The long noncoding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1) is a critical regulator for the development and progression of multiple tumors. Yet, the role of SNHG1 in acute myeloid leukemia (AML) is unknown. The present study demonstrated that SNHG1 expression was upregulated in AML. SNHG1 silencing markedly repressed AML cell growth, whereas SNHG1 overexpression had the opposite effect. MicroRNA-489-3p (miR-489-3p) was identified as a SNHG1-targeting miRNA. SNHG1 knockdown increased miR-489-3p expression. Low expression of miR-489-3p was correlated with high expression of SNHG1 in AML tissues. miR-489-3p overexpression restricted AML cell growth, and SRY-related high-mobility-group box <em>12</em> (SOX<em>12</em>) was identified as a miR-489-3p-targeting gene. SNHG1 inhibition or miR-489-3p overexpression inactivated <em>Wnt</em>/β-catenin signaling through downregulation of SOX<em>12</em>. SOX<em>12</em> overexpression partially reversed the SNHG1 knockdown- or miR-489-3p overexpression-mediated effects. Taken together, these data indicate that suppression of SNHG1 downregulates AML cell growth by inactivating SOX<em>12</em>/<em>Wnt</em>/β-catenin signaling via upregulating miR-489-3p.

<strong class="sub-title"> Keywords: </strong> SNHG1; SOX<em>12</em>; acute myeloid leukemia; miR-489-3p.

Mechanical ventilation may cause ventilator‑induced lung injury (VILI). Canonical <em>Wnt</em> signaling has been reported to serve an important role in the pathogenesis of VILI. Bioinformatics analysis revealed that canonical and non‑canonical <em>Wnt</em> signaling pathways were activated in VILI. However, the role of non‑canonical <em>Wnt</em> signaling in the pathogenesis of VILI remains unclear. The present study aimed to analyze the potential role of non‑canonical <em>Wnt</em> signaling in VILI pathogenesis. Lung injury was assessed via Evans blue albumin permeability and histological scoring, as well as by inflammatory cytokine expression and total protein concentration in bronchoalveolar lavage fluid. The relative protein expression of canonical and non‑canonical <em>Wnt</em> signaling pathway components were examined via western blotting and immunohistochemistry. The results demonstrated that 6 h of mechanical ventilation at low tidal volume (LTV; 6 ml/kg) or moderate tidal volume (MTV; <em>12</em> ml/kg) induced lung injury in sensitive A/J mice. Ventilation with MTV increased the protein levels of <em>Wnt</em>‑induced secreted protein 1 (WISP1), Rho‑associated protein kinase 1 (ROCK1), phosphorylated (p)‑Ras homolog gene family, member A and p‑C‑Jun N‑terminal kinase (JNK). Inhibition of ROCK1 by Y27632 and JNK by SP600<em>12</em>5 attenuated MTV‑induced lung injury and decreased the expression of proteins involved in non‑canonical <em>Wnt</em> signaling, including WISP1. In conclusion, non‑canonical <em>Wnt</em> signaling participates in VILI by modulating WISP1 expression, which has been previously noted as critical for VILI development. Therefore, the non‑canonical <em>Wnt</em> signaling pathway may provide a preventive and therapeutic target in VILI.

Background: This paper aims to identify alternative RNA splicing landscape and its prognostic value in adrenocortical carcinoma.

Methods: The alternative splicing events data with corresponding clinical information data of 79 ACC patients were obtained from the Cancer Genome Atlas and SpliceSeq package. Prognosis-associated AS events by using univariate Cox regression analysis were selected. Gene functional enrichment analysis demonstrated the potential pathways enriched by survival-associated AS. Prognosis-related splicing events were submitted to develop moderate predictors using Lasso regression model.

<strong class="sub-title"> Results: </strong> One thousand five survival-associated alternative splicing events were identified. The prognostic genes included <i>ATXN2L, MEIS1</i>, <i>IKBKB</i>, <i>COX4I1</i>. Functional enrichment analysis suggested that prognostic splicing events are associated with <em>Wnt</em> signaling pathway. A prediction model including <em>12</em> alternative splicing events was constructed by Lasso regression using train set. ROC analysis showed good performance of the prediction model in test set. Then, a nomogram integrating the clinical-pathological factors and riskscore was constructed for predicting 1- and 3-year survival rate.

Conclusion: Our data provide a comprehensive bioinformatics analysis of AS events in ACC, providing biomarkers for disease progression and a potentially rich source of novel therapeutic targets.

Keywords: AUC; adrenocortical carcinoma; alternative splicing; bioinformatics; prognostic model.

Endometriosis is characterized by the presence of functional endometrial tissue in other pelvic organs. This gynecologic problem occurs in 35-50% of women with pain and infertility. Endometriotic cells share some characteristics such as proliferation, migration, and invasion with tumor cells. Pyrvinium pamoate, an anthelmintic drug approved by the Food and Drug Administration, could inhibit the <em>Wnt</em>/β-catenin signaling pathway and its anticancer effects were examined by several researchers. In this study, <em>12</em> ectopic and eutopic endometrial biopsies from females with ovarian endometrioma and <em>12</em> endometrial biopsies from nonendometriotic females were obtained. Ectopic (EESCs), eutopic (EuESCs), and control (CESCs) endometrial stromal cells were isolated. Then, the effect of pyrvinium pamoate on the proliferation and invasiveness of in vitro cultured cells was evaluated. The proliferation of CESCs, EuESCs, and EESCs was significantly decreased after treatment with pyrvinium pamoate. In addition, treatment with pyrvinium pamoate significantly inhibited the invasiveness of CESCs, EuESCs, and EESCs compared to nontreated groups. The results of the present research showed that pyrvinium pamoate inhibits the proliferation and invasion of human endometriotic stromal cells in vitro, further investigations on the therapeutic potential of this compound in endometriosis are required.

Ovarian cancer is a major cause of mortality in women. However, the molecular events underlying the pathogenesis of the disease are yet to be fully elucidated. In the present study, an integrated bioinformatics analysis was performed to identify core genes involved in serous epithelial ovarian cancer. A total of three expression datasets were downloaded from the Gene Expression Omnibus database, and included 46 serous epithelial ovarian cancer and 30 ovarian surface epithelium samples. The three datasets were merged, and batch normalization was performed. The normalized merged data were subsequently analyzed for differentially expressed genes (DEGs). In total, 2,2<em>12</em> DEGs were identified, including 1,300 upregulated and 9<em>12</em> downregulated genes. Gene Ontology analysis revealed that these DEGs were primarily involved in 'regulation of cell cycle', 'mitosis', 'DNA packaging' and 'nucleosome assembly'. The main cellular components included 'extracellular region part', 'chromosome', 'extracellular matrix' and 'condensed chromosome kinetochore', whereas the molecular functions included 'Calcium ion binding', 'polysaccharide binding', 'enzyme inhibitor activity', 'growth factor activity', 'cyclin-dependent protein kinase regulator activity', 'microtubule motor activity' and '<em>Wnt</em> receptor activity'. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that these DEGs were predominantly involved in '<em>Wnt</em> signaling pathway', 'pathways in cancer', 'PI3K-Akt signaling pathway', 'cell cycle', 'ECM-receptor interaction', 'p53 signaling pathway' and 'focal adhesion'. The 20 most significant DEGs were identified from the protein-protein interaction network, and Oncomine analysis of these core genes revealed that 13 were upregulated and two were downregulated in serous epithelial ovarian cancer. Survival analysis revealed that cyclin B1, polo like kinase 1, G protein subunit γ transducin 1 and G protein subunit γ <em>12</em> are key molecules that may be involved in the prognosis of serous epithelial ovarian cancer. These core genes may provide novel treatment targets, although their roles in the carcinogenesis and prognosis of serous epithelial ovarian cancer require further study.

Genotype plays an important role in influencing bone phenotypes, such as bone mineral density, but the role of genotype in determining responses of bone to exercise has yet to be elucidated.

To determine whether 10 SNPs associated with genes in the vicinity of P2X7R, RANK/RANKL/OPG and <em>Wnt</em> Signalling Pathways are associated with bone phenotypes in elite academy footballers (Soccer players) and to determine whether these genotypes are associated with training induced changes in bone. Design, participants, and methods: 99 elite academy footballers volunteered to participate. Peripheral computed tomography of the tibia (4%, 14%, 38% and 66% sites) was performed immediately before and <em>12</em> weeks after an increase in football training volume. Genotypes were determined using proprietary fluorescence-based competitive allele-specific PCR assays.

No significant genotype by time interactions were shown for any of the SNPs analysed (P>> .05). A main effect of genotype was shown. SOST SNP rs1877632 (trabecular density), P2X7R SNPs rs1718119 (cortical thickness and CSA), rs3751143 (SSI, CSA, cortical CSA and periosteal circumference) RANK/RANKL/OPG SNPs rs9594738 (periosteal circumference), rs1021188 (cortical thickness and CSA) and rs9594759 (cortical density) were associated with bone phenotypes (P < .05).

No association was shown between P2X7R, RANK/RANKL/OPG and <em>Wnt</em> Signalling SNPs and a change in bone phenotypes following <em>12</em> weeks of increased training volume in elite academy footballers. However, SNPs were associated with bone phenotypes pre training. These data highlight the complexity of the interaction between SNPs in the vicinity of the RANK/RANKL/OPG, P2X7R and <em>Wnt</em> metabolic regulatory pathways and bone phenotypes in elite academy footballers.

Osterix is a critical transcription factor of mesenchymal stem cell fate, where its loss or loss of <em>Wnt</em> signaling diverts differentiation to a chondrocytic lineage. Intervertebral disc (IVD) degeneration activates the differentiation of prehypertrophic chondrocyte-like cells and inactivates <em>Wnt</em> signaling, but its interactive role with osterix is unclear. First, compared to young-adult (5 mo), mechanical compression of old (18 mo) IVD induced greater IVD degeneration. Aging (5 vs <em>12</em> mo) and/or compression reduced the transcription of osterix and notochordal marker T by 40-75%. Compression elevated the transcription of hypertrophic chondrocyte marker MMP13 and pre-osterix transcription factor RUNX2, but less so in <em>12</em> mo IVD. Next, using an Ai9/td reporter and immunohistochemical staining, annulus fibrosus and nucleus pulposus cells of young-adult IVD expressed osterix, but aging and compression reduced its expression. Lastly, in vivo LRP5-deficiency in osterix-expressing cells inactivated <em>Wnt</em> signaling in the nucleus pulposus by 95%, degenerated the IVD to levels similar to aging and compression, reduced the biomechanical properties by 45-70%, and reduced the transcription of osterix, notochordal markers and chondrocytic markers by 60-80%. Overall, these data indicate that age-related inactivation of <em>Wnt</em> signaling in osterix-expressing cells may limit regeneration by depleting the progenitors and attenuating the expansion of chondrocyte-like cells.

Mechanical load application promotes bone formation, while reduced load leads to bone loss. However, the underlying mechanisms that regulate new bone formation are not fully understood. <em>Wnt</em>/β‑catenin signaling has an important role in bone formation, bone growth and remodeling. The aim of the present study was to investigate whether mechanical stimuli regulated bone formation through the <em>Wnt</em>/β‑catenin signaling pathway. Saos‑2 osteoblastic cells were subjected to mechanical strain using a Flexcell strain loading system. The results demonstrated that <em>12</em>% cyclical tensile stress significantly stimulated Saos‑2 cell proliferation, increased the activity of alkaline phosphatase and promoted the formation of mineralized nodules, as determined by MTT and p‑nitrophenyl phosphate assays and Alizarin Red S staining, respectively. Furthermore, western blot analysis demonstrated that, following mechanical strain, increased phosphorylation of glycogen synthase kinase‑3β and nuclear β‑catenin expression was observed in cells, compared with static control culture cells. Results of reporter gene and reverse transcription‑polymerase chain reaction assays also demonstrated that mechanical strain significantly increased T‑cell factor reporter gene activity and the mRNA expression of cyclooxygenase (COX)‑2, cyclin D1, c‑fos and c‑Jun in Saos‑2 cells. Co‑immunoprecipitation analysis revealed that elongation mechanical strain activated <em>Wnt</em>/β‑catenin signaling and reduced β‑catenin and E‑cadherin interaction in Saos‑2 cells. In conclusion, the results of the current study indicate that mechanical strain may have an important role in the proliferation and differentiation of osteoblasts. The disassociation of the β‑catenin/E‑cadherin complex in the osteoblast membrane under stretch loading and the subsequent translocation of β‑catenin into the nucleus may be an intrinsic mechanical signal transduction mechanism.

Sclerostin is a known inhibitor of the <em>Wnt</em> signaling pathway which is involved in osteogenesis and, when inactivated, stimulates bone formation. To our knowledge, this effect has not been studied in the context of distraction osteogenesis (DO). Tibial DO was conducted on a total of 24 wild-type mice, which were then divided into 2 groups-a saline injection group (control) and an anti-sclerostin (Scl-Ab) injection group (treatment). The mice in the treatment group received 100 mg/kg intravenous injections of the antibody weekly until killing. The <em>12</em> mice in each group were subdivided into four time points according to post-osteotomy time of killing-11 days (mid-distraction), 17 days (late distraction), 34 days (mid-consolidation) and 51 days (late consolidation), with 3 mice per subgroup. After killing, the tibia specimens were collected for immunohistochemical analysis. Our results show that the group injected with anti-sclerostin had an earlier peak (day 11) in the distraction phase of the osteogenic molecules involved in the <em>Wnt</em> signaling pathway in comparison to the placebo group. In addition, downregulation of the inhibitors of this pathway was noted in the treatment group when compared with the placebo group. Furthermore, LRP-5 showed a significant increase in expression in the treatment group. Sclerostin inhibition has a significant effect on the DO process through its effect on the <em>Wnt</em> pathway. This effect was evident through the decreased effect of sclerostin on LRP-5 and earlier upregulation of the osteogenic molecules involved in this pathway.

Endothelial progenitor cells (EPCs) play a vital role in endothelial repair following vascular injury by maintaining the integrity of endothelium. As EPCs home to endothelial injury sites, they may communicate with exposed vascular smooth muscle cells (VSMCs), which are subjected to cyclic stretch generated by blood flow. In this study, the synergistic effect of cyclic stretch and communication with neighboring VSMCs on EPC function during vascular repair was investigated. <i>In vivo</i> study revealed that EPCs adhered to the injury site and were contacted to VSMCs in the Sprague-Dawley (SD) rat carotid artery injury model. <i>In vitro</i>, EPCs were cocultured with VSMCs, which were exposed to cyclic stretch at a magnitude of 5% (which mimics physiological stretch) and a constant frequency of 1.25 Hz for <em>12</em> h. The results indicated that stretched VSMCs modulated EPC differentiation into mature endothelial cells (ECs) and promoted angiogenesis. Meanwhile, cyclic stretch upregulated the mRNA expression and secretion level of connective tissue growth factor (CTGF) in VSMCs. Recombinant CTGF (r-CTGF) treatment promoted endothelial differentiation of EPCs and angiogenesis, and increased their protein levels of FZD8 and β-catenin. CTGF knockdown in VSMCs inhibited cyclic stretch-induced EPC differentiation into ECs and attenuated EPC tube formation via modulation of the FZD8/β-catenin signaling pathway. FZD8 knockdown repressed endothelial differentiation of EPCs and their angiogenic activity. <em>Wnt</em> signaling inhibitor decreased the endothelial differentiation and angiogenetic ability of EPCs cocultured with stretched VSMCs. Consistently, an <i>in vivo</i> Matrigel plug assay demonstrated that r-CTGF-treated EPCs exhibited enhanced angiogenesis; similarly, stretched VSMCs also induced cocultured EPC differentiation toward ECs. In a rat vascular injury model, r-CTGF improved EPC reendothelialization capacity. The present results indicate that cyclic stretch induces VSMC-derived CTGF secretion, which, in turn, activates FZD8 and β-catenin to promote both differentiation of cocultured EPCs into the EC lineage and angiogenesis, suggesting that CTGF acts as a key intercellular mediator and a potential therapeutic target for vascular repair.

Keywords: angiogenesis; connective tissue growth factor; cyclic stretch; differentiation; endothelial progenitor cells; vascular smooth muscle cells.

Olfactory neuroblastoma (ONB) is a rare, locally aggressive, malignant neoplasm originating in the olfactory epithelium in the nasal vault. The recurrence rate of ONB remains high and there are no specific treatment guidelines for recurrent/metastatic ONBs. This study retrospectively evaluated 23 ONB samples profiled at Caris Life Sciences (Phoenix, Arizona) using DNA sequencing (Sanger/NGS [Illumina], n = 15) and gene fusions (Archer FusionPlex, n = 6), whole genome RNA microarray (HumanHT-<em>12</em> v4 beadChip, Illumina, n = 4), gene copy number assays (chromogenic and fluorescent in situ hybridization), and immunohistochemistry. Mutations were detected in 63% ONBs including TP53, CTNNB1, EGFR, APC, cKIT, cMET, PDGFRA, CDH1, FH, and SMAD4 genes. Twenty-one genes were over-expressed and 19 genes under-expressed by microarray assay. Some of the upregulated genes included CD24, SCG2, and IGFBP-2. None of the cases harbored copy number variations of EGFR, HER2 and cMET genes, and no gene fusions were identified. Multiple protein biomarkers of potential response or resistance to classic chemotherapy drugs were identified, such as low ERCC1 [cisplatin sensitivity in 10/<em>12</em>], high TOPO1 [irinotecan sensitivity in <em>12</em>/19], high TUBB3 [vincristine resistance in 13/14], and high MRP1 [multidrug resistance in 6/6 cases]. None of the cases (0/10) were positive for PD-L1 in tumor cells. Overexpression of pNTRK was observed in 67% (4/6) of the cases without underlying genetic alterations. Molecular alterations detected in our study (e.g., <em>Wnt</em> and cKIT/PDGFRA pathways) are potentially treatable using novel therapeutic approaches. Identified protein biomarkers of response or resistance to classic chemotherapy could be useful in optimizing existing chemotherapy treatment(s) in ONBs.

We aimed to evaluate whether applying low magnitude vibration (LMV) in early postmenopausal osteoporosis (PMO) suppresses its progression, and to investigate underlying mechanisms. Rats were randomly divided into Sham (Sham-operated), Sham+V, OVX (ovariectomized), OVX+E2 (estradiol benzoate), OVX+V (LMV at <em>12</em>-20 weeks postoperatively), and OVX+Vi (LMV at 1-20 weeks postoperatively) groups. LMV was applied for 20 min once daily for 5 days weekly. V rats were loaded with LMV at <em>12</em>-20 weeks postoperatively. Vi rats were loaded with LMV at 1-20 weeks postoperatively. Estradiol (E2) rats were intramuscularly injected at <em>12</em>-20 weeks postoperatively once daily for 3 days. The bone mineral densities (BMDs), biomechanical properties, and histomorphological parameters of tibiae were analyzed. <i>In vitro</i>, rat bone marrow-derived mesenchymal stem cells (rBMSCs) were subjected to LMV for 30 min daily for 5 days, or 17β-E2 with or without 1-day pretreatment of estrogen receptor (ER) inhibitor ICI 182,780 (ICI). The mRNA and protein expresion were performed. Data showed that LMV increased BMD, bone strength, and bone mass of rats, and the effects of Vi were stronger than those of E2. <i>In vitro</i>, LMV up-regulated the mRNA and protein expressions of Runx2, Osx, Col I, and OCN and down-regulated PPARγ, compared with E2. The effects of both LMV and E2 on rBMSCs were inhibited by ICI. Altogether, LMV in early PMO suppresses its progression, which is associated with osteogenic differentiation of rBMSCs via up-regulation of ERα and activation of the canonical <em>Wnt</em> pathway. LMV may therefore be superior to E2 for the suppression of PMO progression.

Autologous platelet-rich plasma (PRP) exerts positive therapeutic effects on hair thickness and density in patients with pattern hair loss. The aim of our study was to evaluate the efficacy of intra-perifollicular autologous PRP and polydeoxyribonucleotide (PDRN) injections in treating female pattern hair loss (FPHL). Twenty FPHL patients were treated with a single session of PRP injection, followed by <em>12</em> sessions of PDRN intra-perifollicular injection, along the scalp at weekly intervals. Additionally, another 20 FPHL patients were treated with <em>12</em> sessions of PDRN injection only. Meanwhile, one half of the backs of two rabbits was injected with the PRP preparation, while the other half was injected with phosphate buffered saline as a control. Tissue samples from the rabbits were analyzed by real-time polymerase chain reaction and Western blotting. Compared with baseline values, patients treated with PRP and PDRN injections exhibited clinical improvement in mean hair counts (23.2 ± 15.5%; p < 0.001) and mean hair thickness (16.8 ± 10.8%; p < 0.001). In addition, patients treated with the <em>12</em> sessions of intra-perifollicular PDRN injection alone also showed clinical improvement in mean hair counts (17.9 ± 13.2%; p < 0.001) and mean hair thickness (13.5 ± 10.7%; p < 0.001). Comparison analyses between the two groups revealed that combined therapy with PRP and PDRN induces greater improvement in hair thickness than treatment with PDRN therapy alone (p = 0.031), but not in hair counts (p>> 0.05). The pilot animal study revealed significant up-regulation of <em>WNT</em>, platelet-derived growth factor, and fibroblast growth factor expression in rabbit skin treated with the PRP preparation, compared with control skin. In conclusion, intra-perifollicular injections of autologous PRP and/or PDRN generate improvements in hair thickness and density in FPHL patients.

<AbstractText>Keratoconus (KTCN) is a complex eye disorder resulting in loss of visual function. Its development is affected by genetic and environmental components. The aim of this study was to unravel the role of epigenetic factors in KTCN.</AbstractText><AbstractText>To verify if DNA methylation may play a role in KTCN development, reduced representation bisulfite sequencing of five KTCN and five non-KTCN human corneas was performed.</AbstractText><AbstractText>Multiple KTCN-specific differentially methylated regions were detected and many of them overlap previously identified KTCN linkage loci (3p14.3, 5q35.2, 13q32.3, 15q24.1, and 20p13) and chromosome arms that have been linked to KTCN (2q, 4q, 5p, 9p, 14q, and 17q). Reanalysis of the previously described RNA sequencing dataset of 25 KTCN and 25 non-KTCN human corneas revealed that <em>12</em> genes downregulated in KTCN and 6 upregulated genes overlapped or were located in the near vicinity of the identified differentially methylated regions. Particularly interesting were the DNA methylation changes in WNT3 and WNT5A encoding <em>Wnt</em> ligands, as they provide a potential explanation for the <em>Wnt</em> signaling pathway dysregulation observed in KTCN.</AbstractText><AbstractText>We presented the results of data analysis from the first study of DNA methylation changes in human KTCN corneas compared to non-KTCN samples. We were able to identify genomic regions with distinct patterns of DNA hypo- and hypermethylation and link them to previously found KTCN susceptibility loci as well as transcriptomic disruption of <em>Wnt</em> signaling pathway observed in KTCN.</AbstractText>

<i>Purpose</i>: To appraise the curative effect of ginsenoside Rb1 and trigonelline in diabetic nephropathy and to analyze the expression analysis of microRNAs and their target genes during experimental diabetic renal lesions in rats. <i>Methods</i>: Wistar rats were made diabetic by intraperitoneal injection of 55 mg/kg streptozotocin. According to their fasting blood glucose values and initial body weight, diabetic rats were assigned to specific groups and treated as follows: DN group (tap water, n = 10), A group (ginsenoside Rb1, 40 mg/kg, n = 10), B group (trigonelline, 20 mg/kg, n = 10) and the C group (ginsenoside Rb1 and trigonelline, 60 mg/kg, m(ginsenoside Rb1) : m (trigonelline) = 2:1, n = 10). The control group was treated with tap water (n = 10). All rats were gavaged with drugs or tap water once daily for <em>12</em> weeks. <i>Results</i>: Renal dysfunction, oxidative stress, and pathological alteration were significantly alleviated by a combination of ginsenoside Rb1 and trigonellin (C group). Some miRNAs were expressed differentially in Con, DN, A and C groups. Results of immunohistochemistry and western blotting showed that <em>Wnt</em> and β-catenin were expressed differentially in Con, DN, and C groups. <i>Conclusion</i>: Ginsenoside Rb1 and trigonelline could prevent the development of diabetic renal lesions by regulating the expression of miR-3550 and further associating with the <em>Wnt</em>/β-catenin signaling.

Genetic risk factors for osteoporosis, a prevalent disease associated with aging, have been examined in many genome-wide association studies (GWASs). A major challenge is to prioritize transcription-regulatory GWAS-derived variants that are likely to be functional. Given the critical role of epigenetics in gene regulation, we have used an unusual epigenetics-based and transcription-based approach to identify some of the credible regulatory single-nucleotide polymorphisms (SNPs) relevant to osteoporosis from 38 reported bone mineral density (BMD) GWASs. Using Roadmap databases, we prioritized SNPs based upon their overlap with strong enhancer or promoter chromatin preferentially in osteoblasts relative to <em>12</em> heterologous cell culture types. We also required that these SNPs overlap open chromatin (Deoxyribonuclease I [DNaseI]-hypersensitive sites) and DNA sequences predicted to bind to osteoblast-relevant transcription factors in an allele-specific manner. From >50,000 GWAS-derived SNPs, we identified 14 novel and credible regulatory SNPs (Tier-1 SNPs) for osteoporosis risk. Their associated genes, <i>BICC1</i>, <i>LGR4</i>, <i>DAAM2</i>, <i>NPR3</i>, or <i>HMGA2</i>, are involved in osteoblastogenesis or bone homeostasis and regulate cell signaling or enhancer function. Four of these genes are preferentially expressed in osteoblasts. <i>BICC1</i>, <i>LGR4</i>, and <i>DAAM2</i> play important roles in canonical <em>Wnt</em> signaling, a pathway critical for bone formation and repair. The transcription factors predicted to bind to the Tier-1 SNP-containing DNA sequences also have bone-related functions. We present evidence that some of the Tier-1 SNPs exert their effects on BMD risk indirectly through little-studied long noncoding RNA (lncRNA) genes, which may, in turn, control the nearby bone-related protein-encoding gene. Our study illustrates a method to identify novel BMD-related causal regulatory SNPs for future study and to prioritize candidate regulatory GWAS-derived SNPs, in general. © 2021 The Authors. <i>JBMR Plus</i> published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Keywords: EPIGENETICS; MOLECULAR PATHWAYS – REMODELING; OSTEOBLASTS; OSTEOPOROSIS; Wnt/β‐CATENIN.

Maternal regulatory factors endow the oocyte with developmental competence <i>in vivo</i>, which might be absent in current <i>in vitro</i> maturation (IVM) systems, thereby compromising oocyte quality. In the present study, by employing RNA sequencing data analysis, we expect to identify potential contributing factors to support porcine oocyte maturation through binding to their receptors on the oolemma. Here, C-X-C motif chemokine ligand <em>12</em> (CXCL<em>12</em>), vascular endothelial growth factor A (VEGFA), and Wingless-type MMTV integration site family member 5A (<em>WNT</em>5A), termed CVW, are selected and confirmed to be important maternal cytokines for porcine oocyte maturation. Combined supplementation of CVW promotes the nuclear maturation percentage from 57.2% in controls to 75.9%. More importantly, these maternal cytokines improve the developmental potential of matured oocytes by parthenogenesis, fertilization, and cloning, as their blastocyst formation efficiencies and total cell numbers are increased. CVW supplementation also enlarges perivitelline space and promotes cumulus expansion, which results in a more complete transzonal projection retraction on the zona pellucida, and a reduced incidence of polyspermy in fertilized oocytes. Meanwhile, inhibiting the CVW receptor-mediated signaling pathways severely impairs oocyte meiotic resumption and cumulus expansion during IVM. We further determine that maturation improvement by CVW is achieved through activating the MAPK pathway in advance and inhibiting the canonical <em>WNT</em> pathway at the end of the IVM period. These findings provide a new combination of three cytokines to promote the porcine IVM process, which also holds potential to be used in human assisted reproduction technologies as well as in other species.

<strong class="sub-title"> Keywords: </strong> CXCL<em>12</em>; VEGFA; <em>WNT</em>5A; embryo development; in vitro maturation; porcine oocyte.

Women with polycystic ovary syndrome (PCOS) are at increased risk of cardiovascular diseases (CVD); however, the independent role of PCOS in the incident CVD remains unknown. There are reports that hyperhomocysteinemia (HHcy), a potential cause of CVD, is frequently associated with PCOS. The present study investigates the independent attributes of hyperandrogenemia (HA), the integral associate of PCOS, and HHcy in causing atherogenic dyslipidemia. Twenty-five-day old rats were treated with homocysteine (Hcy) at 50 mg/kg/day dose level for <em>12</em> weeks. The HepG2 cell lines transfected with siRNA directed to PCSK9 were challenged with Hcy, homocysteine thiolactone (HTL), testosterone, 5α-dihydroxytestosterone (5α-DHT), or estradiol for 24 h. Rats administered with Hcy developed HHcy and displayed PCOS-like phenotypes with adversely altered lipid homeostasis and attenuated PI3K-AKT and <em>Wnt</em> signalling cascade. Overexpression of steroidogenic acute regulatory protein (StAR) and down-regulated expression of Aromatase together with elevated testosterone level marked the state of HA. In culture, the HepG2 cells responded independently to Hcy, HTL, testosterone, and 5α-DHT by an overt expression of PCSK9 and down-regulated expression of LDLR. The effect was magnified under the combined influence of Hcy and androgen(s). Estradiol, by contrast, exhibited the reverse effect. The findings suggest that HA may independently attribute to an increased cardiovascular risk in PCOS; however, the coexistence of HHcy catalyzes the risk further.

Plakoglobin (gamma-catenin) and beta-catenin are major components of the adherens junctions and can be localized to the nucleus by activation of the <em>Wnt</em> signalling pathway. In addition, plakoglobin is also found in desmosomes, a vertebrate-specific cell-cell adhesion structure. Plakoglobin expression and localization were examined at the protein level during zebrafish embryonic development by Western blotting and confocal microscopy. Plakoglobin was expressed throughout embryo development at the protein level. Western blotting revealed that embryonic plakoglobin protein content increased between <em>12</em>- and 24-h post-fertilization (hpf). Confocal microscopy showed that at stages up to <em>12</em> hpf, plakoglobin and beta-catenin were co-localized and expressed in both the nucleus and in cell-cell junctions. At 24- and 72-hpf, separate patterns were seen for plakoglobin and beta-catenin. These data indicate that plakoglobin localization in the heart region shifts from adherens junctions to desmosomes during heart chamber development.

OBJECTIVE

Dupuytren's disease is a progressive fibrosis of the hand that often results in debilitating flexion contractures. Its etiology is not completely understood but likely involves both genetic and environmental factors. A recent study performed in Europe identified DNA variants that associate with Dupuytren's disease. Given the likelihood for genetic variation among populations, we planned to validate the genetic variants identified by this study in a North American population.

METHODS

In the Marshfield Clinic's Personalized Medicine Research Project, 296 cases with Dupuytren's disease were identified and matched 3-to-1 to controls without Dupuytren's disease. Clinical data were abstracted from the electronic medical record. The top <em>12</em> single nucleotide polymorphisms (SNPs) from the European study were selected and tested in a multiplex assay using the MassArray Analyzer 4 (Sequenom, Inc., San Diego, CA). Differences in allele frequency were determined, and variants with a P value of <0.004 were considered significant.

RESULTS

We replicated 5 of the <em>12</em> SNPs previously reported to be associated with Dupuytren's disease.

CONCLUSIONS

Our findings support a role for the Wnt signaling pathway in the development of Dupuytren's disease, and suggest that further study of this pathway may result in early diagnosis and non-surgical treatments for Dupuytren's disease.

Type 2 diabetic patients have impaired bone quality, leading to increased fracture risk. Substantial evidence demonstrates that pulsed electromagnetic fields (PEMF) could resist osteopenia/osteoporosis induced by estrogen deficiency and disuse. However, the effects of PEMF on osteopenia/osteoporosis associated with diabetes, especially for more prevalent type 2 diabetes, remain poorly understood. We herein investigated the skeletal effects and mechanisms of PEMF (15 Hz, 20 Gs) on leptin receptor-deficient db/db mice with typical type 2 diabetic symptoms. Our µCT results showed that <em>12</em>-week PEMF exposure significantly improved both cancellous and cortical bone microarchitecture in db/db mice. Three-point bending and biomechanical indentation testing demonstrated that PEMF improved whole-bone structural properties and tissue-level material properties in db/db mice. PEMF significantly promoted bone formation in db/db mice evidenced by increased serum osteocalcin and bone mineral apposition rate, whereas PEMF exerted no observable alteration in bone resorption. Real-time PCR showed that PEMF upregulated tibial gene expression of osteoblastogenesis-related of canonical <em>Wnt</em>/β-catenin signaling but not osteoclastogenesis-related RANKL-RANK signaling in db/db mice. Our findings demonstrate that PEMF improved bone quantity and quality with obvious anabolic activities in db/db mice, and imply that PEMF might become a clinically applicable treatment modality for improving bone quality in type 2 diabetic patients.

Familial adenomatous polyposis (FAP) is caused by germ line mutations in the APC gene. Barrett's esophagus (BE) and Barrett's adenocarcinoma are intestinal type lesions of the esophagus characterized by an early loss of heterozygosity at the APC locus. We hypothesized that patients with FAP are at risk for the early development of BE due to the inherited mutations in the APC gene (haploinsufficiency). Upper gastrointestinal (UGI) tract biopsies from 36 patients with FAP were reviewed to determine the incidence and characteristics of BE in these patients. Twenty-four patients were confirmed carriers of a deleterious germline APC mutation. The other <em>12</em> patients were from FAP families with known APC gene mutations and had clinical manifestations of FAP. The control group consisted of patients who did not have a personal or family history of FAP undergoing UGI endoscopic examination in our institution over a 30 month period of time. The difference in expression of <em>Wnt</em> pathway proteins (APC, β-catenin, E-cadherin and cyclin D1) in BE between BE(+)/FAP(+), BE(-)/FAP(+) and age-matched BE(+)/FAP(-) groups was studied using immunohistochemistry. BE was found in 6 of 36 (6/36 or 16%) patients with FAP and in 266 of 1662 patients (16%) in the control group of symptomatic patients. The average age at the first diagnosis of BE in FAP patients was 37.8 versus 57.5 years in the control group (sporadic BE). When compared to age matched BE(+)/FAP- group (7/334), patients with FAP had a significantly (p = 0.005843, odds ratio 9.2; Fisher exact test) higher incidence of BE. Both classic FAP and attenuated FAP phenotypes were associated with BE .Two types of germ line mutations in APC gene were identified in BE(+)/FAP(+) patients: Five patients had 2-base deletion in exon 4 (426delAT) and one patient had 4-base deletion in exon 15 (3202del4). No difference in <em>Wnt</em> signaling pathway proteins expression was detected between BE(+)/FAP(+) and the age matched group of patients with sporadic BE (BE(+)/FAP(-)). Patients with FAP appear to have increased risk for the development of BE, which on average develops some 20 years earlier than in patients without FAP. This association needs to be taken in account when caring for the patients with FAP.

Bone marrow-derived mesenchymal stem cells (BMSCs) are the most promising seed cells for cell transplant. The proliferation of BMSCs is one of the most important determinants of the efficiency of MSC-based transplant therapy. It has been reported that transforming growth factor-β1 (TGF-β1) activates <em>Wnt</em>/β-catenin signaling and regulates cell proliferation. In this study, we investigated the effect of low concentrations of TGF-β1 on proliferation of BMSCs and the related mechanisms. BMSCs were treated with 0, 1, 5 and 10 ng/L recombinant mouse TGF-β1 for <em>12</em> h. Cell proliferation was measured by cell counting and MTT assay, and the proliferation-related signals p53, Mdm2, Aktl, <em>Wnt</em>3, phospho-Akt and β-catenin were measured by quantitative polymerase chain reaction (qPCR) and/or Western blot. Our results showed that TGF-β1 at low concentrations induced BMSC proliferation and expression of Mdm2, Aktl, phospho-Akt, <em>Wnt</em>3 and β-catenin, and inhibited p53 expression in dose dependent manner. Importantly, β-catenin siRNA significantly inhibited TGF-β1-induced BMSC proliferation. These findings suggest that low concentrations of TGF-β1 can stimulate proliferation of BMSCs, which is at least partially dependent on the activation of <em>Wnt</em>/β-catenin pathway.

SWV mouse embryos collected on gestational days (GD) 9:<em>12</em> and 10:00 following chronic in utero exposure to teratogenic concentrations of phenytoin were utilized for in situ transcription studies of gene expression. The substrate cDNA obtained from the frozen embryo sections was amplified into radiolabelled antisense RNA (RT/aRNA) and used as a probe to screen a panel of 20 cDNA clones representing genes that are important regulators of craniofacial and neural development. The magnitude of alteration in gene expression following phenytoin treatment was determined densitometrically by changes in the hybridization intensity of the aRNA probes to the cDNA clones immobilized to the slot blots. We found that both <em>Wnt</em>-1 and the calcium channel gene were developmentally regulated, as their level of expression decreased significantly between the two collection times. Phenytoin treatment produced a significant downregulation in the level of expression for 25% of the genes examined in the GD 9:<em>12</em> embryos, including the growth factors TGF-beta and NT3, the proto-oncogene <em>Wnt</em>-1, the nicotinic receptor, and the voltage sensitive calcium channel gene. Additional changes in the coordinate expression of several of the growth and transcription factors were observed at both gestational timepoints. The application of RT/aRNA technology has extended our appreciation of the normal patterns of gene expression during craniofacial and neural development, and provided the first demonstration of multiple coordinate changes in transcription patterns following teratogenic insult.