Expression Of Wnt10b Research Articles

Interactive role of Wnt signaling and Zn in regulating testicular function of the common carp, Cyprinus carpio

Wnt signaling is conserved among all species and plays a significant role in various cellular processes including reproduction. The present study identified significant involvement of wnt4a, wnt5b, and wnt8a signaling in the testicular growth of common carp,Cyprinus carpio. Predominant expression of wnt4a, wnt5b, and wnt8a was found in the gonads and Wnt4a was localized in spermatocytes and interstitial cells. Ontogeny and testicular phase-wise analysis signified the importance of wnt isofoms analyzed in this study. Specific pathway activation of Wnt signaling revealed that Wnt4a and Wnt5b act through non-canonical while Wnt8a prefers the canonical pathway. The Wnt signaling regulates several steroidogenic enzyme and testis-related genes which was confirmed by the Wnt blockade experiments. Incidentally, zinc (Zn) is an essential trace element involved in the progression of spermatogenesis in teleosts. In adult male carp, a single administration of Zn at different doses elevated the expression of Wnt and Zn transporter genes and a single-dose (30 μg/g body weight [BW]) of Zn treatment elevated steroidogenic enzyme and testis-related genes which coincided with elevated androgens. Conversely, single-dose administration of Zn chelator to the Zn administered (30 μg/g BW) fish reversed the effects emphasizing a prominent role of Zn in the testicular function perhaps through Wnt signaling. Similar effects were observed in the in vitro experiments using the Zn chelator. Bioaccumulation of Zn and histological analysis revealed the importance of Zn in progression of spermatogenesis and sperm motility. Various assays related to cell viability and proliferation exhibited the role of Zn in promoting spermatogenic cell progression. Flow cytometric analysis confirmed Zn-induced elevation of Wnt and Zn transporter genes in germ and supporting cells. Furthermore, the effects of Zn are dose-related in carp. Taken together, it seems wnt4a, wnt5b, and wnt8a play an important role in testis and exposure of Wnt inhibitor, canonical as well as non-canonical activators, and Zn confirmed that Zn regulates Wnt signaling vis-à-vis promoting spermatogenesis in the common carp.

Runt-related transcription factor-2 (Runx2) is required for bone matrix protein gene expression in committed osteoblasts in mice.

Runt-related transcription factor-2 (Runx2) is an essential transcription factor for osteoblast differentiation. However, its functions after the commitment into osteoblasts are controversial and remain to be clarified. We generated enhanced green fluorescent protein (EGFP)-Cre transgenic mice driven by the 2.3-kilobase (kb) Col1a1 promoter, and Runx2 was deleted in osteoblasts and odontoblasts in Runx2fl/flCre mice. The sutures and fontanelles were more widely opened in Runx2fl/flCre newborns than in Runx2fl/fl newborns. Runx2fl/flCre mice exhibited dwarfism with shorter incisors and 37% had irregularly aligned incisors. The volume of trabecular bone in femurs and vertebrae and their bone mineral density (BMD), in addition to the cortical thickness and BMD were reduced in Runx2fl/flCre mice compared with Runx2fl/fl mice in both sexes. The bone formation of both trabecular and cortical bone, osteoblast number, osteoclast surface, osteoblast proliferation, and the serum levels of procollagen type 1 N-terminal propeptide (P1NP), tartrate-resistant acid phosphatase 5b (TRAP5b), and C-terminal cross-linked telopeptide of type 1 collagen (CTX1) were reduced in Runx2fl/flCre mice. The expression of major bone matrix protein genes, including Col1a1, Col1a2, Spp1, Ibsp, and Bglap&Bglap2, and of Tnfsf11 was lower in Runx2fl/flCre mice than in Runx2fl/fl mice. The expression of Runx2 target genes, including Ihh, Fgfr1, Fgfr2, Fgfr3, Tcf7, Wnt10b, Pth1r, Sp7, and Dlx5, was also reduced. Osteoblasts in Runx2fl/fl mice were cuboidal and contained abundant type I collagen α1 (Col1a1), whereas those in Runx2fl/flCre mice were deflated and contained a small amount of Col1a1. Runx2 activated the reporter activity of the 2.3-kb Col1a1 promoter and bound the region around the Col1a1 transcription start site. The deletion of Runx2 by Cre-expressing adenovirus in Runx2fl/fl primary osteoblasts impaired osteoblast differentiation and the expression of genes encoding major bone matrix proteins, and osteoclastogenesis was inhibited due to the reduction of Tnfsf11 expression in the osteoblasts. This study demonstrated that Runx2 is required for the expression of the major bone matrix protein genes and Tnfsf11 after commitment into osteoblasts in mice. © 2021 American Society for Bone and Mineral Research (ASBMR).

Phosphate restriction impairs mTORC1 signaling leading to increased bone marrow adipose tissue and decreased bone in growing mice.

Bone marrow stromal cells (BMSCs) are multipotent cells that differentiate into cells of the osteogenic and adipogenic lineage. A striking inverse relationship between bone marrow adipose tissue (BMAT) and bone volume is seen in several conditions, suggesting that differentiation of BMSCs into bone marrow adipocytes diverts cells from the osteogenic lineage, thereby compromising the structural and mechanical properties of bone. Phosphate restriction of growing mice acutely decreases bone formation, blocks osteoblast differentiation and increases BMAT. Studies performed to evaluate the cellular and molecular basis for the effects of acute phosphate restriction demonstrate that it acutely increases 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and inhibits mammalian target of rapamycin complex 1 (mTORC1) signaling in osteoblasts. This is accompanied by decreased expression of Wnt10b in BMSCs. Phosphate restriction also promotes expression of the key adipogenic transcription factors, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer binding protein α (CEBPα), in CXCL12 abundant reticular (CAR) cells, which represent undifferentiated BMSCs and are the main source of BMAT and osteoblasts in the adult murine skeleton. Consistent with this, lineage tracing studies reveal that the BMAT observed in phosphate-restricted mice is of CAR cell origin. To determine whether circumventing the decrease in mTORC1 signaling in maturing osteoblasts attenuates the osteoblast and BMAT phenotype, phosphate-restricted mice with OSX-CreERT2 -mediated haploinsufficiency of the mTORC1 inhibitor, TSC2, were generated. TSC2 haploinsufficiency in preosteoblasts/osteoblasts normalized bone volume and osteoblast number in phosphate-restricted mice and attenuated the increase in BMAT observed. Thus, acute phosphate restriction leads to decreased bone and increases BMAT by impairing mTORC1 signaling in osterix-expressing cells. © 2021 American Society for Bone and Mineral Research (ASBMR).

How to Use Online Tools to Generate New Hypotheses for Mammary Gland Biology Research: A Case Study for Wnt7b

An increasing number of ‘-omics’ datasets, generated by labs all across the world, are becoming available. They contain a wealth of data that are largely unexplored. Not every scientist, however, will have access to the required resources and expertise to analyze such data from scratch. Fortunately, a growing number of investigators is dedicating their time and effort to the development of user friendly, online applications that allow researchers to use and investigate these datasets. Here, we will illustrate the usefulness of such an approach. Using regulation of Wnt7b expression as an example, we will highlight a selection of accessible tools and resources that are available to researchers in the area of mammary gland biology. We show how they can be used for in silico analyses of gene regulatory mechanisms, resulting in new hypotheses and providing leads for experimental follow up. We also call out to the mammary gland community to join forces in a coordinated effort to generate and share additional tissue-specific ‘-omics’ datasets and thereby expand the in silico toolbox.

Increased H3K4me3 methylation and decreased miR-7113-5p expression lead to enhanced Wnt/\u03b2-catenin signaling in immune cells from PTSD patients leading to inflammatory phenotype

BackgroundPosttraumatic stress disorder (PTSD) is a psychiatric disorder accompanied by chronic peripheral inflammation. What triggers inflammation in PTSD is currently unclear. In the present study, we identified potential defects in signaling pathways in peripheral blood mononuclear cells (PBMCs) from individuals with PTSD.MethodsRNAseq (5 samples each for controls and PTSD), ChIPseq (5 samples each) and miRNA array (6 samples each) were used in combination with bioinformatics tools to identify dysregulated genes in PBMCs. Real time qRT-PCR (24 samples each) and in vitro assays were employed to validate our primary findings and hypothesis.ResultsBy RNA-seq analysis of PBMCs, we found that Wnt signaling pathway was upregulated in PTSD when compared to normal controls. Specifically, we found increased expression of WNT10B in the PTSD group when compared to controls. Our findings were confirmed using NCBI’s GEO database involving a larger sample size. Additionally, in vitro activation studies revealed that activated but not naïve PBMCs from control individuals expressed more IFNγ in the presence of recombinant WNT10B suggesting that Wnt signaling played a crucial role in exacerbating inflammation. Next, we investigated the mechanism of induction of WNT10B and found that increased expression of WNT10B may result from epigenetic modulation involving downregulation of hsa-miR-7113-5p which targeted WNT10B. Furthermore, we also observed that WNT10B overexpression was linked to higher expression of H3K4me3 histone modification around the promotor of WNT10B. Additionally, knockdown of histone demethylase specific to H3K4me3, using siRNA, led to increased expression of WNT10B providing conclusive evidence that H3K4me3 indeed controlled WNT10B expression.ConclusionsIn summary, our data demonstrate for the first time that Wnt signaling pathway is upregulated in PBMCs of PTSD patients resulting from epigenetic changes involving microRNA dysregulation and histone modifications, which in turn may promote the inflammatory phenotype in such cells.

Stratified layer analysis reveals intrinsic leptin stimulates cryptal mesenchymal cells for controlling mucosal inflammation

Mesenchymal cells in the crypt play indispensable roles in the maintenance of intestinal epithelial homeostasis through their contribution to the preservation of stem cells. However, the acquisition properties of the production of stem cell niche factors by the mesenchymal cells have not been well elucidated, due to technical limitations regarding the isolation and subsequent molecular and cellular analyses of cryptal mesenchymal cells. To evaluate the function of mesenchymal cells located at the large intestinal crypt, we established a novel method through which cells are harvested according to the histologic layers of mouse colon, and we compared cellular properties between microenvironmental niches, the luminal mucosa and crypts. The gene expression pattern in the cryptal mesenchymal cells showed that receptors of the hormone/cytokine leptin were highly expressed, and we found a decrease in Wnt2b expression under conditions of leptin receptor deficiency, which also induced a delay in cryptal epithelial proliferation. Our novel stratified layer isolation strategies thus revealed new microenvironmental characteristics of colonic mesenchymal cells, including the intrinsic involvement of leptin in the control of mucosal homeostasis.

Hair growth promotion by Necrostatin-1s

Necrostatins (Necs) have been developed as a receptor-interacting protein kinase 1 (RIPK1) inhibitor, thus inhibiting necroptosis. In this current study, we have investigated the possible involvement of necroptosis in the hair cycle regulation and further examined its underlying molecular mechanisms. Diverse RIPK1/3 inhibitors and siRNA were tested in the human outer-root sheath (ORS) cells and animal models. The expression and hair cycle-dependent expression of RIPK 1, respectively, were investigated in the hair follicles (HF) of human, pig, and the mouse. Resulting from the experiment, Nec-1s was most effective in the hair growth promotion among several inhibitors. Nec-1s induced the ORS cell proliferation and migration, and increased the HF length in mouse and pig organ cultures. In addition, it accelerated the telogen-to-anagen transition and elongated the anagen period in the mouse model. Both apoptosis and necroptosis were detected in hair cycle. RIPK1 and RIPK3 were highly expressed in ORS cells during the hair regression period. Nec-1s upregulated the mRNA expression of Wnt3a and Wnt5b, and the activity of β-catenin. Collectively, Nec-1s promotes hair growth through inhibiting necroptosis and activating the Wnt/β-catenin pathway. Necroptosis is involved in hair cycle regression, and Nec-1s is a promising target for hair-loss treatment.

Canonical Wnts Mediate CD8+ T Cell Noncytolytic Anti-HIV-1 Activity and Correlate with HIV-1 Clinical Status.

CD8+ T cells do not rely solely on cytotoxic functions for significant HIV control. Moreover, the noncytotoxic CD8+ T cell antiviral response is a primary mediator of natural HIV control such as that seen in HIV elite controllers and long-term nonprogressors that does not require combined antiretroviral therapy. In this study, we investigated the biological factors contributing to the noncytotoxic control of HIV replication mediated by primary human CD8+ T cells. We report that canonical Wnt signaling inhibits HIV transcription in an MHC-independent, noncytotoxic manner and that mediators of this pathway correlate with HIV controller clinical status. We show that CD8+ T cells express all 19 Wnts and CD8+ T cell-conditioned medium (CM) induced canonical Wnt signaling in infected recipient cells while simultaneously inhibiting HIV transcription. Antagonizing canonical Wnt activity in CD8+ T cell CM resulted in increased HIV transcription in infected cells. Further, Wnt2b expression was upregulated in HIV controllers versus viremic patients, and in vitro depletion of Wnt2b and/or Wnt9b from CD8+ CM reversed HIV inhibitory activity. Finally, plasma concentration of Dkk-1, an antagonist of canonical Wnt signaling, was higher in viremic patients with lower CD4 counts. This study demonstrates that canonical Wnt signaling inhibits HIV and significantly correlates with HIV controller status.

The epigenetic treatment remodel genome-wide histone H4 hyper-acetylation patterns and affect signaling pathways in acute promyelocytic leukemia cells

Although majority of acute promyelocytic leukemia (APL) patients achieve complete remission after the standard treatment, 5–10% of patients are shown to relapse or develop resistance to treatment. In such cases, medications that target epigenetic processes could become an appealing supplementary approach. In this study, we tested the anti-leukemic activity of histone deacetylase inhibitor Belinostat (PXD101) and histone methyltransferase inhibitor 3-Deazaneplanocin A combined with all-trans retinoic acid in APL cells NB4, promyelocytes resembling HL-60 cells and APL patients' cells. After HL-60 and NB4 cell treatment, ChIP-sequencing was performed using antibodies against hyper-acetylated histone H4. Hyper-acetylated histone H4 distribution peaks were compared in treated vs untreated HL-60 and NB4 cells. Results demonstrated that in treated HL-60 cells, the majority of peaks were distributed within the regions of proximal promoters, whereas in treated NB4 cells, hyper-acetylated histone H4 peaks were mainly localized in gene body regions. Further ChIP-seq data analysis revealed the changes in histone H4 hyper-acetylation in promoter/gene body regions of genes involved in cancer signaling pathways. In addition, quantitative gene expression analysis proved changes in various cellular pathways important for carcinogenesis. Epigenetic treatment down-regulated the expression of MTOR, LAMTOR1, WNT2B, VEGFR3, FGF2, FGFR1, TGFA, TGFB1, TGFBR1, PDGFA, PDGFRA and PDGFRB genes in NB4, HL-60 and APL patients’ cells. In addition, effect of epigenetic treatment on protein expression of aforementioned signaling pathways was confirmed with mass spectrometry analysis. Taken together, these results provide supplementary insights into molecular changes that occur during epigenetic therapy application in in vitro promyelocytic leukemia cell model.

Epithelial WNT2B and Desert Hedgehog Are Necessary for Human Colonoid Regeneration after Bacterial Cytotoxin Injury.

SummaryIntestinal regeneration and crypt hyperplasia after radiation or pathogen injury relies on Wnt signaling to stimulate stem cell proliferation. Mesenchymal Wnts are essential for homeostasis and regeneration in mice, but the role of epithelial Wnts remains largely uncharacterized. Using the enterohemorrhagic E. coli-secreted cytotoxin EspP to induce injury to human colonoids, we evaluated a simplified, epithelial regeneration model that lacks mesenchymal Wnts. Here, we demonstrate that epithelial-produced WNT2B is upregulated following injury and essential for regeneration. Hedgehog signaling, specifically activation via the ligand Desert Hedgehog (DHH), but not Indian or Sonic Hedgehog, is another driver of regeneration and modulates WNT2B expression. These findings highlight the importance of epithelial WNT2B and DHH in regulating human colonic regeneration after injury.

Wnt5b/Ryk-mediated membrane trafficking of P2X3 receptors contributes to bone cancer pain

Wnt5b, a member of Wnt family, plays multiple roles in tumor progression and metastasis. However, whether Wnt5b contributes to the sensitization of dorsal root ganglia (DRG) neurons and pathogenesis of bone cancer pain still remains unclear. Here, we found that the protein expression of Wnt5b and its atypical tyrosine protein kinase receptor Ryk was upregulated in ipsilateral DRGs in tumor-bearing mice. Application of Wnt5b evoked an increased discharge frequency in isolated DRG neurons and pain hypersensitivity in naïve mice which were almost completely prevented by anti-Ryk antibody. Moreover, intrathecal injection of anti-Ryk antibody to tumor-bearing mice significantly inhibited bone cancer-induced mechanic allodynia and thermal hyperalgesia. Subsequently, we also demonstrated that application of Wnt5b to cultured DRG neurons could enhance membrane P2X3 receptors and α,β-meATP-induced currents. Intrathecal injection of calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 or P2X3 receptors antagonist A317491 almost completely abolished Wnt5b-induced mechanical allodynia and thermal hyperalgesia in mice. Meanwhile, pretreatment with anti-Ryk antibody or CaMKII inhibitor KN93 can attenuate bone-cancer induced the upregulation of P2X3 membrane protein as well as pain hypersensitivity. These findings suggested that Wnt5b/Ryk promoted the trafficking of P2X3 receptors to the membrane via the activation of CaMKII in primary sensory neurons, resulting in peripheral sensitization and bone cancer-induced pain. Our results may offer a potential therapeutic strategy for bone cancer pain.

Molecular Pathways Involved in Promoting Activity of Timosaponin BII on Hair Growth in C57BL/6 Mice.

Hair loss is a common disease in dermatology, while the approved drugs may have unpredictable side effects. In this study, the effect of timosaponin BII extracted from Anemarrhena asphodeloides on hair growth of C57BL/6 mice was investigated by measuring the hair follicle morphology, hair growth length and area in C57BL/6 male mice, and the immunohistochemical analysis of β-catenin, Wnt3a, and Wnt10b in the dorsal skins of mice after topical application with minoxidil and timosaponin BII for 15 days. The decrease in skin brightness, the increase in the regrowing area of hair and hair follicles numbers, and the improvement of hair follicle morphology in the group applied with 0.5% timosaponin BII indicated an induction of the anagen phase in telogenic mice skin, which were comparative to the 2% minoxidil treatment. The immunohistochemical analysis detected an increase in the expression of β-catenin and Wnt10b, supporting the theory of the activation of the β-catenin/Wnt pathway was one of the pathways that are related to anagen phase induction. Anemarrhena asphodeloides is a herb commonly used for metabolic disorders in China. The present study is the first to show that the timosaponin BII, which is present at a high concentration in A. asphodeloides, promotes hair growth in C57BL/6 male mice. The results indicate that timosaponin BII may be a potential promoting agent for hair growth.

The Role of Wnt/β-Catenin Pathway Mediators in Aortic Valve Stenosis.

Aortic valve stenosis (AVS) is a prevailing and life-threatening cardiovascular disease in adults over 75 years of age. However, the molecular mechanisms governing the pathogenesis of AVS are yet to be fully unraveled. With accumulating evidence that Wnt signaling plays a key role in the development of AVS, the involvement of Wnt molecules has become an integral study target in AVS pathogenesis. Thus, we hypothesized that the Wnt/β-catenin pathway mediators, SFRP2, DVL2, GSK3β and β-catenin are dysregulated in patients with AVS. Using immunohistochemistry, Real-Time qPCR and Western blotting, we investigated the presence of SFRP2, GSK-3β, DVL2, and β-catenin in normal and stenotic human aortic valves. Markedly higher mRNA and protein expression of GSK-3β, DVL2, β-catenin and SFRP2 were found in stenotic aortic valves. This was further corroborated by observation of their abundant immunostaining, which displayed strong immunoreactivity in diseased aortic valves. Proteomic analyses of selective GSK3b inhibition in calcifying human aortic valve interstitial cells (HAVICs) revealed enrichment of proteins involved organophosphate metabolism, while reducing the activation of pathogenic biomolecular processes. Lastly, use of the potent calcification inhibitor, Fetuin A, in calcifying HAVICs significantly reduced the expression of Wnt signaling genes Wnt3a, Wnt5a, Wnt5b, and Wnt11. The current findings of altered expression of canonical Wnt signaling in AVS suggest a possible role for regulatory Wnts in AVS. Hence, future studies focused on targeting these molecules are warranted to underline their role in the pathogenesis of the disease.

Calcitonin Induces Bone Formation by Increasing Expression of Wnt10b in Osteoclasts in Ovariectomy-Induced Osteoporotic Rats.

Calcitonin is a small peptide hormone secreted from the parafollicular cells of the thyroid gland in response to an increase in serum calcium. The inhibition of osteoclastic resorption is the main mechanism by which calcitonin quickly decreases circulating calcium levels. Although calcitonin pharmacologically acts on osteoclasts to prevent bone resorption, the results of studies on genetically modified animals have shown that the physiological effect of calcitonin is in the inhibition of osteoblastic bone formation. Because the calcitonin receptor is only expressed in osteoclasts, the effect of calcitonin on osteoblasts maybe indirect and mediated via osteoclasts. Wnt ligands are involved in various aspects of skeletal biology, including bone remodeling and endochondral bone formation. Wnt10b has recently been recognized as a clastokine, and is potentially a therapeutic target for treating bone disorders. However, the extent to which Wnt signaling is involved in bone physiology and disease is not yet fully understood. We hypothesize that calcitonin indirectly increases osteoblastic bone formation by inducing Wnt10b expression in osteoclasts. Micro-CT analysis revealed reduced bone loss in calcitonin-treated ovariectomized rats. The serum of animals treated with calcitonin had decreased TRAP5b and CTX-1 but increased osteocalcin, P1NP, and Wnt10b. Immunohistochemistry staining showed that the level of Wnt10b in the femur was increased in calcitonin-treated groups as compared with control groups. Hematopoietic mononuclear cells were separated from rat femur and tibia bone marrow, and were induced into osteoclasts following treatment with M-CSF and RANKL. In these cells, immunoconfocal microscopy and Western blot analysis showed that calcitonin induced an increase in Wnt10b expression. In a culture of osteoblasts isolated from neonatal rat calvariae, the calcitonin-treated osteoclast supernatant showed an increase in mineralization, as indicated by ALP and alizarin red staining. Taken together, these results indicate that calcitonin induces bone formation by increasing the expression of Wnt10b in osteoclasts in ovariectomy-induced osteoporotic rats. The present study provides in-depth information about the effects of calcitonin on bone remodeling and will thus help in the development of future potential therapeutic strategies for postmenopausal osteoporosis.

Platelet factor 4 inhibits human hair follicle growth and promotes androgen receptor expression in human dermal papilla cells.

Platelet-rich plasma (PRP) has been reported recently as a potential therapeutic approach for alopecia, such as androgenetic alopecia, but the exact mechanisms and effects of specific components of this recipe remain largely unknown. In this study, we identified that platelet factor 4 (PF4), a component of PRP, significantly suppressed human hair follicle growth and restrained the proliferation of human dermal papilla cells (hDPCs). Furthermore, our results showed that PF4 upregulated androgen receptor (AR) in human dermal papilla cells in vitro and via hair follicle organ culture. Among the hair growth-promoting and DP-signature genes investigated, PF4 decreased the expression of Wnt5a, Wnt10b, LEF1, HEY1 and IGF-1, and increased DKK1 expression, but did not affect BMP2 and BMP4 expression. Collectively, Our data demonstrate that PF4 suppresses human hair follicle growth possibly via upregulating androgen receptor signaling and modulating hair growth-associated genes, which provides thought-provoking insights into the application and optimization of PRP in treating hair loss.

Gene expression in regenerating and scarring tails of lizard evidences three main key genes (wnt2b, egfl6, and arhgap28) activated during the regulated process of tail regeneration.

We have analyzed the expression of key genes orchestrating tail regeneration in lizard under normal and scarring conditions after cauterization. At 1-day post-cauterization (1dpc), the injured blastema contains degenerating epithelial and mesenchymal cells, numerous mast cells, and immune cells. At 3 and 7dpc, a stratified wound epidermis is forming while fibrocytes give rise to a scarring connective tissue. Oncogenes such as wnt2b, egfl6, wnt6, and mycn and the tumor suppressor arhgap28 are much more expressed than other oncogenes (hmga2, rhov, fgf8, fgfr4, tert, shh) and tumor suppressors (apcdd1, p63, rb, fat2, bcl11b) in the normal blastema and at 7dpc. Blastemas at 3dpc feature the lowest upregulation of most genes, likely derived from damage after cauterization. Immunomodulator genes nfatc4 and lef1 are more expressed at 7dpc than in normal blastema and 3dpc suggesting the induction of immune response favoring scarring. Balanced over-expression of oncogenes, tumor suppressor genes, and immune modulator genes determines regulation of cell proliferation (anti-oncogenic), of movement (anti-metastatic), and immunosuppression in the normal blastema. Significant higher expression of oncogenes wnt2b and egfl6 in normal blastema and higher expression of the tumor suppressor arhgap28 in the 7dpc blastema indicate that they are among the key/master genes that determine the regulated regeneration of the tail.

LncRNA-599547 contributes the inductive property of dermal papilla cells in cashmere goat through miR-15b-5p/Wnt10b axis

The lncRNA-599547 (619-nt in length) is identified in secondary hair follicle (SHF) of cashmere goat, but its functional roles in regulating the inductive property of dermal papilla cells (DPCs) remains unknown. We found that lncRNA-599547 had significantly higher expression in dermal papilla of cashmere goat SHF at anagen than its counterpart at telogen. The overexpression of lncRNA-599547 led to a significant increase of ALP and LEF1 expression in DPCs (p < 0.05), whereas, the siLncRNA-1 mediated silencing of lncRNA-599547 significantly down-regulated the expression of ALP and LEF1 in DPCs (p < 0.05). Based on biotin-labeled RNA pull-down assay, we found that lncRNA-599547 directly interacted with chi-miR-15b-5p in DPCs. Based on both overexpression and silencing analysis of lncRNA-599547, our results indicate that lncRNA-599547 promotes the expression of Wnt10b in DPCs but without modulating its promoter methylation level. Using the mRNA-3′UTR fragments of goat Wnt10b containing the predicted binding sites of chi-miR-15b-5p in Dual-luciferase Reporter Assays, we show that lncRNA-599547 modulates the expression of Wnt10b at the chi-miR-15b-5p mediated post-transcriptional level. Taken together, our results indicate that lncRNA-599547 sponges miR-15b-5p to positively regulate the expression of Wnt10 gene, and thereby contributes the inductive property of DPCs in cashmere goat.

Extracellular vesicles engineered with valency-controlled DNA nanostructures deliver CRISPR/Cas9 system for gene therapy.

Extracellular vesicles (EVs) hold great promise for transporting CRISPR–Cas9 RNA-guided endonucleases (RNP) throughout the body. However, the cell-selective delivery of EVs is still a challenge. Here, we designed valency-controlled tetrahedral DNA nanostructures (TDNs) conjugated with DNA aptamer, and loaded the valency-controlled TDNs on EV surface via cholesterol anchoring for specific cell targeting. The targeting efficacy of different ratios of aptamer/cholesterol from 1:3 to 3:1 in TDNs on decorating EVs was investigated. TDNs with one aptamer and three cholesterol anchors (TDN1) efficiently facilitated the tumor-specific accumulation of the EVs in cultured HepG2 cells and human primary liver cancer-derived organoids, as well as xenograft tumor models. The intracellular delivery of RNP by TDN1-EVs successfully realized its subsequent genome editing, leading to the downregulation of GFP or WNT10B in specific cells. This system was ultimately applied to reduce the protein expression of WNT10B, which presented remarkable tumor growth inhibition in vitro, ex vivo and in vivo, and could be extended to other therapeutic targets. The present study provides a platform for the directional display of aptamer on surface labeling and the EVs-based Cas9 delivery, which provides a meaningful idea for future cell-selective gene editing.

Abstract 498: Targeting EZH2/DAB2IP/C-JUN axis in ovarian cancer stem cells

Abstract Ovarian cancer (OC) is the leading cause of death from gynecologic malignancies. The persistence of quiescent OC stem cells (OCSCs) not eliminated by chemotherapy plays a key role in tumor relapse and metastasis. In the current study, we tested the hypothesis that targeting DAB2IP will inhibit OCSCs and prevent disease recurrence. Subpopulations of CSC and non-CSC were isolated from OC cell lines by fluorescence-activated cell sorting (FACS) based on aldehyde dehydrogenase (ALDH) activity, a consistent CSC marker. Expression of DAB2IP in ALDH(+) cells was lower (P&amp;lt;0.05) compared to non-CSC ALDH(-) cells. Chromatin immunoprecipitation (ChIP) analysis demonstrated enrichment (P&amp;lt;0.05) of H3K27me3 at DAB2IP promoter loci in CSC compared to non-CSC. Furthermore, inhibiting EZH2 decreased H3K27me3 and increased (P&amp;lt;0.05) DAB2IP expression in OC cells, demonstrating that DAB2IP downregulation in CSC was due to EZH2. Knocking out DAB2IP using CRISPR/Cas9 system in OC cell lines upregulated (P&amp;lt;0.05) expression of stemness-related genes. Enforced overexpression of DAB2IP decreased (P&amp;lt;0.05) the number of ALDH(+) cells and inhibited (P&amp;lt;0.05) spheroid formation. Transcriptome analysis revealed that DAB2IP overexpression significantly (FDR &amp;lt; 0.05, fold change &amp;gt; 2) altered expression of 449 genes, including down-regulation of ALDH1A1, LGR5, PROM1 and TWIST1, markers strongly associated with CSC phenotypes. Ingenuity Pathway Analysis for upstream regulators of differentially expressed genes revealed WNT-signaling as a dominant pathway mediating the anti-OCSC effects of DAB2IP. Based on RNA-seq analysis, WNT5B expression decreased (P&amp;lt;0.05) by 3.83 fold, indicating that DAB2IP may negatively regulate Wnt signaling pathway by repressing WNT5B. Furthermore, WNT5B recombinant protein significantly increased (P&amp;lt;0.05) the OCSC population, and Reverse Phase Protein Array (RPPA) analysis demonstrated activation of non-canonical WNT signaling via C-JUN as a possible downstream target of WNT5B. Collectively, our data revealed that EZH2-regulated DAB2IP is a critical tumor suppressor regulating cancer stemness-related phenotypes, via inhibiting WNT5B-induced C-JUN activation, which could be abolished by RAC1 inhibitor. Based on these novel findings, we are testing novel combination treatment strategies targeting OCSCs, with the goal of inhibiting tumor relapse and overcoming chemoresistance. Importantly, we showed that co-administration of EZH2 inhibitor and RAC1 inhibitor inhibits OCSC survival in vitro, impedes tumor growth and augments chemoresponse in vivo. These findings establish that targeting EZH2/DAB2IP/C-JUN axis could be considered as adjuvant therapy for platinum-based chemotherapy to prevent OC recurrence. Citation Format: Xingyue Zong, Ali Ozes, Weini Wang, Fang Fang, Kenneth P. Nephew. Targeting EZH2/DAB2IP/C-JUN axis in ovarian cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 498.

Contrary Roles of Wnt/β-Catenin Signaling in BMP9-Induced Osteogenic and Adipogenic Differentiation of 3T3-L1 Preadipocytes.

Our previous study revealed that 3T3-L1 preadipocytes can differentiate to either osteoblasts or adipocytes in response to bone morphogenic protein 9 (BMP9). In the present study, we try to further investigate whether the Wnt/β-catenin signaling plays a crucial role in this process. It was found that BMP9 effectively activated the Wnt/β-catenin signaling, and induced the expression levels of certain canonical Wnt ligands and their receptors in preadipocytes. Exogenous expression of β-catenin, Wnt1, Wnt3a, and Wnt10b potentiated BMP9-induced alkaline phosphatase (ALP) activity, while β-catenin knockdown or Dickkopf 1 (Dkk1) diminished BMP9-induced ALP activity. Moreover, it was demonstrated that β-catenin overexpression promoted BMP9-induced mineralization, and increased the expression levels of late osteogenic markers osteopontin and osteocalcin. Furthermore, β-catenin inhibited BMP9-induced lipid accumulation and the adipogenic marker adipocyte fatty acid binding protein (aP2). The cell-implantation assay results identified that β-catenin not only augmented BMP9-induced ectopic bone formation, but also blocked adipogenesis in vivo. Mechanistically, it was found that β-catenin and BMP9 synergistically stimulated the osteogenic transcription factors runt-related transcription factor 2 (Runx2) and Osterix (OSX). However, BMP9-induced adipogenic transcription factors, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT enhancer-binding protein α (C/EBPα), were inhibited by β-catenin. Therefore, these findings suggested that the Wnt/β-catenin signaling, potentially via the modulation of osteogenic and adipogenic transcriptional factors, exerts an opposite effect on BMP9-induced osteogenic and adipogenic differentiation in preadipocytes.