expression of Wnt2b and Wnt5b proteins, as assessed by immunohistochemistry and Western blot analysis, was significantly up-regulated by BaP in WT mice; while in PARG+/− mice it was not statistically affected. Our work provides initial evidence that PARG silencing suppresses BaP induced lung cancer and stabilizes the expression of Wnt ligands, PARG gene and Wnt ligands may provide new options for the diagnosis and treatment of lung cancer.

Inflammation in PTSD is a consequence of dysregulated WNT signaling orchestrated by long non-coding RNA LINC00926 and lysine methyltransferase MLL1

Abstract Posttraumatic Stress Disorder patients experience chronic inflammation. It is not known whether immune dysregulation causes PTSD or the psychiatric imbalance occurs first. Moreover, little is known about the molecular mechanisms involved in the immune dysregulation. Our past work indicates the involvement of epigenetic mechanisms such as histone methylation in the regulation of inflammation in PTSD. By employing RNA-Seq, we identified upregulated expression of WNT10B and LINC00926 in the PBMCs of PTSD. Thus, we hypothesized that LINC00926 overexpression in PTSD results in increased H3K4me3 (gene activating histone methylation) around the promoter of WNT10B leading to its upregulation which in turn, regulated the elevated expression of inflammatory cytokines like IFNg and IL17. Thus, we first confirmed the interaction between LINC00926 and the histone H3K4 methyltransferase, MLL1. Then, by in vitro knockdown of LINC00926 we confirmed lowered expression of WNT10B implying that the interaction between LINC00926 and MLL1 possibly leads to increased H3K4me3 on WNT10B promoter and thereby its upregulation. Following this, we confirmed increased H3K4me3 around WNT10B promotor of PTSD patients by ChIP-Seq. Finally, we confirmed that upregulated WNT10B indeed contributed to the elevated expression of IFNg and IL17. In conclusion, our data implies that upregulation of LINC00926 in PTSD leads to inflammatory state by a mechanism that involves interaction with MLL1 resulting in increased H3K4me3 around WNT10B promotor and, eventually leads to elevated expression of proinflammatory cytokines. Our report is the first to show that inflammation in PTSD is a result of altered epigenetic mechanism involving LINC00926 and WNT10B.

Wnt and RUNX2 mediate cartilage breakdown by osteoarthritis synovial fibroblast-derived ADAMTS-7 and -12.

Failure of therapeutic approaches for the treatment of osteoarthritis (OA) based on the inhibition of metalloproteinases, might be because of their constitutive expression in homeostasis, together with their network complexity. The knowledge of this network would contribute to selective target pathological conditions. In this sense, blockade of mediators produced by neighbouring joint cells, such as synovial fibroblasts (SF), would prevent cartilage damage. Thus, we studied the contribution of ADAMTS‐7 and ‐12 from SF to cartilage oligomeric matrix protein (COMP) degradation, and the signalling pathways involved in their expression. We report for the first time in SF, the involvement of ERK‐Runx2 axis and Wnt/β‐catenin signalling in ADAMTS‐12 and ADAMTS‐7 expressions, respectively, with the subsequent consequences in COMP degradation from cartilage extracellular matrix. After stimulation with IL‐1β or fibronectin fragments, we showed that ERK inhibition decreased Runx2 activation and ADAMTS‐12 expression in OA‐SF, also reducing Fn‐fs‐induced COMP degradation. Blockage of Wnt signalling by DKK1 reduced ADAMTS‐7 and COMP degradation in OA‐SF as well. In addition, Wnt7B expression was induced by IL‐1β and by itself, also increasing ADAMTS‐7. Our results could contribute to the development of disease‐modifying OA drugs targeting ADAMTS‐7 and ‐12 for the prevention of extracellular matrix components degradation like COMP.

Wnt Production in Dental Epithelium Is Crucial for Tooth Differentiation

The Wnt ligands display varied spatiotemporal expression in the epithelium and mesenchyme in the developing tooth. Thus far, the actions of these differentially expressed Wnt ligands on tooth development are not clear. Shh expression specifies the odontogenic epithelium during initiation and is consistently restricted to the dental epithelium during tooth development. In this study, we inactivate Wntless (Wls), the key regulator for Wnt trafficking, by Shh-Cre to investigate how the Wnt ligands produced in the dental epithelium lineage act on tooth development. We find that conditional knockout of Wls by Shh-Cre leads to defective ameloblast and odontoblast differentiation. WlsShh-Cre teeth display reduced canonical Wnt signaling activity in the inner enamel epithelium and the underlying mesenchyme at the early bell stage, as exhibited by target gene expression and BAT-gal staining. The expression of Wnt5a and Wnt10b is not changed in WlsShh-Cre teeth. By contrast, Wnt10a expression is significantly increased in response to epithelial Wls deficiency. In addition, the expression of Hedgehog signaling pathway components Shh, Gli1, and Patched1 was greatly decreased in WlsShh-Cre teeth. Epithelial Wls loss of function in Shh lineage also leads to aberrant cell proliferation in dental epithelium and mesenchyme at embryonic day 16.5; however, the cell apoptosis is unaffected. Moreover, we find that Decorin and Col1a1, the key markers for odontoblast differentiation that are downregulated in WlsShh-Cre teeth, act as direct downstream targets of the canonical Wnt signaling pathway by chromatin immunoprecipitation analysis. Additionally, Decorin and Col1a1 expression can be increased by lithium chloride (LiCl) treatment in the in vitro tooth explants. Taken together, our results suggest that the spatial expression of Wnt ligands within the dental epithelial lineage regulates the differentiation of tooth structures in later stages.

Met-enkephalin inhibits ROS production through Wnt/β-catenin signaling in the ZF4 cells of zebrafish

Opioid neuropeptides are developed early in the course of a long evolutionary process. As the endogenous messengers of immune system, opioid neuropeptides participate in regulating immune response. In this study, the mechanism that Met-enkephalin (M-ENK) inhibits ROS production through Wnt/β-catenin signaling was investigated in the ZF4 cells of zebrafish. ZF4 cells were exposed to 0, 10, 20, 40, 80, and 160 μM Met-enkephalin (M-ENK) for 24 h, and the cell viability was detected with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The cell viability was significantly increased by 10, 20, 40, 80, and 160 μM M-ENK. After ZF4 cells were exposed to 0, 20, 40, and 80 μM M-ENK for 24 h, the mRNA expression of Wnt10b, β-catenin, and CCAAT/enhancer binding protein α (C/EBPα) was significantly increased by 40 and 80 μM M-ENK. However, the mRNA and protein expression of GSK-3β was significantly decreased by 40 and 80 μM M-ENK. The protein expression of β-catenin was significantly induced by 40 and 80 μM M-ENK, while the protein expression of p-β-catenin was significantly decreased by 20, 40, and 80 μM M-ENK. In addition, the mRNA expression of CAT, SOD, and GSH-PX was significantly increased by 40 and 80 μM M-ENK. The levels of H2O2, ·OH, and O2·- were significantly decreased, but the activity of CAT, SOD, and GSH-PX was significantly increased by 40 and 80 μM M-ENK. The fluorescence intensity of reactive oxygen species (ROS) was decreased, and that of mitochondrial membrane potential (MMP) was increased with the increase of M-ENK concentration in ZF4 cells. The results showed that M-ENK could induce Wnt/β-catenin signaling, which further inhibited ROS production through the induction of C/EBPα, MMP, and the activities of antioxidant enzymes.

Analysis of the wnt1 regulatory chromosomal landscape.

One of the earliest patterning events in the vertebrate neural plate is the specification of mes/r1, the territory comprising the prospective mesencephalon and the first hindbrain rhombomere. Within mes/r1, an interface of gene expression defines the midbrain-hindbrain boundary (MHB), a lineage restriction that separates the mesencephalon and rhombencephalon. wnt1 is critical to mes/r1 development and functions within the MHB as a component of the MHB gene regulatory network (GRN). Despite its importance to these critical and early steps of vertebrate neurogenesis, little is known about the factors responsible for wnt1 transcriptional regulation. In the zebrafish, wnt1 and its neighboring paralog, wnt10b, are expressed in largely overlapping patterns, suggesting co-regulation. To understand wnt1 and wnt10b transcriptional control, we used a comparative genomics approach to identify relevant enhancers. We show that the wnt1-wnt10b locus contains multiple cis-regulatory elements that likely interact to generate the wnt1 and wnt10b expression patterns. Two of 11 conserved enhancers tested show activity restricted to the midbrain and MHB, an activity that is conserved in the distantly related spotted gar orthologous elements. Three non-conserved elements also play a likely role in wnt1 regulation. The identified enhancers display dynamic modes of chromatin accessibility, suggesting controlled deployment during embryogenesis. Our results suggest that the control of wnt1 and wnt10b expression is under complex regulation involving the interaction of multiple enhancers.

Sinusoidal Electromagnetic Fields Increase Peak Bone Mass in Rats by Activating Wnt10b/β-Catenin in Primary Cilia of Osteoblasts

Extremely low-frequency electromagnetic fields have been considered a potential candidate for the prevention and treatment of osteoporosis; however, their action mechanism and optimal magnetic flux density (intensity) parameter are still elusive. The present study found that 50-Hz sinusoidal electromagnetic fields (SEMFs) at 1.8 mT increased the peak bone mass of young rats by increasing bone formation. Gene array expression studies with femoral bone samples showed that SEMFs increased the expression levels of collagen-1α1 and Wnt10b, a critical ligand of the osteogenic Wnt/β-catenin pathway. Consistently, SEMFs promoted osteogenic differentiation and maturation of rat calvarial osteoblasts (ROBs) in vitro through activating the Wnt10b/β-catenin pathway. This osteogenesis-promoting effect of SEMFs via Wnt10b/β-catenin signaling was found to depend on the functional integrity of primary cilia in osteoblasts. When the primary cilia were abrogated by small interfering RNA (siRNA) targeting IFT88, the ability of SEMFs to promote the osteogenic differentiation of ROBs through activating Wnt10b/β-catenin signaling was blocked. Although the knockdown of Wnt10b expression with RNA interference had no effect on primary cilia, it significantly suppressed the promoting effect of SEMFs on osteoblastic differentiation/maturation. Wnt10b was normally localized at the bases of primary cilia, but it disappeared (or was released) from the cilia upon SEMF treatment. Interestingly, primary cilia were elongated to different degrees by different intensities of 50-Hz SEMFs, with the window effect observed at 1.8 mT, and the expression level of Wnt10b increased in accord with the lengths of primary cilia. These results indicate that 50-Hz 1.8-mT SEMFs increase the peak bone mass of growing rats by promoting osteogenic differentiation/maturation of osteoblasts, which is mediated, at least in part, by Wnt10b at the primary cilia and the subsequent activation of Wnt/β-catenin signaling. © 2019 American Society for Bone and Mineral Research.

Expression of the Wnt ligands gene family and its relationship to prognosis in hepatocellular carcinoma

BackgroundThe Wnt gene family members are known to participate regulating various normal and pathological processes including tumorigenesis. However, the association between Wnt ligands gene family and prognosis in hepatocellular carcinoma has not been systematically studied. Therefore, we evaluated the role of Wnt ligands gene family in hepatocellular carcinoma using publicly available data from The Cancer Genome Atlas (TCGA).MethodsClinical information and RNA-Seq mRNA expression data were derived from TCGA hepatocellular carcinoma cohort. Differences in overall survival (OS) and disease-free survival (DFS) between increased and decreased expression groups (defined by X-tile analyses) of Wnt ligands gene family were compared using Kaplan–Meier method and Cox regression model, with p-values calculated via log-rank test. Gene Set Enrichment Analysis (GSEA) was performed.ResultsMultivariate analysis adjusted for patient age, sex, BMI, tumor grade, and TMN stage revealed that Wnt1, Wnt3 and Wnt5B expressions were independent prognostic factors for OS and DFS (OS: HR = 0.58, P = 0.006; HR = 0.65, P = 0.03; HR = 0.56, P = 0.023, respectively; DFS: HR = 0.52, P < 0.001; HR = 1.93, P = 0.003; HR = 0.59, P = 0.011, respectively). Furthermore, expression of Wnt1 and Wnt5B was significantly associated with TMN stage (P = 0.02 and P = 0.03 for OS; P = 0.02 and P = 0.02 for DFS). GSEA showed that nucleotide excision repair was differentially enriched in Wnt1 low expression phenotype and aminoacyl trna biosynthesis and basal transcription factors were differentially enriched in Wnt5B low expression phenotype.ConclusionsOur results identified associations of several Wnt ligands with prognosis of HCC patients, indicating that these genes could serve as prognostic biomarkers of HCC.

Single-cell analysis reveals congruence between kidney organoids and human fetal kidney

BackgroundHuman kidney organoids hold promise for studying development, disease modelling and drug screening. However, the utility of stem cell-derived kidney tissues will depend on how faithfully these replicate normal fetal development at the level of cellular identity and complexity.MethodsHere, we present an integrated analysis of single cell datasets from human kidney organoids and human fetal kidney to assess similarities and differences between the component cell types.ResultsClusters in the combined dataset contained cells from both organoid and fetal kidney with transcriptional congruence for key stromal, endothelial and nephron cell type-specific markers. Organoid enriched neural, glial and muscle progenitor populations were also evident. Major transcriptional differences between organoid and human tissue were likely related to technical artefacts. Cell type-specific comparisons revealed differences in stromal, endothelial and nephron progenitor cell types including expression of WNT2B in the human fetal kidney stroma.ConclusionsThis study supports the fidelity of kidney organoids as models of the developing kidney and affirms their potential in disease modelling and drug screening.

Long noncoding RNA OIP5-AS1 targets Wnt-7b to affect glioma progression via modulation of miR-410.

The present study was undertaken to investigate the underlying mechanisms of long noncoding RNA OIP5-AS1 via regulating miR-410 to modulate Wnt-7b in the progression of glioma. To address this problem, we measured the expression of OIP5-AS1 and miR-410 in glioma tissues by qRT-PCR. Glioma U87 cells were transfected with OIP5-AS1 siRNA or miR-410 inhibitors. The targeting relationships among miR-410, OIP5-AS1 and Wnt-7b were verified by luciferase reporter assays. Western blotting was employed to determine the expression of Wnt-7b/β-catenin pathway-related proteins, while MTT, flow cytometry, Transwell assays and wound-healing assays were used to measure the biological characteristics of glioma cells. The results showed that OIP5-AS1 expression was higher and miR-410 was lower in glioma tissues. Luciferase reporter assays confirmed a targeting relationship between OIP5-AS1 and miR-410, as well as between miR-410 and Wnt-7b. Silencing OIP5-AS1 reduced cell proliferation, invasion and migration of glioma U87 cells and led to depressed expression levels of miR-410, Wnt-7b, p-β-catenin, GSK-3β-pS9, c-Myc and cyclin D1. Furthermore, down-regulation of OIP5-AS1 induced G0/G1 phase cell cycle arrest and apoptosis of glioma cells. Inhibitors of miR-410 abolished the biological effects of OIP5-AS1 siRNA in glioma cells. In vivo, OIP5-AS1 knockdown also inhibited tumor growth. Taken together, this research suggested that silencing OIP5-AS1 may specifically block the Wnt-7b/β-catenin pathway via targeted up-regulating miR-410, thereby inhibiting growth, invasion and migration while promoting apoptosis in glioma cells.

MiR-338-3p Enhances Ovarian Cancer Cell Sensitivity to Cisplatin by Downregulating WNT2B.

PurposeChemoresistance is a concern in ovarian cancer patients, in whom survival remains. MicroRNA, a novel class of small RNAs, have frequently been found to be dysregulated in human malignancies and to act as negative regulators of gene expression. This study aimed to explore the function of miR-338-3p in cisplatin resistance in ovarian cancer and potential molecular mechanisms thereof.Materials and MethodsThe expression levels of miR-338-3p and WNT2B in ovarian cancer tissues and cells were estimated by real-time quantitative polymerase chain reaction (RT-qPCR). In addition, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT), transwell, and flow cytometry assays were used to assess biological role of miR-338-3p in vitro. Western blot assay was conducted to measure protein expression of WNT2B, epithelial-mesenchymal transition (EMT)-related proteins, and apoptosis-related proteins. The relationship between miR-338-3p and WNT2B was confirmed by dual-luciferase reporter. Finally, a xenograft tumor model was developed to explore the effects of overexpression of miR-338-3p on tumor growth in ovarian cancer in vivo.ResultsMiR-338-3p was downregulated in cisplatin resistant ovarian cancer tissues and cells. Mechanistically, high expression of miR-338-3p enhanced cell sensitivity to cisplatin by inhibiting proliferation, motility, and EMT and by promoting apoptosis via targeting WNT2B expression in vitro. Furthermore, overexpression of miR-338-3p increased cisplatin sensitivity among ovarian cancer in an in vivo xenograft tumor model.ConclusionMiR-338-3p enhances the sensitivity of ovarian cancer cells to cisplatin by downregulating WNT2B.

WISP1 silencing confers protection against epithelial-mesenchymal transition of renal tubular epithelial cells in rats via inactivation of the wnt/β-catenin signaling pathway in uremia.

Uremia can affect hepatic metabolism of drugs by regulating the clearance of drugs, but it has not been clarified whether gene silencing could modulate the epithelial-mesenchymal transition (EMT) process in uremia. Hence, we investigated the effect of WISP1 gene silencing on the renal tubular EMT in uremia through the wnt/β-catenin signaling pathway. Initially, microarray-based gene expression profiling of uremia was used to identify differentially expressed genes. Following theestablishment of uremia rat model, serum creatinine, and urea nitrogen of rats were detected. Renal tubular epithelial cells (TECs) were transfected with shRNA-WISP1 lentivirus interference vectors and LiCI (the wnt/β-catenin signaling pathway activator) to explore the regulatory mechanism of WISP1 in uremia in relation to the wnt/β-catenin signaling pathway. Then, expression of WISP1, wnt2b, E-cadherin, α-SMA, c-myc, Cyclin D1, MMP-2, and MMP-9 was determined. Furthermore, TEC migration and invasion were evaluated. Results suggested that WISP1 and the wnt/β-catenin signaling pathway were associated with uremia. Uremic rats exhibited increased serum creatinine and urea nitrogen levels, upregulated WISPl, and activated wnt/β-catenin signaling pathway. Subsequently, WISP1 silencing decreased wnt2b, c-myc, Cyclin D1, α-SMA, MMP-2, and MMP-9 expression but increased E-cadherin expression, whereas LiCI treatment exhibited the opposite trends. In addition, WISP1 silencing suppressed TEC migration and invasion, whereas LiCI treatment promoted TEC migration and invasion. The findings indicate that WISP1 gene silencing suppresses the activation of the wnt/β-catenin signaling pathway, thus reducing EMT of renal TECs in uremic rats.

Retracted: Downregulation of microRNA-367 promotes osteoblasts growth and proliferation of mice during fracture by activating the PANX3-mediated Wnt/β-catenin pathway.

A majority of people suffering from bone fractures fail to heal and develop a nonunion, which is a challenging orthopedic complication requiring complex and expensive treatment. Previous data showed the inhibition of some microRNAs (miRNAs or miRs) can enhance fracture healing. The objective of the present study is to explore effects of miR-367 on the osteoblasts growth and proliferation of mouse during fracture via the Wnt/β-catenin pathway by targeting PANX3. Primarily, the femur fracture model was successfully established in 66 (C57BL/6) 6-week-old male mice. To verify whether miR-367 target PANX3, we used the target prediction program and performed luciferase activity determination. Subsequently, to figure out the underlying regulatory roles of miR-367 in fracture, osteoblasts were elucidated by treatment with miR-367 mimic, miR-367 inhibitor, or siRNA against PANX3 to determine the expression of miR-367, siPANX3, β-catenin, and Wnt5b as well as cell proliferation and apoptosis. The results demonstrated that PANX3 was verified as a target gene of miR-367. MiR-367 was found to highly expressed but PANX3, β-catenin, and Wnt5b were observed poorly expressed in fracture mice. downregulated miR-367 increased the mRNA and protein expression of PANX3, β-catenin, and Wnt5b, increased cell growth, proliferation, and migration, while decreased cell apoptosis in osteoblasts. Altogether, our study demonstrates that the downregulation of miR-367 may promote osteoblasts growth and proliferation in fracture through the activation of the PANX3-dependent Wnt/β-catenin pathway.

Enhancer Trapping and Annotation in Zebrafish Mediated with Sleeping Beauty, piggyBac and Tol2 Transposons.

Although transposon-mediated enhancer trapping (ET) is successfully applied in diverse models, the efficiency of various transposon systems varies significantly, and little information is available regarding efficiency of enhancer trapping by various transposons in zebrafish. Most potential enhancers (Ens) still lack evidence of actual En activity. Here, we compared the differences in ET efficiency between sleeping beauty (SB), piggyBac (PB) and Tol2 transposons. Tol2 represented the highest germline transfer efficiencies at 55.56% (NF0 = 165), followed by SB (38.36%, NF0 = 151) and PB (32.65%, NF0 = 149). ET lines generated by the Tol2 transposon tended to produce offspring with a single expression pattern per line, while PB and SB tended to generate embryos with multiple expression patterns. In our tests, 10 putative Ens (En1–10) were identified by splinkerette PCR and comparative genomic analysis. Combining the GFP expression profiles and mRNA expression patterns revealed that En1 and En2 may be involved in regulation of the expression of dlx1a and dlx2a, while En6 may be involved in regulation of the expression of line TK4 transgene and rps26, and En7 may be involved in the regulation of the expression of wnt1 and wnt10b. Most identified Ens were found to be transcribed in zebrafish embryos, and their regulatory function may involve eRNAs.

The Microbial Metabolite Butyrate Stimulates Bone Formation via T Regulatory Cell-Mediated Regulation of WNT10B Expression

SUMMARYNutritional supplementation with probiotics can prevent pathologic bone loss. Here we examined the impact of supplementation with Lactobacillus rhamnosus GG (LGG) on bone homeostasis in eugonadic young mice. Micro-computed tomography revealed that LGG increased trabecular bone volume in mice, which was due to increased bone formation. Butyrate produced in the gut following LGG ingestion, or butyrate fed directly to germ-free mice, induced the expansion of intestinal and bone marrow (BM) regulatory T (Treg) cells. Interaction of BM CD8+ T cells with Treg cells resulted in increased secretion of Wnt10b, a bone anabolic Wnt ligand. Mechanistically, Treg cells promoted the assembly of a NFAT1-SMAD3 transcription complex in CD8+ cells, which drove expression of Wnt10b−/−. Reducing Treg cell numbers, or reconstitution of TCRβ−/− mice with CD8+ T cells from Wnt10b−/− mice, prevented butyrate-induced bone formation and bone mass acquisition. Thus, butyrate concentrations regulate bone anabolism via Treg cell-mediated regulation of CD8+ T cell Wnt10b production.

MiR-148a suppressed cell invasion and migration via targeting WNT10b and modulating β-catenin signaling in cisplatin-resistant colorectal cancer cells

Cancer stem cells (CSCs) are suggested to be responsible for high recurrence rate and metastasis of colorectal cancer (CRC). Identifying novel targets that can suppress CSCs proliferation and metastasis may provide novel approach to combat against CRC. In the present study, we examined the role of miR-148a in cisplatin-resistant CRC cells with enhanced stem cell marker expression and explored the underlying mechanisms. In this study, we used cisplatin to selectively enrich cisplatin-resistant CRC cells from SW480 cell line, and these selected cisplatin-resistant SW480 cells were with significantly enhanced expression of stem cell markers and increased chemoresistance. MicroRNA (miRNA) array and qRT-PCR assay identified the down-regulation of miR-148a in cisplatin-resistant SW480 cells. Overexpression of miR-148a suppressed expression of stem cell markers, inhibited sphere formation, invasion and migration, induced apoptosis, and reduced chemo-resistance in cisplatin-resistant SW480 cells. Bioinformatics prediction and luciferase reporter assay revealed that WNT10b was a downstream target of miR-148a, and overexpression of miR-148a suppressed WNT10b expression and β-catenin signaling activities. Enforced expression WNT10b attenuated the effects of miR-148a on cisplatin-resistant SW480 cells sphere formation, invasion and migration. Further study showed that overexpression of miR-148a also suppressed in vivo tumor growth, and WNT10b expression and β-catenin signaling activities in tumor tissues were suppressed by miR-148a overexpression. In the clinical samples, miR-184a was found to be down-regulated in CRC tissues, down-regulation of miR-148a predicted poor prognosis in CRC patients. In conclusion, our study for the first time enriched the cisplatin-resistant CRC cells with enhanced stem cell marker expression from sphere-forming and chemo-resistant SW480-derived tumor xenografts in immune-deficient mice, and miR-148a suppressed the expression of stem cell markers, increased chemo-sensitivity, cell invasion and migration at least partly via regulating WNT10b and β-catenin signaling pathway.

Runx1 regulates osteogenic differentiation of BMSCs by inhibiting adipogenesis through Wnt/β-catenin pathway

ObjectiveBone marrow stem cells (BMSCs) can commit to both adipocyte and osteoblast lineages. However, the mechanism underlying how transcription factors regulate this process remains elusive. Our aims were to determine the role of runt-related transcription factor 1 (Runx1) in BMSCs lineage determination and the underlying mechanisms. Study designBMSCs from mouse femur bone marrow were harvested and cultured in osteogenic medium. Runx1 was knocked down in BMSCs using lentivirus. Alkaline phosphatase (ALP), Von Kossa and Oil Red O staining were performed on the Runx1-transduced BMSCs and control cells to see the differences of osteogenic and adipogenic differentiation in these groups. Real-time quantitative PCR and Western blot were performed to analyse the expression levels of osteogenic and adipogenic factors regulated by Runx1 at gene and protein levels. ResultsIn BMSCs with Runx1 knockdown, the expression levels of osteogenic-related genes decreased significantly while the adipogenic genes C/EBPα, PPARγ and Fabp4 increased by 12-fold, 10-fold, and 30-fold, respectively, compared with the control cells. ALP activity and Von kossa staining were greatly decreased in Runx1-transfected cells while the Oil Red O staining was comparable to that in the control groups. Canonical Wnt signaling was investigated in the Runx1-deficient BMSCs, and a 50% decrease in the expression of active β-catenin in these cells was found. Lef1 and Tcf1, which are regulated by β-catenin were also decreased in Runx1-deficient cells compared with the levels in controls. Moreover, although there was no difference in the expression of Wnt3a among the three groups of cells, the expression of Wnt10b decreased by 80% in Runx1-deficient BMSCs compared with the levels in the other two groups. ConclusionsOur results show Runx1 promotes the capacity of osteogenesis in BMSCs while inhibits their adipogenesis through canonical Wnt/β-catenin pathway, which provides new insights into osteoblast development.