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It has been evidenced that ROR2 influences the growth of many tumors, including non‐small cell lung cancer, osteosarcoma, and breast cancer. This research examined the effect of the WNT1/ROR2 signaling pathway on the progression of triple‐negative breast cancer (TNBC). Bioinformatics analysis results demonstrated that ROR2 had a higher messenger RNA (mRNA) expression level in TNBC tissues and was positively correlated with poor patient prognosis. Western blot analysis (WB) and quantitative reverse transcription polymerase chain reaction demonstrated that TNBC cells had relatively higher ROR2 mRNA and protein levels than normal cell lines. Transwell and Cell Counting Kit‐8 (CCK‐8) assay further proved that downregulating ROR2 expression dramatically slowed the MDA‐MB‐231 cell progression. WB detection of epithelial‐mesenchymal transition (EMT)‐related proteins suggested that knocking down ROR2 could alleviate the EMT tendency of cancer cells. The WNT1/ROR2 signaling pathway could be inhibited by the WNT inhibitor pyrvinium pamoate (PP). Experiments on in vitro cell functional recovery have demonstrated that PP could restore malignant phenotypes caused by ROR2 overexpression. Finally, the mouse model experiments further validated the anticancer effect of PP on TNBC. Generally speaking, the malignant progression of TNBC could be stimulated by the WNT1/ROR2 signaling pathway which can be inhibited by PP, suggesting the potential value of PP in controlling TNBC. [ABSTRACT FROM AUTHOR]

Skeletal growth, modeling, and remodeling are regulated by various molecules, one of them being the recently identified osteoanabolic factor WNT1. We have previously reported that WNT1 transcriptionally activates the expression of Omd, encoding Osteomodulin (OMD), in a murine mesenchymal cell line, which potentially explained the skeletal fragility of mice with mutational WNT1 inactivation, since OMD has been shown to regulate type I collagen fibril formation in vitro. In this study we confirmed the strong induction of Omd expression in a genome-wide expression analysis of transfected cells, and we obtained further evidence for Omd being a direct target gene of WNT1. To assess the in vivo relevance of this regulation, we crossed Omd-deficient mice with a mouse line harboring an inducible, osteoblast-specific Wnt1 transgene. After induction of Wnt1 expression for 1 or 3 weeks, the osteoanabolic potency of WNT1 was not impaired despite the Omd deficiency. Since current knowledge regarding the in vivo physiological function of OMD is limited, we next focused on skeletal phenotyping of wild-type and Omd-deficient littermates, in the absence of a Wnt1 transgene. Here we did not observe an impact of Omd deficiency on trabecular bone parameters by histomorphometry and μCT either. Importantly, however, male and female Omd-deficient mice at the ages of 12 and 24 weeks displayed a slender bone phenotype with significantly smaller long bones in the transversal dimension, while the longitudinal bone growth remained unaffected. Although mechanical testing revealed no significant changes explained by impaired bone material properties, atomic force microscopy of the femoral bone surface of Omd-deficient mice revealed moderate changes at the nanostructural level, indicating altered regulation of collagen fibril formation and aggregation. Taken together, our data demonstrate that, although OMD is dispensable for the osteoanabolic effect of WNT1, its deficiency in mice specifically modulates transversal cortical bone morphology. Lay Summary: We explored the physiological relevance of the protein Osteomodulin (OMD) that we previously found to be induced by the osteoanabolic molecule WNT1. While other studies have shown that OMD is involved in the regulation of collagen fibril formation in vitro, its function in vivo has not been investigated. We confirmed that OMD is directly regulated by WNT1 but surprisingly, when we bred mice lacking OMD with mice engineered to highly express WNT1, we found that the osteoanabolic effect of WNT1 was unaffected by the absence of OMD. Interestingly, mice lacking OMD did show differences in the shape of their bones, particularly in their width, despite no significant changes in bone density or length. Investigation of the bone matrix of mice lacking OMD at the nanostructural level indicated moderate differences in the organization of collagen fibrils. This study provided further insights into the effect of WNT1 on bone metabolism and highlighted a specific function of OMD in skeletal morphology. [ABSTRACT FROM AUTHOR]

Osteogenesis imperfecta Type XV, first reported in 2013, is caused by WNT1 gene mutations and is characterized by moderate to severe bone fragility and deformities with osteoporosis. We reported three cases of patients with WNT1-related osteogenesis imperfecta, who presented in early infancy and subsequently developed multiple fractures and bone deformities but with different extra-skeletal manifestations, including one patient with neonatal supraventricular tachycardia with Wolf-Parkinson-White syndrome, which was not reported in previous literature. [ABSTRACT FROM AUTHOR]

Abstract Background The roles of low molecular mass polypeptide 2 (LMP2) and transporter-associated with antigen processing (TAP2) in tumorigenesis are controversial. Here we aimed to explore the effect of LMP2 and TAP2 on the oncogenesis and metastasis of cervical cancer cells. Methods The expressions of LMP2 and TAP2 in cervical cancer and normal tissues were determined by qPCR. Plate colony formation, cell counting kit-8 analysis and in vivo tumor xenograft assays were used to detect the tumor growth. Wound healing and transwell assays were used to detect the metastasis of cervical cancer. Gelatin zymography and western blotting assays were used to detect the effect of LMP2 and TAP2 on the EMT and Wnt/β-catenin pathway in cervical cancer cells. Results In the present study, we reported that LMP2 and TAP2 levels were overexpressed in cervical cancer. Overexpression of LMP2 and TAP2 impaired the proliferation of Hela cells. In vivo studies substantiated that LMP2 and TAP2 antagonized tumor growth. Likewise, LMP2 and TAP2 overexpression decreased the migration and invasion ability of Hela cells by regulating the process of epithelial-mesenchymal transition (EMT). Mechanically, LMP2 and TAP2 subverted the protein abundance of Wnt1 and β-catenin, thereby downregulating their downstream targets Cyclin D1 and c-Myc. In addition, Wnt1 overexpression partially rescued the observed consequences of ectopic expression of LMP2 and TAP2 in cervical cancer cells. Taken together, our study revealed that LMP2 and TAP2 suppress the oncogenesis and metastasis of cervical cancer cells by Wnt/β-catenin pathway and altering EMT. Conclusion LMP2 and TAP2 may inhibit the oncogenesis and metastasis of cervical cancer cells by inhibiting the process of EMT and the Wnt/β-catenin signaling pathway, which may provide important insight into prospective targets for the treatment of cervical cancer.

Introduction: Odontogenic lesions of the maxillofacial region constitute a complex group of lesions with diverse histopathologic types and clinical behaviour. Early diagnosis is important to minimize the need for radical surgery and to improve quality of life of the patients. Tumour markers play an essential role in the molecular level understanding of Odontogenic lesions and also used for early diagnosis and target therapies which improves the quality of life of the patients. Patched, a tumour suppressor gene encodes the transmembrane protein PTCH and is a receptor for the morphogen Sonic Hedgehog. It is evident that PTCH gene mutations occur in odontogenic keratocysts and the Hedgehog signalling pathway has an important role during tooth formation. WNT 1 is a key signal molecule that controls cell growth and proliferation. WNT pathway abnormalities are reported to induce tumour occurrence. Hence, my study was to determine the presence of WNT1 and PTCH in peripheral blood of patients with Odontogenic lesions using quantitative RT-PCR. Materials and Methods: In this cross-sectional study, two groups were included: Group 1—blood samples from 8 individuals with odontogenic cysts and tumours, and Group 2—blood samples of 8 individuals without Odontogenic lesions. 2 ml of blood sample was collected from radial veins into PAX gene tubes containing RNA stabilizing agent and stored at a temperature of 2 to 4 degrees and transported to Enable Biolabs India Pvt Ltd., Chennai. PAX gene tubes were subjected to centrifugation at 8000 rpm to separate plasma fraction. Reverse transcription of mRNA was performed using miScript II RT Kit (Cat#218161, Qiagen, Germany) to synthesize cDNA. GAPDH house-keeping gene used as control. Results: The study group had 3 males and 5 females (n = 8) with a mean age group of 32.6 years and the control group had 2 males and 6 females (n = 8) with mean age of 35.2 years. Group I (study group) showed 37.5% positive expression of WNT1 gene with a p value of 0.055 (p > 0.05) and 50% positive expression of PTCH with a p value of 0.021 (p < 0.05) (Figs. 3 and 4) which was statistically significant when compared with control group. Group II (control group) showed 100% negative expression for WNT1 and PTCH genes. Conclusion: WNT1 and PTCH genes were expressed in peripheral blood of patients with odontogenic lesions. WNT1 and PTCH genes may be potential predictors in individuals who would develop odontogenic lesions. Further studies on expression of WNT1 and PTCH genes with larger number of samples might give a future scope for target therapy in odontogenic lesions. [ABSTRACT FROM AUTHOR]

Background: The roles of low molecular mass polypeptide 2 (LMP2) and transporter-associated with antigen processing (TAP2) in tumorigenesis are controversial. Here we aimed to explore the effect of LMP2 and TAP2 on the oncogenesis and metastasis of cervical cancer cells. Methods: The expressions of LMP2 and TAP2 in cervical cancer and normal tissues were determined by qPCR. Plate colony formation, cell counting kit-8 analysis and in vivo tumor xenograft assays were used to detect the tumor growth. Wound healing and transwell assays were used to detect the metastasis of cervical cancer. Gelatin zymography and western blotting assays were used to detect the effect of LMP2 and TAP2 on the EMT and Wnt/β-catenin pathway in cervical cancer cells. Results: In the present study, we reported that LMP2 and TAP2 levels were overexpressed in cervical cancer. Overexpression of LMP2 and TAP2 impaired the proliferation of Hela cells. In vivo studies substantiated that LMP2 and TAP2 antagonized tumor growth. Likewise, LMP2 and TAP2 overexpression decreased the migration and invasion ability of Hela cells by regulating the process of epithelial-mesenchymal transition (EMT). Mechanically, LMP2 and TAP2 subverted the protein abundance of Wnt1 and β-catenin, thereby downregulating their downstream targets Cyclin D1 and c-Myc. In addition, Wnt1 overexpression partially rescued the observed consequences of ectopic expression of LMP2 and TAP2 in cervical cancer cells. Taken together, our study revealed that LMP2 and TAP2 suppress the oncogenesis and metastasis of cervical cancer cells by Wnt/β-catenin pathway and altering EMT. Conclusion: LMP2 and TAP2 may inhibit the oncogenesis and metastasis of cervical cancer cells by inhibiting the process of EMT and the Wnt/β-catenin signaling pathway, which may provide important insight into prospective targets for the treatment of cervical cancer. [ABSTRACT FROM AUTHOR]

Abstract Background Osteoblastic skeletal metastasis is frequently observed in prostate cancer. An effective therapy has not been developed due to the unclear molecular mechanism. The Wnt family is involved in various biological phenomena including bone metabolism. There is no direct evidence that the family causes osteoblastic skeletal metastasis. Aims The present study aims to evaluate whether overexpressed Wnt induces osteoblastic bone metastasis in a well‐established osteolytic bone metastatic model. Methods and Results The breast cancer‐derived 5a‐D‐Luc‐ZsGreen cells were transfected with Wnt1, Wnt3A, and Wnt5A expression vectors, producing stably highly expressing cells. These cells were intracardially transplanted in nude mice. Bone metastasis development was confirmed by fluorescence imaging. Hind‐limb bones including metastasis were dissected and visualized through micro‐CT imaging. After imaging, sections were stained with hematoxylin and eosin (H&E), and immunohistochemically stained with an anti‐SATB2 antibody. Luminescent imaging confirmed mice with bone metastases in the hind limbs. Micro‐CT imaging found an osteoblastic change only in bone metastasis of mice transplanted with Wnt1‐expressing cells. This was confirmed on H&E‐stained sections. SATB2 immunostaining showed differentiated osteoblasts were at the site of bone metastases in the diaphysis. SATB2 in the Wnt/β‐catenin pathway activated by overexpressed Wnt1 could induce osteoblastic change. Conclusion Our findings provided direct evidence Wnt1 is involved in osteoblastic bone metastasis development. Our model would be a powerful tool for further elucidating molecular mechanisms underlying the disease and developing effective therapies.

Limb problems are one of the most common problems with fast-growing meat-type chickens. Various bone abnormalities, which can lead to limping, bone weakness, or even fractures, bring overall discomfort to birds and a loss of production. Genetic aspects are often associated with these side effects on bone stability and are also cited as the dominant cause. These points to a close negative relationship of genetic selection for rapid growth with traits involved in bone integrity. Due to the assumption of an additive genetic background, improvements through genetic tools can be used. Our study is focused on selected genes of important signaling pathways for bone metabolism. We tried to detect polymorphisms that would show associations with selected bone parameters in a total of 48 broilers. Those were fast-growing Ross 308 hybrids and slow-growing Hubbard M22BxJA87A hybrids. The TNFRSF11A and WISP1 genes were tested. A total of fourteen polymorphisms were found, three of them were synonymous and five in the intron. In the case of four polymorphisms found in exons of the TNFRSF11A gene (c.11G > T, c.31G > A, c.37C > G, c.514G > A), associations with the observed bone parameters (bone strength, bone dimensions and bone mass) were demonstrated. The genetic architecture of bone traits is not fully understood, therefore the present study and the knowledge gained can help to increase the potential in poultry breeding processes and thus reduce the death of individuals.

Pathogenic variants in the LRP5, PLS3, or WNT1 genes can significantly affect bone mineral density, causing monogenic osteoporosis. Much remains to be discovered about the phenotype and medical care needs of these patients. The purpose of this study was to examine the use of medical care among Dutch individuals identified between 2014 and 2021 with a pathogenic or suspicious rare variant in LRP5, PLS3, or WNT1. In addition, the aim was to compare their medical care utilization to both the overall Dutch population and the Dutch Osteogenesis Imperfecta (OI) population. The Amsterdam UMC Genome Database was used to match 92 patients with the Statistics Netherlands (CBS) cohort. Patients were categorized based on their harbored variants: LRP5, PLS3, or WNT1. Hospital admissions, outpatient visits, medication data, and diagnosis treatment combinations (DTCs) were compared between the variant groups and, when possible, to the total population and OI population. Compared to the total population, patients with an LRP5, PLS3, or WNT1 variant had 1.63 times more hospital admissions, 2.0 times more opened DTCs, and a greater proportion using medication. Compared to OI patients, they had 0.62 times fewer admissions. Dutch patients with an LRP5, PLS3, or WNT1 variant appear to require on average more medical care than the total population. As expected, they made higher use of care at the surgical and orthopedic departments. Additionally, they used more care at the audiological centers and the otorhinolaryngology (ENT) department, suggesting a higher risk of hearing-related problems. [ABSTRACT FROM AUTHOR]

To explore the influence of the long-non coding RNA-H19/microRNA-140-5p/Wnt1 axis on the osteogenic differentiation in bone marrow mesenchymal stem cells. First, we used dual-luciferase reporter gene detection to verify long-non coding-H19/miRNA-140-5p/Wnt1 regulatory axis targeting relationship. Then long-non coding-H19 was overexpressed or silenced in bone marrow mesenchymal stem cells and then long-non coding-H19, microRNA-140-5p and Wnt1 expression were assessed using quantitative real-time polymerase chain reaction and cell activity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide. Then the osteogenic differentiation culture was performed, formation of calcium nodules were assessed via Alizarin red staining, while the expression of osteocalcin and runt-related transcription factor 2 detected via Western blot. Wild type long-non coding RNA-H19 group and wild type-Wnt1 group showed significantly decreased luciferase activity in dual-luciferase reporter assays (p<0.05). After overexpressed long-non coding RNA-H19, the microRNA-140-5p expression decreased and Wnt1 expression increased (p<0.05), and while silenced the long-non coding RNA-H19 expression, the opposite result was found (p<0.05). In terms of cell viability, there were no significant differences between groups after long-non coding RNA-H19 was overexpressed or silenced (p>0.05). After overexpressed long-non coding RNA-H19, calcium nodules formation, mineralized bone matrix level, the expression of osteocalcin and runt-related transcription factor 2 protein significantly increased (p<0.05), and while silenced the long-non coding RNA-H19 expression, the opposite result was found (p<0.05). Long-non coding RNA-H19 overexpression promotes the bone marrow mesenchymal stem cells osteogenic differentiation via targeting microRNA-140-5p/Wnt1 signaling pathway. [ABSTRACT FROM AUTHOR]

The CRISPR/Cas9 system has been successfully applied in dozens of diverse species; although the screening of successful CRISPR/Cas9 editing events remains particularly laborious, especially for those that occur at relatively low frequency. Recently, a co‐CRISPR strategy was proved to enrich the desired CRISPR events. Here, the co‐CRISPR strategy was developed in the Fall armyworm, Spodoptera frugiperda, with kynurenine 3‐monooxygenase gene (kmo) as a marker. The kmo mosaics induced by single‐guide RNAs (sgRNAs)/Cas9 displayed the darker green color phenotype in larvae, compared with wild type (brown), and mosaic‐eye adults were significantly acquired from the mosaic larvae group. In the kmo knockout strain, no significant difference was observed in larval development and adult reproduction. Acetylcholinesterase 2 (ace2) and Wnt1 were selected as target genes to construct the co‐CRISPR strategy using kmo marker. By co‐injection of kmo and ace2 sgRNAs, the mutant efficiency of ace2 was significantly increased in the kmo mosaic (larvae or adults) groups. Similarly, more malformed pupae with Wnt1 mutations were observed in the darker green larvae group. Taken together, these results demonstrated that kmo was a suitable visible marker gene for the application and extension of co‐CRISPR strategy in Fall armyworm. Using darker green color in larvae or mosaic‐eye in adults from kmo knockout as a marker, the mutant efficiency of a target gene could be enriched in a Fall armyworm group consisting of marked individuals. The co‐CRISPR strategy is helpful for gene function studies by the knockout technique with no or lethal phenotypes. [ABSTRACT FROM AUTHOR]

This essay reexamines molecular evidence supporting the existence of the 'preisthmus', a caudal midbrain domain present in vertebrates (studied here in the mouse). It is thought to derive from the embryonic m2 mesomere and appears intercalated between the isthmus (caudally) and the inferior colliculus (rostrally). Among a substantial list of gene expression mappings examined from the Allen Developing and Adult Brain Atlases, a number of quite consistent selective positive markers, plus some neatly negative markers, were followed across embryonic stages E11.5, E13.5, E15.5, E18.5, and several postnatal stages up to the adult brain. Both alar and basal subdomains of this transverse territory were explored and illustrated. It is argued that the peculiar molecular and structural profile of the preisthmus is due to its position as rostrally adjacent to the isthmic organizer, where high levels of both FGF8 and WNT1 morphogens must exist at early embryonic stages. Isthmic patterning of the midbrain is discussed in this context. Studies of the effects of the isthmic morphogens usually do not attend to the largely unknown preisthmic complex. The adult alar derivatives of the preisthmus were confirmed to comprise a specific preisthmic sector of the periaqueductal gray, an intermediate stratum represented by the classic cuneiform nucleus, and a superficial stratum containing the subbrachial nucleus. The basal derivatives, occupying a narrow retrorubral domain intercalated between the oculomotor and trochlear motor nuclei, include dopaminergic and serotonergic neurons, as well as a variety of peptidergic neuron types. [ABSTRACT FROM AUTHOR]

Despite considerable improvement in fracture care, 5%–10% of all fractures still heal poorly or result in nonunion formation. Therefore, there is an urgent need to identify new molecules that can be used to improve bone fracture healing. One activator of the Wnt‐signaling cascade, Wnt1, has recently gained attention for its intense osteoanabolic effect on the intact skeleton. The aim of the present study was to investigate whether Wnt1 might be a promising molecule to accelerate fracture healing both in skeletally healthy and osteoporotic mice that display a diminished healing capacity. Transgenic mice for a temporary induction of Wnt1 specifically in osteoblasts (Wnt1‐tg) were subjected to femur osteotomy. Non‐ovariectomized and ovariectomized Wnt1‐tg mice displayed significantly accelerated fracture healing based on a strong increase in bone formation in the fracture callus. Transcriptome profiling revealed that Hippo/yes1‐associated transcriptional regulator (YAP)‐signaling and bone morphogenetic protein (BMP) signaling pathways were highly enriched in the fracture callus of Wnt1‐tg animals. Immunohistochemical staining confirmed increased activation of YAP1 and expression of BMP2 in osteoblasts in the fracture callus. Therefore, our data indicate that Wnt1 boosts bone formation during fracture healing via YAP/BMP signaling both under healthy and osteoporotic conditions. To further test a potential translational application of Wnt1, we applied recombinant Wnt1 embedded into a collagen gel during critical‐size bone‐defect repair. Mice treated with Wnt1 displayed increased bone regeneration compared to control mice accompanied by increased YAP1/BMP2 expression in the defect area. These findings are of high clinical relevance because they indicate that Wnt1 could be used as a new therapeutic agent to treat orthopedic complications in the clinic. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR). [ABSTRACT FROM AUTHOR]

Wnt and R-spondin (Rspo) proteins are two major types of endogenous Wnt/β-catenin signaling agonists. While Wnt/β-catenin signaling is greatly diminished in Alzheimer's disease (AD), it remains to be elucidated whether the inhibition of this pathway is associated with dysregulation of Wnt and Rspo proteins. By analyzing temporal cortex RNA-seq data of the human postmortem brain samples, we found that WNT1 and RRPO2 were significantly downregulated in human AD brains. In addition, the expression of Wnt acyltransferase porcupine (PORCN), which is essential for Wnt maturation and secretion, was greatly deceased in these human AD brains. Interestingly, the lowest levels of WNT1, PORCN, and RSPO2 expression were found in human AD brains carrying two copies of APOE4 allele, the strongest genetic risk factor of late-onset AD. Importantly, there were positive correlations among the levels of WNT1, PORCN, and RSPO2 expression in human AD brains. Supporting observations in humans, Wnt1, PORCN, and Rspo2 were downregulated and Wnt/β-catenin signaling was diminished in the 5xFAD amyloid model mice. In human APOE-targeted replacement mice, downregulation of WNT1, PORCN, and RSPO2 expression was positively associated with aging and APOE4 genotype. Finally, WNT1 and PORCN expression and Wnt/β-catenin signaling were inhibited in human APOE4 iPSC-derived astrocytes when compared to the isogenic APOE3 iPSC-derived astrocytes. Altogether, our findings suggest that the dysregulations of Wnt1, PORCN, and Rspo2 could be coordinated together to diminish Wnt/β-catenin signaling in aging- and APOE4-dependent manners in the AD brain. [ABSTRACT FROM AUTHOR]

R‐spondins (RSPOs) are influential signaling molecules that promote the Wnt/β‐catenin pathway and self‐renewal of stem cells. Currently, RSPOs are emerging as clinically relevant oncogenes, being linked to cancer development in multiple organs. Although this has instigated the rapid development and testing of therapeutic antibodies targeting RSPOs, functional evidence that RSPO causally drives cancer has focused primarily on the intestinal tract. Here, we assess the oncogenic capacity of RSPO in breast cancer in a direct fashion by generating and characterizing a novel mouse model with conditional Rspo3 expression in the mammary gland. We also address the prevalence of RSPO gene alterations in breast cancer patients. We found that a quarter of breast cancer patients harbor RSPO2/RSPO3 copy number amplifications, which are associated with lack of steroid hormone receptor expression and reduced patient survival. Foremost, we demonstrate the causal oncogenic capacity of RSPO3 in the breast, as conditional Rspo3 overexpression consistently drives the development of mammary adenocarcinomas in our novel Rspo3 breast cancer model. RSPO3‐driven mammary tumors typically show poor differentiation, areas of epithelial‐to‐mesenchymal transition, and metastatic potential. Given the reported interplay in the Wnt/β‐catenin pathway, we comparatively analyzed RSPO3‐driven mouse mammary tumors versus classical WNT1‐driven analogues. This revealed that RSPO3‐driven tumors are distinct, as the poorly differentiated tumor morphology and metastatic potential were observed in RSPO3‐driven tumorigenesis exclusively, further substantiated by differentiating gene expression profiles. Co‐expression of Rspo3 and Wnt1 transduced mammary tumors with a mixed phenotype harboring morphological features characteristic of both transgenes. In summary, we report that a quarter of breast cancer patients harbor RSPO2/RSPO3 copy number gains, and these patients have a worse prognosis, whilst providing in vivo evidence that RSPO3 drives poorly differentiated invasive breast cancer in mice. Herewith, we establish RSPO3 as a driver of breast cancer with clinical relevance, proposing RSPO3 as a novel candidate target for therapy in breast cancer. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Background: The pathogenic mechanisms of early-onset osteoporosis caused by WNT1 and PLS3 mutations are incompletely understood and diagnostic biomarkers of these disorders are limited. Recently, lipocalin-2 has been recognized as an osteokine involved in bone development and homeostasis. However, the role of lipocalin-2 in WNT1 and PLS3 osteoporosis is unknown. Objective: We aimed to investigate if plasma lipocalin-2 could be utilized as a biomarker for WNT1 and PLS3 osteoporosis and to evaluate the association between lipocalin-2 and other parameters of bone metabolism. Methods: We measured plasma lipocalin-2 in 17 WNT1 and 14 PLS3 mutationpositive patients and compared them to those of 34 mutation-negative (MN) healthy subjects. We investigated possible associations between lipocalin-2 and several bone biomarkers including collagen type I cross-linked Ctelopeptide (CTX), alkaline phosphatase (ALP), type I procollagen intact Nterminal propeptide (PINP), intact and C-terminal fibroblast growth factor 23 (FGF23), dickkopf-1 (DKK1) and sclerostin as well as parameters of iron metabolism (iron, transferrin, transferrin saturation, soluble transferrin receptor and ferritin). Results: We found no differences in plasma lipocalin-2 levels in WNT1 or PLS3 patients compared with MN subjects. However, lipocalin-2 was associated with C-terminal FGF23 in WNT1 patients (r=0.62; p=0.008) and PLS3 patients (r=0.63, p=0.017), and with intact FGF23 in PLS3 patients (r=0.80; p<0.001). In addition, lipocalin-2 correlated with serum transferrin in WNT1 patients (r=0.72; p=0.001). Conclusion: We conclude that plasma lipocalin-2 is not altered in WNT1 or PLS3 mutation-positive subjects but is associated with FGF23 in abnormal WNT1 or PLS3 signaling and with iron status in abnormal WNT1 signaling. [ABSTRACT FROM AUTHOR]

Abstract To explore the effect and underlying molecular mechanism of long non‐coding RNA (lncRNA)‐H19 on ovarian cancer (OC) cells, a total of 41 cases of OC and adjacent normal tissues were collected. H19 and microRNA (miR)‐140 expressions in OC tissues and cells were detected using quantitative real‐time polymerase chain reaction (qRT‐RCR). The correlation between H19 expression and prognosis of OC patient was analyzed. siRNA (si)‐H19 and si‐negative control (NC) were transfected into OC cells. Cell proliferation was checked by cell counting kit‐8 assay and colony formation assay, and cell migration and invasion were analyzed via Transwell assay. The targeted binding relationship between H19 and miR‐140 was predicted and verified, miR‐140 downstream gene was predicted and Wnt1 was screened out. The impact of in‐miR‐140 on the si‐H19‐induced decreased OC cell proliferation and migration was evaluated. H19 expression was upregulated in OC tissues and cells, and its overexpression was associated with a poor prognosis of OC. si‐H19 remarkably reduced OC cell proliferation and migration. H19 upregulated Wnt1 expression through targeting miR‐140 in OC cells. Altogether, miR‐140 was notably downregulated in OC, and in‐miR‐140 partially inhibited the si‐H19‐induced decrease of OC cell proliferation and migration. H19 competitively bound to miR‐140 to upregulate Wnt1, thereby promoting OC cell proliferation and migration.

Abstract Background This study aimed to investigate the exact regulatory mechanisms of exosomal miR-34c in mediating communication between cholangiocarcinoma cells and fibroblasts. Methods Exosomes were isolated from HuCCT-1 and HIBEC cells using differential ultracentrifugation and identified by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) method. Real-time quantitative PCR (qRT-PCR) and western blotting analyses were performed to assess the levels of pro-inflammatory factors, and fibroblast-related proteins and Wnt-linked signaling pathway proteins, respectively. Exosome-tracking was performed with confocal microscopy. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and Transwell assays were used to measure cell proliferation and migration, respectively. Further, the oncogenicity of cholangiocarcinoma cells was analyzed in nude mice transplanted tumor model. Results The analysis suggested that the expression of miR-34c was decreased in exosomes from HuCCT-1 cells. Moreover, miR-34c in exosomes mediated fibroblast activation by directly targeting WNT1. Additionally, cancer-associated fibroblasts (CAFs) activated by downregulation of exosomal miR-34c promoted cholangiocarcinoma progression. Conclusions Thus, miR-34c in exosomes was found to be a key player in regulating intercellular communication between tumor cells and fibroblasts.

BackgroundThe pathogenic mechanisms of early-onset osteoporosis caused by WNT1 and PLS3 mutations are incompletely understood and diagnostic biomarkers of these disorders are limited. Recently, lipocalin-2 has been recognized as an osteokine involved in bone development and homeostasis. However, the role of lipocalin-2 in WNT1 and PLS3 osteoporosis is unknown.ObjectiveWe aimed to investigate if plasma lipocalin-2 could be utilized as a biomarker for WNT1 and PLS3 osteoporosis and to evaluate the association between lipocalin-2 and other parameters of bone metabolism.MethodsWe measured plasma lipocalin-2 in 17 WNT1 and 14 PLS3 mutation-positive patients and compared them to those of 34 mutation-negative (MN) healthy subjects. We investigated possible associations between lipocalin-2 and several bone biomarkers including collagen type I cross-linked C-telopeptide (CTX), alkaline phosphatase (ALP), type I procollagen intact N-terminal propeptide (PINP), intact and C-terminal fibroblast growth factor 23 (FGF23), dickkopf-1 (DKK1) and sclerostin as well as parameters of iron metabolism (iron, transferrin, transferrin saturation, soluble transferrin receptor and ferritin).ResultsWe found no differences in plasma lipocalin-2 levels in WNT1 or PLS3 patients compared with MN subjects. However, lipocalin-2 was associated with C-terminal FGF23 in WNT1 patients (r=0.62; p=0.008) and PLS3 patients (r=0.63, p=0.017), and with intact FGF23 in PLS3 patients (r=0.80; p

Objective Zearalenone (ZEA) has estrogen-like effects. Our previous study has shown that ZEA (0.5 to 1.5 mg/kg) could induce abnormal uterine proliferation through transforming growth factor signaling pathway. To further study the other regulatory networks of uterine hypertrophy caused by ZEA, the potential mechanism of ZEA on porcine endometrial epithelial cells (PECs) was explored by the Illumina Hiseq 2000 sequencing system. Methods The PECs were treated with ZEA at 0 (ZEA0), 5 (ZEA5), 20 (ZEA20), and 80 (ZEA80) μmol/L for 24 h. The collected cells were subjected to cell cycle, RNA-seq, real-time quantitative polymerase chain reaction, immunofluorescence, and western blot analysis. Results The proportion of cells in the S and G2 phases decreased (p

Abstract Background WNT1 c.110 T>C and c.505G>T missense mutations have been identified in patients with osteogenesis imperfecta (OI). Whether these mutations affect osteoblast differentiation remains to be determined. This study aimed to investigate the effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast function, gene expression, and pathways involved in OI. Methods Empty vector (negative control), wild-type WNT1, WNT1 c.110 T>C, WNT1 c.505G>T, and WNT1 c.884C>A (positive control) mutant plasmids were constructed and transfected into preosteoblast (MC3T3-E1) cells to investigate their effect on osteoblast differentiation. The expressions of osteoblast markers, including BMP2, RANKL, osteocalcin, and alkaline phosphatase (ALP), were determined using quantitative real-time polymerase chain reaction (RT-qPCR), western blotting (WB), enzyme-linked immunosorbent assay, and ALP staining assay, respectively. The mRNA and protein expression levels of WNT1 or the expression levels of the relevant proteins involved in the WNT1/β-catenin signaling pathway were also determined using RT-qPCR, WB, and immunofluorescence (IF) assays after the different plasmids were transfected into MC3T3-E1 cells. Results Compared with those in the wild-type group, in the mutation groups, the mRNA and protein expression levels of BMP2 were suppressed, the expressions of osteocalcin and ALP were inhibited, and the mRNA and protein expression levels of RANKL were enhanced in MC3T3-E1 cells. WB and IF assays revealed that the protein expression levels of WNT1 in MC3T3-E1 cells were downregulated in the mutation groups compared with those in the wild-type WNT1 group. Furthermore, the expression levels of nonphosphorylated β-catenin (non-p-β-catenin) and phosphorylated GSK-3β (p-GSK-3β) were downregulated in the mutation groups compared with those in the wild-type group. However, no significant changes in the expression level of non-p-β-catenin or p-GSK-3β were observed in the mutation groups. Conclusions WNT1 c.110 T>C and c.505G>T mutations may alter the proliferation and osteogenic phenotype of MC3T3-E1 linked to the progression of OI via the inhibition of the WNT1/β-catenin signaling pathway. This is the first study to confirm the effect of WNT1 c.110 T>C and c.505G>T missense mutations on osteoblast differentiation and propose a new molecular mechanism for OI development.

Osteogenesis imperfecta (OI) is a group of severe genetic bone disorders characterized by congenital low bone mass, deformity, and frequent fractures. Type XV OI is a moderate to severe form of skeletal dysplasia caused by WNT1 variants. In this cohort study from southern China, we summarized the clinical phenotypes of patients with WNT1 variants and found that the proportion of type XV patients was around 10.3% (25 out of 243) with a diverse spectrum of phenotypes. Functional assays indicated that variants of WNT1 significantly impaired its secretion and effective activity, leading to moderate to severe clinical manifestations, porous bone structure, and enhanced osteoclastic activities. Analysis of proteomic data from human skeleton indicated that the expression of SOST (sclerostin) was dramatically reduced in type XV patients compared to patients with COL1A1 quantitative variants. Single-cell transcriptome data generated from human tibia samples of patients diagnosed with type XV OI and leg-length discrepancy, respectively, revealed aberrant differentiation trajectories of skeletal progenitors and impaired maturation of osteocytes with loss of WNT1, resulting in excessive CXCL12+ progenitors, fewer mature osteocytes, and the existence of abnormal cell populations with adipogenic characteristics. The integration of multi-omics data from human skeleton delineates how WNT1 regulates the differentiation and maturation of skeletal progenitors, which will provide a new direction for the treatment strategy of type XV OI and relative low bone mass diseases such as early onset osteoporosis. Lay Summary: Osteogenesis imperfecta is a rare disease characterized by low bone mass, frequent fractures, and long bone deformity. Type XV osteogenesis imperfect is an autosomal recessive disorder caused by WNT1 variants, while heterozygous variants of WNT1 result in early onset osteoporosis. In this cohort study, we summarized the clinical features of 25 patients diagnosed with type XV osteogenesis imperfect. The WNT1 variants were confirmed by genetic test. Molecular assays were conducted to reveal the impact of variants on WNT1 protein activity and bone structure. We then compared the protein levels in bone tissues isolated from the type XV patients and patients with mild deformity using proteomic method, and found that the expression of SOST, mainly produced by mature osteoblasts and osteocytes, was dramatically reduced in type XV patients. We further compared the global mRNA expression levels in the skeletal cells using single-cell RNA sequencing. Analyses of these data indicated that more immature progenitors were identified and maturation of osteocytes was impaired with WNT1 loss-of-function. Our study helps to understand the underlying pathogenesis of type XV osteogenesis imperfecta. [ABSTRACT FROM AUTHOR]

Gastric cancer (GC) remains one of the prevalent causes of cancer-related deaths globally. Long non-coding RNAs (lncRNAs) have been associated with different cancers. The polarization of macrophages towards the M2 (alternatively activated) phenotype promotes immunologic tolerance and can induce gastric tumorigenesis. Thus far, lncRNAs have been shown to modulate the differentiation of immune cells. Here, we investigated the biological effects of LINC00665 on the progression of GC and explored the mechanisms underlying its ability to mediate the polarization of macrophages towards the M2 phenotype. We report that the levels of LINC00665 were increased in GC tissues. Furthermore, this increase in LINC00665 expression could be associated with decreased overall survival (OS), progression-free survival (PFS), and post-progression survival (PPS). Using cell-based macrophage polarization models, we demonstrated that LINC00665 upregulation in GC cells facilitated the polarization of macrophages towards the M2 but not M1 (classically activated) phenotype. Furthermore, the loss of LINC00665 prevented the M2 polarization of macrophages. Mechanically, we identified that Wnt1 was the downstream target of LINC00665. Additionally, LINC00665 could directly interact with the transcription factor BTB domain and CNC homology 1 (BACH1). The interaction between LINC00665 and BACH1 resulted in the activation and binding of BACH1 to the Wnt1 promoters. Furthermore, BACH1 silencing could inhibit GC progression, which highlighted a crucial role for BACH1 in LINC00665-mediated Wnt1 activation. In addition, genetic Wnt1 overexpression effectively abolished the repression of Wnt signaling after BACH1 depletion and mediated GC development by supporting M2 macrophage polarization. In conclusion, we report that LINC00665 modulates M2 macrophage polarization and suggest that it may facilitate macrophage-dependent GC progression. • LINC00665 level was increased in GC tissues. • M2 showed an increased expression of LINC00665 and LINC00665 contributed to M2-like polarization of macrophages. • LINC00665 binding to BACH1 increased its occupancy in Wnt1 promoter. [ABSTRACT FROM AUTHOR]

Loss-of-function studies have identified Porcupine (PORCN) and Wntless (WLS) as essential mediators of Wnt secretion and signaling. Whereas PORCN is thought to palmitoylate Wnt proteins, WLS is believed to transport palmitoylated Wnt proteins to the cell surface. However, little is known about how these two proteins cooperate to regulate Wnt palmitoylation, trafficking, secretion, and signaling. We first investigated possible interactions between PORCN, WLS, and WNT1, by carrying out co-immunoprecipitation studies. These studies demonstrate the existence of a complex containing PORCN and WLS. They further show that PORCN and WLS compete for binding to WNT1. Then, we used gain-of-function studies to investigate the cooperation between PORCN and WLS as well as possible biochemical interactions between PORCN, WLS, and WNT1. Consistent with the proposed roles for PORCN and WLS, we show that overexpression of PORCN promotes palmitoylation of WNT1 while overexpression of WLS does not. Overexpression of PORCN enhances the ability of WLS to promote WNT1 trafficking to the cell surface as well as secretion, but decreases the ability of WLS to activate WNT1 signaling in target cell. These observations suggest that the levels of WNT1 on the cell surface and in the media are not the sole determinants of the activation of Wnt signaling in target cells.

A crucial event during the development of the central nervous system (CNS) is the early subdivision of the neural tube along its anterior-to-posterior axis to form neuromeres, morphogenetic units separated by transversal constrictions and programed for particular genetic cascades. The narrower portions observed in the developing neural tube are responsible for relevant cellular and molecular processes, such as clonal restrictions, expression of specific regulatory genes, and differential fate specification, as well as inductive activities. In this developmental context, the gradual formation of the midbrain-hindbrain (MH) constriction has been an excellent model to study the specification of two major subdivisions of the CNS containing the mesencephalic and isthmo-cerebellar primordia. This MH boundary is coincident with the common Otx2 -(midbrain)/ Gbx2 -(hindbrain) expressing border. The early interactions between these two pre-specified areas confer positional identities and induce the generation of specific diffusible morphogenes at this interface, in particular FGF8 and WNT1. These signaling pathways are responsible for the gradual histogenetic specifications and cellular identity acquisitions with in the MH domain. This review is focused on the cellular and molecular mechanisms involved in the specification of the midbrain/hindbrain territory and the formation of the isthmic organizer. Emphasis will be placed on the chick/quail chimeric experiments leading to the acquisition of the first fate mapping and experimental data to, in this way, better understand pioneering morphological studies and innovative gain/loss-of-function analysis. [ABSTRACT FROM AUTHOR]

Complexes of p24 proteins act as cargo receptors for the transport of COPII vesicles from the endoplasmic reticulum (ER). The major cargos of p24 complexes are hydrophilic proteins tethered to the ER membrane via a covalently attached glycosylphosphatidylinositol (GPI) or fatty acid. Each p24 complex is known to contain members from all four p24 subfamilies (p24α, p24β, p24γ and p24δ). However, it remains unclear how the cargo specificities of p24 complexes are influenced by member stoichiometry. Here, we report the subunit compositions of mammalian p24 complexes involved in the transport of GPI-anchored proteins and Wnt1. We show that at least one p24α is required for the formation of p24 complexes and that a p24 complex consisting of p24α2, p24β1, p24γ2 and p24δ1 is required for the efficient transport of GPI-anchored proteins. On the other hand, a p24 complex containing p24α2, p24α3, p24β1, p24γ and p24δ1 is involved in the transport of Wnt1. Further, interactions between p24α2 and p24α3 are critical for Wnt1 transport. Thus, p24α and p24γ subfamily members are important for cargo selectivity. Lastly, our data fit with an octamer, rather than a tetramer, model of p24 complexes, where each complex consists of two proteins from each p24 subfamily. [ABSTRACT FROM AUTHOR]

Abstract Background NEK2, a serine/threonine kinase involved in mitosis, has been found to function in chromosome instability, tumor progression and metastasis, but its role in cervical cancer radioresistance remains unknown. Methods We detected the protein levels of NEK2 in cervical carcinoma tissues and paired paracarcinoma tissues by immunohistochemistry. The roles of NEK2 in oncogenesis were examined using cell growth and colony formation assays, EdU assay, apoptosis assay as well as in vivo mouse model. γ-H2AX and Rad51 foci formation, neutral comet assay and clonogenic cell survival assay were applied to determine the radiosensitivity of cervical cancer cells. RNA-seq was performed to identify the downstream effector of NEK2. The gene expression levels were measured by Real-time PCR. Results We report that NEK2 protein level is overexpressed and correlated with the tumor stage and lymph node metastasis in cervical cancer tissues. Furthermore, we provided evidence that depletion of NEK2 impairs oncogenesis and enhances radiosensitivity in cervical cancer. Using RNA sequencing, we identify Wnt1 as a key downstream effector of NEK2. Knockdown of NEK2 downregulates the mRNA and protein levels of Wnt1, thereby inhibiting the activation of the Wnt/β-catenin signaling pathway. More importantly, the observed consequences induced by NEK2 depletion in cervical cancer cells can be partially rescued by Wnt1 overexpression. Conclusions Our results demonstrate that NEK2 activates the Wnt/β-catenin signaling pathway via Wnt1 to drive oncogenesis and radioresistance in cervical cancer, indicating that NEK2 may be a promising target for the radiosensitization of cervical cancer.

Abstract The highly conserved and ubiquitously expressed transcription factor Yin Yang 1 (Yy1), was named after its dual functions of both activating and repressing gene transcription. Yy1 plays complex roles in various fundamental biological processes such as the cell cycle progression, cell proliferation, survival, and differentiation. Patients with dominant Yy1 mutations suffer from central nervous system (CNS) developmental defects. However, the role of Yy1 in mammalian CNS development remains to be fully elucidated. The isthmus organizer locates to the mid-hindbrain (MHB) boundary region and serves as the critical signaling center during midbrain and cerebellar early patterning. To study the function of Yy1 in mesencephalon/ rhombomere 1 (mes/r1) neuroepithelium development, we utilized the tissue-specific Cre-LoxP system and generated a conditional knockout mouse line to inactivate Yy1 in the MHB region. Mice with Yy1 deletion in the mes/r1 region displayed cerebellar agenesis and dorsal midbrain hypoplasia. The Yy1 deleted neuroepithelial cells underwent cell cycle arrest and apoptosis, with the concurrent changes of cell cycle regulatory genes expression, as well as activation of the p53 pathway. Moreover, we found that Yy1 is involved in the transcriptional activation of Wnt1 in neural stem cells. Thus, our work demonstrates the involvement of Yy1 in cerebellar agenesis and the critical function of Yy1 in mouse early MHB neuroepithelium maintenance and development.

Abstract Background Breast cancer is a heterogeneous disease. Hence, stratification of patients based on the subtype of breast cancer is key to its successful treatment. Among all the breast cancer subtypes, basal-like breast cancer is the most aggressive subtype with limited treatment options. Interestingly, we found focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase, is highly overexpressed and activated in basal-like breast cancer. Methods To understand the role of FAK in this subtype, we generated mice with conditional deletion of FAK and a knock-in mutation in its kinase domain in MMTV-Wnt1-driven basal-like mammary tumors. Tumor initiation, growth, and metastasis were characterized for these mice cohorts. Immunohistochemical and transcriptomic analysis of Wnt1-driven tumors were also performed to elucidate the mechanisms underlying FAK-dependent phenotypes. Pharmacological inhibition of FAK and mTOR in human basal-like breast cancer cell lines was also tested. Results We found that in the absence of FAK or its kinase function, growth and metastasis of the tumors were significantly suppressed. Furthermore, immunohistochemical analyses of cleaved caspase 3 revealed that loss of FAK results in increased tumor cell apoptosis. To further investigate the mechanism by which FAK regulates survival of the Wnt1-driven tumor cells, we prepared an isogenic pair of mammary tumor cells with and without FAK and found that FAK ablation increased their sensitivity to ER stress-induced cell death, as well as reduced tumor cell migration and tumor sphere formation. Comparative transcriptomic profiling of the pair of tumor cells and gene set enrichment analysis suggested mTOR pathway to be downregulated upon loss of FAK. Immunoblot analyses further confirmed that absence of FAK results in reduction of AKT and downstream mTOR pathways. We also found that inhibition of FAK and mTOR pathways both induces apoptosis, indicating the importance of these pathways in regulating cell survival. Conclusions In summary, our studies show that in a basal-like tumor model, FAK is required for survival of the tumor cells and can serve as a potential therapeutic target.

Purpose: This study tested if the protective anti-remodeling effect of GLP-1 agonist Exendin-4 after an acute myocardial infarction (MI) in rats involves inhibition of the Wnt1/β-catenin signaling pathway. Methods: Rats were divided into sham, sham + Exendin-4 (10 μg/day, i.p), MI, and MI + Exendin-4. MI was introduced to rats by permanent left anterior descending coronary artery (LAD) ligation. Results: On day 7 post-infraction, MI rats showed LV dysfunction with higher serum levels of cardiac markers. Their remote myocardia showed increased mRNA and protein levels of collagen I/III with higher levels of reactive oxygen species (ROS) and inflammatory cytokines, as well as protein levels of Wnt1, phospho-Akt, transforming growth factor (TGF-β1), Smad, phospho-Smad3, α-SMA, caspase-3, and Bax. They also showed higher protein levels of phospho-glycogen synthase kinase-3β (p-GSK3β), as well as total, phosphorylated, and nuclear β-catenin with a concomitant decrease in the levels of cyclic adenosine monophosphate (cAMP), mRNA of manganese superoxide dismutase (MnSOD), and protein levels of Bcl-2, β-arrestin-2, and protein phosphatase-2 (PP2A). Administration of Exendin-4 to MI rats reduced the infarct size and reversed the aforementioned signaling molecules without altering protein levels of TGF-1β and Wnt1 or Akt activation. Interestingly, Exendin-4 increased mRNA levels of MnSOD, protein levels of β-arrestin-2 and PP2A, and β-catenin phosphorylation but reduced the phosphorylation of GSK3β and Smad3, and total β-catenin levels in the LV of control rats. Conclusion: Exendin-4 inhibits the remodeling in the remote myocardium of rats following acute MI by attenuating β-catenin activation and activating β-arrestin-2, PP2A, and GSK3β. [ABSTRACT FROM AUTHOR]

Studies suggest that Wnt/β-catenin agonists are beneficial in the treatment of acute kidney injury (AKI); however, it remains elusive about its role in the prevention of AKI and its progression to chronic kidney disease (CKD). In this study, renal Wnt/β-catenin signaling was either activated by overexpression of exogenous Wnt1 or inhibited by administration with ICG-001, a small molecule inhibitor of β-catenin signaling, before mice were subjected to ischemia/reperfusion injury (IRI) to induce AKI and subsequent CKD. Our results showed that in vivo expression of exogenous Wnt1 before IR protected mice against AKI, and impeded the progression of AKI to CKD in mice, as evidenced by both blood biochemical and kidney histological analyses. In contrast, pre-treatment of ICG-001 before IR had no effect on renal Wnt/β-catenin signaling or the progression of AKI to CKD. Mechanistically, in vivo expression of exogenous Wnt1 before IR suppressed the expression of proapoptotic proteins in AKI mice, and reduced inflammatory responses in both AKI and CKD mice. Additionally, exogenous Wnt1 inhibited apoptosis of tubular cells induced by hypoxia-reoxygenation (H/R) treatment in vitro. To conclude, the present study provides evidences to support the preventive effect of Wnt/β-catenin activation on IR-related AKI and its subsequent progression to CKD. [ABSTRACT FROM AUTHOR]

The role of Wnt family proteins, E-cadherin, and β-catenin in non-small cell lung cancer (NSCLC) is unclear. In this study, we assessed the expression of these proteins as well as their reciprocal interaction and clinical relevance in NSCLC. Immunohistochemical expression of Wnt1, Wnt2, E-cadherin, and β-catenin was assessed in 208 patients with NSCLC who underwent curative pulmonary resection. Expression of Wnt1, Wnt2, and E-cadherin was found in 49.5%, 22.3%, and 37.4% of the patients, respectively, whereas expression of membranous and cytoplasmic β-catenin was found in 23.7% and 34.8% of the patients, respectively. The expression of Wnt1 and E-cadherin was lower in squamous cell carcinoma than in adenocarcinoma and large cell carcinoma, and the expression of both Wnt proteins, E-cadherin, and membranous β-catenin was lower in poorly differentiated compared with well-differentiated tumors. None of the analyzed proteins was associated with relapse-free or overall survival. Expression of Wnt1, Wnt2, E-cadherin, and β-catenin is a common occurrence in NSCLC and is related to tumor histology and grade. However, these proteins have no prognostic role in operable NSCLC: [ABSTRACT FROM AUTHOR]

Studies suggest that Wnt/β-catenin agonists are beneficial in the treatment of acute kidney injury (AKI); however, it remains elusive about its role in the prevention of AKI and its progression to chronic kidney disease (CKD). In this study, renal Wnt/β-catenin signaling was either activated by overexpression of exogenous Wnt1 or inhibited by administration with ICG-001, a small molecule inhibitor of β-catenin signaling, before mice were subjected to ischemia/reperfusion injury (IRI) to induce AKI and subsequent CKD. Our results showed that in vivo expression of exogenous Wnt1 before IR protected mice against AKI, and impeded the progression of AKI to CKD in mice, as evidenced by both blood biochemical and kidney histological analyses. In contrast, pre-treatment of ICG-001 before IR had no effect on renal Wnt/β-catenin signaling or the progression of AKI to CKD. Mechanistically, in vivo expression of exogenous Wnt1 before IR suppressed the expression of proapoptotic proteins in AKI mice, and reduced inflammatory responses in both AKI and CKD mice. Additionally, exogenous Wnt1 inhibited apoptosis of tubular cells induced by hypoxia-reoxygenation (H/R) treatment in vitro. To conclude, the present study provides evidences to support the preventive effect of Wnt/β-catenin activation on IR-related AKI and its subsequent progression to CKD.

Wnt1 is one of the morphogenes that controls the specification and differentiation of neuronal populations in the developing central nervous system. The habenula is a diencephalic neuronal complex located in the most dorsal aspect of the thalamic prosomere. This diencephalic neuronal population is involved in the limbic system and its malfunction is related with several psychiatric disorders. Our aim is to elucidate the Wnt1 role in the habenula and its main efferent tract, the fasciculus retroflexus, development. In order to achieve these objectives, we analyzed these structures development in a Wnt1 lack of function mouse model. The habenula was generated in our model, but it presented an enlarged volume. This alteration was due to an increment in habenular neuroblasts proliferation rate. The fasciculus retroflexus also presented a wider and disorganized distribution and a disturbed final trajectory toward its target. The mid-hindbrain territories that the tract must cross were miss-differentiated in our model. The specification of the habenula is Wnt1 independent. Nevertheless, it controls its precursors proliferation rate. Wnt1 expressed in the isthmic organizer is vital to induce the midbrain and rostral hindbrain territories. The alteration of these areas is responsible for the fasciculus retroflexus axons misroute.

试验研究植物甾醇对猪生产性能、免疫指标及Wnt1 基因表达的影响。选择240 头"杜×长×大"三 元猪, 公、母各半, 将试验猪随机分为4 组, 每组6 个重复, 每个重复10 头试验猪。分别在日粮中添加0、100、 200、300 g/t 植物甾醇, 按照植物甾醇添加量依次命名为A （对照组）、B、C、D 组。试验期50 d。结果显示, B、 C、D 组试验猪末重显著高于对照组（P<0.05）, 平均日增重（ADG）、平均日采食量（ADFI） 均极显著高于对照 组（P<0.01）, 但料重比（F/G） 显著低于对照组（P<0.05）。眼肌面积（LMA） 随着植物甾醇的添加量提高而极 显著升高（P<0.01）。与对照组对比, 试验组血清免疫球蛋白A （IgA） 显著提高（P<0.05）, 免疫球蛋白G （IgG） 含量极显著提高（P<0.01）, 免疫球蛋白M （IgM） 含量无显著差异（P>0.05）。试验组猪血清中总超氧化物歧化 酶（T-SOD） 活性极显著高于对照组（P<0.01）, 谷胱甘肽过氧化物酶（GSH-Px） 活性、总抗氧化能力 （T-AOC） 显著高于对照组（P<0.05）, 血清丙二醛（MDA） 含量显著降低。试验组育肥猪的肝脏与背最长肌中 Wnt1 基因的相对表达量均极显著高于对照组（P<0.01）, 并且随着植物甾醇添加量的提高呈上升趋势。研究表 明, 本试验条件下, 建议在猪日粮中添加100~200 g/t 植物甾醇. [ABSTRACT FROM AUTHOR]

During embryonic stages, vascular endothelial cells (ECs) originate from the mesoderm, at specific extraembryonic and embryonic regions, through a process called vasculogenesis. In the adult, EC renewal/replacement mostly depend on local resident ECs or endothelial progenitor cells (EPCs). Nevertheless, contribution from circulating ECs/EPCs was also reported. In addition, cells lacking from EC/EPC markers with in vitro extended plasticity were shown to originate endothelial-like cells (ELCs). Most of these cells consist of mesenchymal stromal progenitors, which would eventually get mobilized from the bone marrow after injury. Based on that, current knowledge on different mouse and human bone marrow stromal cell (BM-SC) subpopulations, able to contribute with mesenchymal stromal/stem cells (MSCs), is herein reviewed. Such analyses underline an unexpected heterogeneity among sinusoidal LepR+ stromal/CAR cells. For instance, in a recent report a subgroup of LepR+ stromal/CAR progenitors, which express GLAST and is traced in Wnt1Cre;R26RTom mice, was found to contribute with ELCs in vivo. These GLAST ​+ ​Wnt1+ BM-SCs were shown to get mobilized to the peripheral blood and to contribute with liver regeneration. Other sources of ELCs, such as adipose, neural and dental pulp tissues, were also published. Finally, mechanisms likely involved in the enhanced cellular plasticity properties of bone marrow/adipose tissue stromal cells, able to originate ELCs, are assessed. In the future, strategies to analyze the in vivo expression profile of stromal cells, with MSC properties, in combination with screening of active genomic regions at the single cell-level, during early postnatal development and/or after injury, will likely help understanding properties of these ELC sources. • Extraembryonic as well as embryonic vessels origins are first discussed. • Most adult endothelial cells derive from local endothelial progenitor cells. • Human and mouse bone marrow contain similar subpopulations of stromal progenitors. • GLAST ​+ ​Wnt1+ bone marrow stromal cells contribute to few distant endothelial cells. • Mechanisms probably involved in the increased plasticity are addressed. [ABSTRACT FROM AUTHOR]

To explore the effect and underlying molecular mechanism of long non‐coding RNA (lncRNA)‐H19 on ovarian cancer (OC) cells, a total of 41 cases of OC and adjacent normal tissues were collected. H19 and microRNA (miR)‐140 expressions in OC tissues and cells were detected using quantitative real‐time polymerase chain reaction (qRT‐RCR). The correlation between H19 expression and prognosis of OC patient was analyzed. siRNA (si)‐H19 and si‐negative control (NC) were transfected into OC cells. Cell proliferation was checked by cell counting kit‐8 assay and colony formation assay, and cell migration and invasion were analyzed via Transwell assay. The targeted binding relationship between H19 and miR‐140 was predicted and verified, miR‐140 downstream gene was predicted and Wnt1 was screened out. The impact of in‐miR‐140 on the si‐H19‐induced decreased OC cell proliferation and migration was evaluated. H19 expression was upregulated in OC tissues and cells, and its overexpression was associated with a poor prognosis of OC. si‐H19 remarkably reduced OC cell proliferation and migration. H19 upregulated Wnt1 expression through targeting miR‐140 in OC cells. Altogether, miR‐140 was notably downregulated in OC, and in‐miR‐140 partially inhibited the si‐H19‐induced decrease of OC cell proliferation and migration. H19 competitively bound to miR‐140 to upregulate Wnt1, thereby promoting OC cell proliferation and migration. [ABSTRACT FROM AUTHOR]

Background: This study aimed to investigate the exact regulatory mechanisms of exosomal miR-34c in mediating communication between cholangiocarcinoma cells and fibroblasts. Methods: Exosomes were isolated from HuCCT-1 and HIBEC cells using differential ultracentrifugation and identified by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) method. Real-time quantitative PCR (qRT-PCR) and western blotting analyses were performed to assess the levels of pro-inflammatory factors, and fibroblast-related proteins and Wnt-linked signaling pathway proteins, respectively. Exosome-tracking was performed with confocal microscopy. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and Transwell assays were used to measure cell proliferation and migration, respectively. Further, the oncogenicity of cholangiocarcinoma cells was analyzed in nude mice transplanted tumor model. Results: The analysis suggested that the expression of miR-34c was decreased in exosomes from HuCCT-1 cells. Moreover, miR-34c in exosomes mediated fibroblast activation by directly targeting WNT1. Additionally, cancer-associated fibroblasts (CAFs) activated by downregulation of exosomal miR-34c promoted cholangiocarcinoma progression. Conclusions: Thus, miR-34c in exosomes was found to be a key player in regulating intercellular communication between tumor cells and fibroblasts. [ABSTRACT FROM AUTHOR]

Background: Circular RNAs (circRNAs) play important roles in the progression of gastric cancer (GC). The Wnt1/β-catenin pathway can promote the proliferation of GC cells. This study aimed to explore whether circRNA_0044516 can regulate the proliferation of GC cells by modulating the Wnt1/β-catenin pathway. Methods: The expression of circRNA_0044516, miR-149, Wnt1, and β-catenin in GC tissues or cells was detected by qRT-PCR and western blot. Cell viability and apoptosis were measured by CCK-8 and flow cytometry assays, respectively. The interaction between circRNA_0044516 and miR-149 was determined by luciferase reporter and RNA pull-down assays. Results: Upregulated circRNA_0044516 was found in GC tissues and cell lines. Downregulating circRNA_0044516 inhibited the viability and promoted apoptosis of GC cells. CircRNA_0044516 targeted miR-149, and its downregulation elevated miR-149 level in GC cells. Mechanistically, silencing circRNA_0044516 reduced the protein level of Wnt1 and β-catenin through miR-149, and finally suppressed viability and contributed to apoptosis of GC cells. Moreover, circRNA_0044516 knockdown inhibited the tumor growth of HGC-27 cells in nude mice. Conclusions: Our results indicated an important role of circRNA_0044516 in GC and elucidated that downregulation of circRNA_0044516 inhibits the proliferation of GC cells through miR-149/Wnt1/β-catenin. [ABSTRACT FROM AUTHOR]

• The interplay between macrophages and tumor cells in the TME promotes HCC progression. • M2 macrophages secrete Wnt1 to ignite β-catenin pathway in tumor cells for HCC growth. • Bufalin suppresses Wnt1 secretion by blocking gene transcription in M2 macrophages. • Bufalin induces HCC regression through the immunomodulatory effect on M2 macrophages. The interplay of tumor-associated macrophages (TAMs) and tumor cells plays a key role in the development of hepatocellular carcinoma (HCC) and provides an important target for HCC therapy. The communication between them is still on the investigation. Bufalin, the active component derived from the traditional Chinese medicine (TCM) Chansu , has been evidenced to possess anti-HCC activity by directly suppressing tumor cells, while its immunomodulatory effect on the tumor microenvironment (TME) is unclear. To explore the mechanism of M2 TAM-governed tumor cell proliferation and the inhibitory effect of bufalin on HCC growth by targeting M2 macrophages. Morphology and marker proteins were detected to evaluate macrophage polarization via microscopy and flow cytometry. Cellular proliferation and malignant transformation of HCC cells cultured with macrophage conditioned medium (CM) or bufalin-primed M2-CM, were assessed by cell viability, colony formation and soft agar assays. Regulations of gene transcription and protein expression and release were determined by RT-qPCR, immunoblotting, immunoprecipitation, ELISA and immunofluorescence. Tumorigenicity upon bufalin treatment was verified in orthotopic and diethylnitrosamine-induced HCC mouse model. In this study, we first verified that M2 macrophages secreted Wnt1, which acted as a mediator to trigger β-catenin activation in HCC cells, leading to cellular proliferation. Bufalin suppressed HCC cell proliferation and malignant transformation by inhibiting Wnt1 release in M2 macrophages, and dose-dependently inhibited HCC progression in mice. Mechanistically, bufalin specially targeted to block Wnt1 transcription, thus inactivating β-catenin signaling cascade in HCC cells and leading to tumor regression in HCC mouse model. These results clearly reveal a novel potential of bufalin to suppress HCC through immunomodulation, and shed light on a new M2 macrophage-based modality of HCC immunotherapy, which additively enhances direct tumor-inhibitory efficacy of bufalin. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract Background MicroRNAs (miRNAs) play a key role in regulating cell differentiation. In the present study, we aimed to explore the role of miR-140-5p in odontoblastic differentiation of dental pulp stem cells (DPSCs). Methods DPSCs from normal human impacted third molars were isolated and cultured. After overexpression or silencing of miR-140-5p in DPSCs, activity, proliferation, and odontoblastic differentiation of DPSCs were evaluated. The possible target gene of miR-140-5p was verified by luciferase reporter gene assay. Using gene transfection technology, RT-CPR, and Western blot to confirm miR-140-5p regulates the odontoblastic differentiation of DPSCs through Wnt1/β-catenin signaling. Results We found the expression of miR-140-5p decreased in the differentiated DPSCs for odontoblastic cells, and at the same time, the expressions of Wnt1 and β-catenin increased. Wnt1 was the target gene of miR-140-5p which was confirmed by luciferase reporter gene system. miR-140-5p overexpression suppressed the expression of Wnt1. miR-140-5p inhibitor could promote the odontoblastic differentiation of DPSCs. miR-140-5p mimic could weaken the odontoblastic differentiation of DPSCs, which could be reversed by the overexpression of Wnt1. Conclusion Our data demonstrated that miR-140-5p regulates the odontoblastic differentiation of DPSCs via targeting Wnt1/β-catenin signaling. Therefore, miR-140-5p might be a molecular target to regulate the odontoblastic differentiation for the therapeutic agents in dental medicine.

Background Aspirin, an anti‐inflammatory drug, has been widely investigated in the treatment of many cancer types, including colorectal, ovarian, breast, and prostate cancers. MicroRNAs (miRNAs) are the most well studied noncoding RNAs in cancers. In the current study, we were interested in defining the function of aspirin in lung cancer treatment and the related noncoding RNAs involved in this process. Methods The function of aspirin in lung cancer growth was evaluated by cell viability and colony formation assays. Screening of miRNAs affected by aspirin was performed through quantitative real‐time PCR. Prediction of miR‐98 targeting WNT1 was performed using online bioinformatics software and was further confirmed by luciferase reporter gene analysis. The levels of miR‐98 and WNT1 were tested through immunoblotting and quantitative real‐time PCR analysis in lung cancer cells under aspirin treatment. Results Cell viability was sharply suppressed in lung cancer cells with an increasing dose of aspirin. Aspirin markedly weakened the malignant colony formation ability of lung cancer cells. One out of six tumor suppressor miRNAs could be obviously regulated by aspirin in lung cancer cells. The inhibition of miR‐98 on the luciferase activities of wild‐type 3' untranslated region vectors of WNT1 was clearly revealed in lung cancer cells. Meanwhile, the inhibitor of miR‐98 increased the luciferase activities of wild‐type 3' untranslated region vectors of WNT1. After treatment with aspirin the expression of miR‐98 was induced and then its target gene, WNT1, was depressed in the cells. Conclusion Aspirin targets the miR‐98/WNT1 axis to ameliorate lung cancer development.

Background: WNT1 c.110 T>C and c.505G>T missense mutations have been identified in patients with osteogenesis imperfecta (OI). Whether these mutations affect osteoblast differentiation remains to be determined. This study aimed to investigate the effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast function, gene expression, and pathways involved in OI. Methods: Empty vector (negative control), wild-type WNT1, WNT1 c.110 T>C, WNT1 c.505G>T, and WNT1 c.884C>A (positive control) mutant plasmids were constructed and transfected into preosteoblast (MC3T3-E1) cells to investigate their effect on osteoblast differentiation. The expressions of osteoblast markers, including BMP2, RANKL, osteocalcin, and alkaline phosphatase (ALP), were determined using quantitative real-time polymerase chain reaction (RT-qPCR), western blotting (WB), enzyme-linked immunosorbent assay, and ALP staining assay, respectively. The mRNA and protein expression levels of WNT1 or the expression levels of the relevant proteins involved in the WNT1/β-catenin signaling pathway were also determined using RT-qPCR, WB, and immunofluorescence (IF) assays after the different plasmids were transfected into MC3T3-E1 cells. Results: Compared with those in the wild-type group, in the mutation groups, the mRNA and protein expression levels of BMP2 were suppressed, the expressions of osteocalcin and ALP were inhibited, and the mRNA and protein expression levels of RANKL were enhanced in MC3T3-E1 cells. WB and IF assays revealed that the protein expression levels of WNT1 in MC3T3-E1 cells were downregulated in the mutation groups compared with those in the wild-type WNT1 group. Furthermore, the expression levels of nonphosphorylated β-catenin (non-p-β-catenin) and phosphorylated GSK-3β (p-GSK-3β) were downregulated in the mutation groups compared with those in the wild-type group. However, no significant changes in the expression level of non-p-β-catenin or p-GSK-3β were observed in the mutation groups. Conclusions: WNT1 c.110 T>C and c.505G>T mutations may alter the proliferation and osteogenic phenotype of MC3T3-E1 linked to the progression of OI via the inhibition of the WNT1/β-catenin signaling pathway. This is the first study to confirm the effect of WNT1 c.110 T>C and c.505G>T missense mutations on osteoblast differentiation and propose a new molecular mechanism for OI development. [ABSTRACT FROM AUTHOR]

Objective: In this study, we investigated the potential and mechanism of odontogenic ameloblast‐associated protein (ODAM) in the promoting junctional epithelium‐related gene expression in an ameloblast‐like cell line ALC. Background: ODAM is expressed in ameloblasts and JE and acts as a component of the inner basal lamina (IBL) and intercellular matrix of JE. ODAM KO mice showed destruction of the integrity of the JE, which detaches from teeth. ODAM was confirmed to regulate the cytoskeleton through the ODAM‐ARHGEF5‐RhoA signaling pathway of the JE. Whether ODAM contributes to the regulation of ameloblast differentiation in JE remains unclear. After the formation of enamel, the ameloblast undergoes a series of morphological changes. Whether ODAM will affect the biological behavior of ameloblasts making them have the characteristics of JE is unclear. Methods: A murine ameloblast‐like cell line, ALC, was used to investigate the effects of ODAM on the JE‐like changes of ALC cells in an epithelium‐induced environment by generating ODAM overexpression and ODAM knockdown cells through a lentivirus transduction approach. The biomarkers of junctional epithelium CK19, SLPI, and ODAM and the potential regulatory gene WNT1 were investigated by real‐time PCR, western blot, immunocytochemistry, immunostaining, luciferase reporter, and rescue assays. Results: ODAM, CK19, and SLPI were significantly upregulated after epithelial induction. Overexpression of ODAM in ALC cells markedly increased CK19 and SLPI expression, while knockdown of ODAM in ALC cells clearly decreased CK19 and SLPI expression. A reporter luciferase assay showed that ODAM activated the WNT signaling pathway, especially through WNT1. Exogenous overexpression of ODAM upregulated WNT1 expression, while knockdown of ODAM reversed this effect. The WNT1 inhibition assay further confirmed the above results and showed that the WNT1 pathway was positively correlated with biomarkers of junctional epithelium CK19 and SLPI expression. Rescue studies showed that knocking down WNT1 in the ODAM‐overexpressing ALC cells decreased the expression of CK19 and SLPI. Immunocytochemistry showed that ODAM colocalized with CK19, SLPI, and WNT1 in the cells. Conclusion: In conclusion, the research work showed that ODAM promotes junctional epithelium‐related gene expression in ALC via the ODAM‐WNT1 axis, which may provide new insight into the function of ODAM and JE formation. [ABSTRACT FROM AUTHOR]

Gallic acid is a phenolic compound with biological and pharmacological activities. Therefore, our study aimed to examine whether gallic acid has a beneficial effect against type 2-induced diabetic hepatic injury in rats and attempt to discover its possible intracellular pathways. Adult male rats were subdivided into six groups: Control, DM (diabetes mellitus), GA (gallic acid)+DM, DM+GA, DM+MET (metformin) and DM+GA+MET. Type 2 diabetes mellitus (T2DM) induced a significant increase in the blood glucose, HOMA-IR, liver enzymes, fetuin-A, hepatic triglycerides content with diminished serum insulin and hepatic glycogen content associated with impairment of cellular redox balance. Administration of gallic acid successfully restored all these alterations which was confirmed by marked improvement of the histopathological changes of the liver. Significantly, gallic acid increased the expression of glucagon-like peptide-1 (GLP-1) immunoreactive cells in the terminal ileum with negative correlation observed between fetuin-A and GLP-1 cells. Furthermore, our results discovered that gallic acid could diminish the DM-induced hepatic damage via upregulated hepatic mRNA expression of GLUT-4, Wnt1 and β-catenin with inhibitory effects on the elevated expression of ERK1/2/NF-κB. In conclusion, this study suggests that gallic acid provides a significant protection against T2DM-mediated liver injury. The use of gallic acid with traditional anti-diabetic drug enhanced its efficiency compared with traditional drug alone. [ABSTRACT FROM AUTHOR]

Alcohol use disorder is associated with pathophysiological changes in the dopaminergic system. Orthodenticle homeobox 2 (OTX2) is a transcription factor important for the development of dopaminergic neurons residing in the ventral tegmental area (VTA), a critical region of the brain involved in drug reinforcement. Previous studies have demonstrated that ethanol exposure during embryonic development reduces Otx2 mRNA levels in the central nervous system. We hypothesized that levels of OTX2 would be altered by binge-like ethanol consumption in adult animals. To test this, Otx2 mRNA and protein levels in the mouse VTA were measured by quantitative real-time PCR and western blotting, respectively, after mice drank ethanol for 4 days in a procedure that elicits binge levels of ethanol consumption (drinking in the dark). Expression of known and putative OTX2 transcriptional target genes (Sema3c, Wnt1, and Mdk) were also measured in the VTA after ethanol drinking. Otx2 mRNA and protein levels were elevated in the VTA 24 hours after the fourth drinking session and there was a corresponding increase in the expression of Mdk transcript. Interestingly, Wnt1 transcript was elevated in the VTA immediately after the fourth drinking session but returned to control levels 24 hours later. We next investigated if viral-mediated reduction of Otx2 in the mouse VTA would alter ethanol or sucrose intake. Lentiviral vectors expressing a shRNA targeting Otx2 or a control shRNA were injected into the VTA and mice were tested in the drinking in the dark protocol for ethanol and sucrose drinking. Reducing levels of OTX2 in the VTA did not alter ethanol or sucrose consumption. One limitation is that the extent of OTX2 reduction may not have been sufficient. Although OTX2 in the VTA may not play a role in binge-like drinking in adult mice, OTX2 could contribute to ethanol-induced transcriptional changes in this region. [ABSTRACT FROM AUTHOR]

Osteogenesis imperfecta (OI) is a rare heritable skeletal disorder which is mainly caused by defected type I collagen. Autosomal recessive OI (AR-OI) is caused by mutations of genes that are responsible for type I collagen modification and folding, and is often associated with more severe phenotypes. Due to the limited number of recessive OI patients, it has been difficult to study the mutation spectrum as well as the correlation of genotype and phenotype. This study recruited a Chinese cohort of 74 AR-OI families, aiming to establish the mutation spectrum and to examine the genotypic and phenotypic correlation. We identified 82 variants including 25 novel variants and 57 HGMD reported variants in these AR-OI patients, using whole exome sequencing/panel sequencing combined with Sanger sequencing. Pathogenic mutations were found at WNT1 (n = 30, 40.54%), SERPINF1 (n = 22, 29.73%), FKBP10 (n = 10, 13.51%), CRTAP (n = 3, 4.05%), P3H1 (n = 3, 4.05%), SERPINH1 (n = 2, 2.70%), SEC24D (n = 3, 4.05%), and PLOD2 (n = 1, 1.35%) respectively. Thus, WNT1 represents the most frequent pathogenic gene of AR-OI in Chinese population. The most common clinical manifestations of AR-OI patients include walking problem (72.86%), scoliosis (65.28%) and frequent fractures (fractures ≥2/year) (54.05%). Interestingly, ptosis represents a unique phenotype of patients carrying WNT1 variants, and it was rare in patients harboring other pathogenic genes. Our study expanded the mutation spectrum of AR-OI and enriched the knowledge of genotypic and phenotypic correlation in Chinese cohort with AR-OI.

Abstract Background Wnt signaling pathway plays an important role in promoting ostergenesis. WNT1 mutations have been considered as a major cause of ostergenesis imperfect (OI). We identified an OI patient with pathogenic consanguineous‐derived homozygous WNT1 missense mutation. Methods We designed and applied a panel of known 261 genes associated with hereditary bone diseases for targeted next‐generation sequencing to examine clinically diagnosed OI patients. Detected mutations were confirmed by Sanger sequencing. Results The female proband presented with severe OI with low bone density, multiple long bone fractures, short stature, and absence of dentinogenesis imperfect and brain malformation. She had congenital ptosis and exotropia with her left eye, and absence of blue sclera. The proband came from a consanguineous family and had a homozygous WNT1 missense mutation (c.677C>T, (p.S226L)). In addition, three other compound heterozygous mutations (c.1729C>T in FKBP10, c.1958A>C in FGFR3, c.760G>C in TRPV4) were also detected in her family members. Conclusion We report the first identified case of consanguineous derived homozygous WNT1 mutation leading to severe osteogenesis imperfecta with congenital ptosis and exotropia.