ΔNp63 regulates Sfrp1 expression to direct salivary gland branching morphogenesis.

Branching morphogenesis is a complex process shared by many organs including the lungs, kidney, prostate, as well as several exocrine organs including the salivary, mammary and lacrimal glands. This critical developmental program ensures the expansion of an organ's surface area thereby maximizing processes of cellular secretion or absorption. It is guided by reciprocal signaling from the epithelial and mesenchymal cells. While signaling pathways driving salivary gland branching morphogenesis have been relatively well-studied, our understanding of the underlying transcriptional regulatory mechanisms directing this program, is limited. Here, we performed in vivo and ex vivo studies of the embryonic mouse submandibular gland to determine the function of the transcription factor ΔNp63, in directing branching morphogenesis. Our studies show that loss of ΔNp63 results in alterations in the differentiation program of the ductal cells which is accompanied by a dramatic reduction in branching morphogenesis that is mediated by dysregulation of WNT signaling. We show that ΔNp63 modulates WNT signaling to promote branching morphogenesis by directly regulating Sfrp1 expression. Collectively, our findings have revealed a novel role for ΔNp63 in the regulation of this critical process and offers a better understanding of the transcriptional networks involved in branching morphogenesis.

Novel 14q32.2 paternal deletion encompassing the whole DLK1 gene associated with Temple syndrome.

BACKGROUND: Temple syndrome (TS14) is a rare imprinting disorder caused by maternal UPD14, imprinting defects or paternal microdeletions which lead to an increase in the maternal expressed genes and a silencing the paternally expressed genes in the 14q32 imprinted domain. Classical TS14 phenotypic features include pre- and postnatal short stature, small hands and feet, muscular hypotonia, motor delay, feeding difficulties, weight gain, premature puberty along and precocious puberty. METHODS: An exon array comparative genomic hybridization was performed on a patient affected by psychomotor and language delay, muscular hypotonia, relative macrocephaly, and small hand and feet at two years old. At 6 years of age, the proband presented with precocious thelarche. Genes dosage and methylation within the 14q32 region were analyzed by MS-MLPA. Bisulfite PCR and pyrosequencing were employed to quantification methylation at the four known imprinted differentially methylated regions (DMR) within the 14q32 domain: DLK1 DMR, IG-DMR, MEG3 DMR and MEG8 DMR. RESULTS: The patient had inherited a 69 Kb deletion, encompassing the entire DLK1 gene, on the paternal allele. Relative hypermethylation of the two maternally methylated intervals, DLK1 and MEG8 DMRs, was observed along with normal methylation level at IG-DMR and MEG3 DMR, resulting in a phenotype consistent with TS14. Additional family members with the deletion showed modest methylation changes at both the DLK1 and MEG8 DMRs consistent with parental transmission. CONCLUSION: We describe a girl with clinical presentation suggestive of Temple syndrome resulting from a small paternal 14q32 deletion that led to DLK1 whole-gene deletion, as well as hypermethylation of the maternally methylated DLK1-DMR.

Effect of Regulation of Chemerin/Chemokine-like Receptor 1/Stimulator of Interferon Genes Pathway on Astrocyte Recruitment to Aß Plaques.

Recruitment and accumulation of reactive astrocytes around senile plaques are common pathological features of Alzheimer's disease (AD), with unclear mechanisms. Chemerin, an adipokine implicated in neuroinflammation, acts through its receptor, chemokine-like receptor 1 (CMKLR1), which also functions as a receptor for amyloid ß (Aß). The impact of the chemerin/CMKLR1 axis on astrocyte migration towards Aß plaques is unknown. Here we investigated the effect of CMKLR1 on astrocyte migration around Aß deposition in APP/PS1 mice with Cmklr1 knockout (APP/PS1-Cmklr1-/-). CMKLR1-expressed astrocytes were upregulated in the cortices and hippocampi of 9-month-old APP/PS1 mice. Chemerin mainly co-localized with neurons, and its expression was reduced in the brains of APP/PS1 mice, compared to WT mice. CMKLR1 deficiency decreased astrocyte colocalization with Aß plaques in APP/PS1-Cmklr1-/- mice, compared to APP/PS1 mice. Activation of the chemerin/CMKLR1 axis promoted the migration of primary cultured astrocytes and U251 cells, and reduced astrocyte clustering induced by Aß42. Mechanistic studies revealed that chemerin/CMKLR1 activation induced STING phosphorylation. Deletion of STING attenuated the promotion of the chemerin/CMKLR1 axis relative to astrocyte migration and abolished the inhibitory effect of chemerin on Aß42-induced astrocyte clustering. These findings suggest the involvement of the chemerin/CMKLR1/STING pathway in the regulation of astrocyte migration and recruitment to Aß plaques/Aß42.

Inhibition of PRMT5-mediated regulation of DKK1 sensitizes colorectal cancer cells to chemotherapy.

The Dickkopf family proteins (DKKs) are strong Wnt signaling antagonists that play a significant role in colorectal cancer (CRC) development and progression. Recent work has shown that DKKs, mainly DKK1, are associated with the induction of chemoresistance in CRC and that DKK1 expression in cancer cells correlates with that of protein arginine N-methyltransferase 5 (PRMT5). This points to the presence of a regulatory loop between DKK1 and PRMT5. Herein, we addressed the question of whether PRMT5 contributes to DKK1 expression in CRC and hence CRC chemoresistance. Both in silico and in vitro approaches were used to explore the relationship between PRMT5 and different DKK members. Our data demonstrated that DKK1 expression is significantly upregulated in CRC clinical samples, KRAS-mutated CRC in particular and that the levels of DKK1 positively correlate with PRMT5 activation. Chromatin immunoprecipitation (ChIP) data indicated a possible epigenetic role of PRMT5 in regulating DKK1, possibly through the symmetric dimethylation of H3R8. Knockdown of DKK1 or treatment with the PRMT5 inhibitor CMP5 in combination with doxorubicin yielded a synergistic anti-tumor effect in KRAS mutant, but not KRAS wild-type, CRC cells. These findings suggest that PRMT5 regulates DKK1 expression in CRC and that inhibition of PRMT5 modulates DKK1 expression in such a way that reduces CRC cell growth.

Identifying high-risk neurological phenotypes in adult-onset classic monogenic autoinflammatory diseases: when should neurologists consider testing?

BACKGROUND: Monogenic autoinflammatory disorders result in a diverse range of neurological symptoms in adults, often leading to diagnostic delays. Despite the significance of early detection for effective treatment, the neurological manifestations of these disorders remain inadequately recognized. METHODS: We conducted a systematic review searching Pubmed, Embase and Scopus for case reports and case series related to neurological manifestations in adult-onset monogenic autoinflammatory diseases. Selection criteria focused on the four most relevant adult-onset autoinflammatory diseases-deficiency of deaminase 2 (DADA2), tumor necrosis factor receptor associated periodic fever syndrome (TRAPS), cryopyrin associated periodic fever syndrome (CAPS), and familial mediterranean fever (FMF). We extracted clinical, laboratory and radiological features to propose the most common neurological phenotypes. RESULTS: From 276 records, 28 articles were included. The median patient age was 38, with neurological symptoms appearing after a median disease duration of 5 years. Headaches, cranial nerve dysfunction, seizures, and focal neurological deficits were prevalent. Predominant phenotypes included stroke for DADA2 patients, demyelinating lesions and meningitis for FMF, and meningitis for CAPS. TRAPS had insufficient data for adequate phenotype characterization. CONCLUSION: Neurologists should be proactive in diagnosing monogenic autoinflammatory diseases in young adults showcasing clinical and laboratory indications of inflammation, especially when symptoms align with recurrent or chronic meningitis, small vessel disease strokes, and demyelinating lesions.

N-cadherin directs the collective Schwann cell migration required for nerve regeneration through Slit2/3-mediated contact inhibition of locomotion.

Collective cell migration is fundamental for the development of organisms and in the adult for tissue regeneration and in pathological conditions such as cancer. Migration as a coherent group requires the maintenance of cell-cell interactions, while contact inhibition of locomotion (CIL), a local repulsive force, can propel the group forward. Here we show that the cell-cell interaction molecule, N-cadherin, regulates both adhesion and repulsion processes during Schwann cell (SC) collective migration, which is required for peripheral nerve regeneration. However, distinct from its role in cell-cell adhesion, the repulsion process is independent of N-cadherin trans-homodimerisation and the associated adherens junction complex. Rather, the extracellular domain of N-cadherin is required to present the repulsive Slit2/Slit3 signal at the cell surface. Inhibiting Slit2/Slit3 signalling inhibits CIL and subsequently collective SC migration, resulting in adherent, nonmigratory cell clusters. Moreover, analysis of ex vivo explants from mice following sciatic nerve injury showed that inhibition of Slit2 decreased SC collective migration and increased clustering of SCs within the nerve bridge. These findings provide insight into how opposing signals can mediate collective cell migration and how CIL pathways are promising targets for inhibiting pathological cell migration.

Effect of miR-590-3p/DKK1 Axis on the Progression of Wilms' Tumour.

BACKGROUND: Wilms' tumour is the most prevalent abdominal malignancy in children. This study focused on the mechanism of the miR-590-3p/Dickkopf 1 (DKK1) axis in Wilms' tumour. METHODS: The mRNA levels of miR-590-3p and DKK1 in 49 pairs of Wilms' tumour pathological specimens and normal tissues were determined using real-time quantitative polymerase chain reaction (RT-qPCR). Wilms' tumour cells' invasion ability and proliferative ability were assessed using a Transwell assay and Cell Counting Kit-8 (CCK-8) assay, respectively. Dual-luciferase assay was performed to evaluate the potential relationship between miR-590-3p and DKK1 in Wilms tumour. Furthermore, a mouse transplanted tumour model was constructed to explore the function of miR-590-3p inhibitor on Wilms' tumour growth in vivo. RESULTS: DKK1 emerged as a target gene of miR-590-3p in Wilms' tumour. DKK1 expression was downregulated (p < 0.01), but miR-590-3p was overexpressed (p < 0.01) in Wilms' tumour tissues compared to normal tissues. miR-590-3p overexpression accelerated Wilms' tumour invasive ability and cell proliferation (p < 0.01). Additionally, DKK1 partially reversed miR-590-3p-induced proliferation (p < 0.05) and invasion ability (p < 0.01). Furthermore, downregulation of miR-590-3p restrained the growth rate of transplanted tumours in nude mice (p < 0.01). CONCLUSIONS: Through the regulation of DKK1, miR-590-3p accelerated the invasion and proliferation of Wilms' tumour. The study suggests that the miR-590-3p/DKK1 axis represents a novel mechanism in Wilms' tumour.

DKK3 promotes renal fibrosis by increasing MFF-mediated mitochondrial dysfunction in Wnt/ß-catenin pathway-dependent manner.

BACKGROUND: Chronic kidney disease (CKD) lacks effective treatments and renal fibrosis (RF) is one of CKD's outcomes. Dickkopf 3 (DKK3) has been identified as an agonist in CKD. However, the underlying mechanisms of DKK3 in CKD are not fully understood. METHODS: H2O2-treated HK-2 cells and ureteric obstruction (UUO) mice were used as RF models. Biomarkers, Masson staining, PAS staining, and TUNEL were used to assess kidney function and apoptosis. Oxidative stress and mitochondria function were also evaluated. CCK-8 and flow cytometry were utilized to assess cell viability and apoptosis. Western blotting, IHC, and qRT-PCR were performed to detect molecular expression levels. Immunofluorescence was applied to determine the subcellular localization. Dual luciferase assay, MeRIP, RIP, and ChIP were used to validate the m6A level and the molecule interaction. RESULTS: DKK3 was upregulated in UUO mouse kidney tissue and H2O2-treated HK-2 cells. Knockdown of DKK3 inhibited oxidative stress, maintained mitochondrial homeostasis, and alleviated kidney damage and RF in UUO mice. Furthermore, DKK3 silencing suppressed HK-2 cell apoptosis, oxidative stress, and mitochondria fission. Mechanistically, DKK3 upregulation was related to the high m6A level regulated by METTL3. DKK3 activated TCF4/ß-catenin and enhanced MFF transcriptional expression by binding to its promoter. Overexpression of MFF reversed in the inhibitory effect of DKK3 knockdown on cell damage. CONCLUSION: Upregulation of DKK3 caused by m6A modification activated the Wnt/ß-catenin pathway to increase MFF transcriptional expression, leading to mitochondrial dysfunction and oxidative stress, thereby promoting RF progression.

Long non-coding RNA H19 mediates osteogenic differentiation of bone marrow mesenchymal stem cells through the miR-29b-3p/DKK1 axis.

Single immobilization theory cannot fully account for the extensive bone loss observed after spinal cord injury (SCI). Bone marrow mesenchymal stem cells (BMSCs) are crucial in bone homeostasis because they possess self-renewal capabilities and various types of differentiation potential. This study aimed to explore the molecular mechanism of long non-coding RNA H19 in osteoporosis after SCI and provide new research directions for existing prevention strategies. We used small interfering RNA to knockdown H19 expression and regulated miR-29b-2p expression using miR-29b-3p mimetics and inhibitors. Western blotting, real-time fluorescence quantitative PCR, Alizarin red staining, alkaline phosphatase staining and double-luciferase reporter gene assays were used to assess gene expression, osteogenic ability and binding sites. lncRNA H19 was upregulated in BMSCs from the osteoporosis group, whereas miR-29b-3p was downregulated. We identified the binding sites between miR-29b-3p and lncRNAs H19 and DKK1. H19 knockdown promoted BMSCs' osteogenic differentiation, whereas miR-29b-3p inhibition attenuated this effect. We discovered potential binding sites for miR-29b-3p in lncRNAs H19 and DKK1. Our findings suggest that long non-coding RNA H19 mediates BMSCs' osteogenic differentiation in osteoporosis after SCI through the miR-29b-3p/DKK1 axis and by directly inhibiting the ß-catenin signalling pathway.

LncRNA DLG5-AS1 facilitates breast cancer cell proliferation and invasion by promoting EZH2-mediated transcriptional silencing of SFRP1.

Rapid proliferation and metastasis of breast cancer contributed to poor clinical prognosis. Accumulating evidence revealed that the dysregulation of long noncoding RNAs (lncRNAs) was associated with breast cancer progression. However, the role of lncRNA DLG5-AS1 in breast cancer has not been established. Here, we investigated the mechanisms of DLG5-AS1 in the development of breast cancer. We found that the expression of DLG5-AS1 was significantly upregulated in breast cancer tissues and cell lines. DLG5-AS1 interference markedly restrained AU565 cell proliferation, invasion, the expression of apoptosis related (caspase3 and caspase8) and Wnt/ß-catenin pathway related proteins (wnt5a, ß-Catenin and c-Myc), as well as promoted cell apoptosis, whereas DLG5-AS1 overexpression showed an opposite effects. In addition, DLG5-AS1 could directly bind with miR-519 b-3p. We also found that enhancer of zeste homolog 2 (EZH2) is a direct target of miR-519 b-3p, and DLG5-AS1 upregulated EZH2 expression by inhibiting the expression of miR-519 b-3p. EZH2 restrained secreted frizzled related protein 1 (SFRP1) expression through inducing H3 histone methylation in its promoter. MiR-519 b-3p overexpression or SFRP1 knockdown memorably reversed the effects of DLG5-AS1 overexpression on cell functions and Wnt/ß-Catenin pathway related protein expression. Finally, in vivo experiments demonstrated that silencing of DLG5-AS1 inhibited xenograft tumor development in mice. Taken together, these findings demonstrated that DLG5-AS1 facilitated cell proliferation and invasion by promoting EZH2-mediated transcriptional silencing of SFRP1 in breast cancer.

Combination of 308-nm excimer laser and piperine promotes melanocyte proliferation, migration, and melanin content production via the miR-328/SFRP1 axis.

OBJECTIVE: Both piperine and a 308-nm excimer laser have significant curative effects on vitiligo. This study mainly explored the molecular mechanism of a 308-nm excimer combined with piperine in regulating melanocyte proliferation. METHODS: Epidermal melanocytes were cultured in piperine solution, and the cells were irradiated by an XTRAC excimer laser treatment system at 308-nm output monochromatic light. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were for detecting the expression levels of genes or proteins. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Transwell method was for assessing cell viability and migration capacity. The content of melanin was also detected. RESULTS: The combination of the 308-nm excimer laser and piperine enhanced the cell proliferation, migration, and melanin production of melanocytes and upregulated the level of miR-328, and restraint of miR-328 reversed the influence of the 308-nm excimer laser and piperine. Secreted frizzled-related protein 1 (SFRP1) is a direct target gene of miR-328, and miR-328 can inhibit the expression of SFRP1 and elevate the protein level of the Wnt/ß-catenin signaling pathway. CONCLUSION: The 308-nm excimer laser combined with piperine may be more efficient than piperine alone in the remedy of vitiligo, and the miR-328/SFRP1 and Wnt/ß-catenin pathways are participated in the proliferation, migration, and melanin synthesis of melanocytes.

Colorectal cancer cells secreting DKK4 transform fibroblasts to promote tumour metastasis.

Wnt/ß-catenin signalling is aberrantly activated in most colorectal cancer (CRC) and is one key driver involved in the initiation and progression of CRC. However, mutations of APC gene in CRC patients retain certain activity of APC protein with decreased ß-catenin signalling and DKK4 expression significantly upregulates and represses Wnt/ß-catenin signalling in human CRC tissues, suggesting that a precisely modulated activation of the Wnt/ß-catenin pathway is essential for CRC formation and progression. The underlying reasons why a specifically reduced degree, not a fully activating degree, of ß-catenin signalling in CRC are unclear. Here, we showed that a soluble extracellular inhibitor of Wnt/ß-catenin signalling, DKK4, is an independent factor for poor outcomes in CRC patients. DKK4 secreted from CRC cells inactivates ß-catenin in fibroblasts to induce the formation of stress fibre-containing fibroblasts and myofibroblasts in culture conditions and in mouse CRC xenograft tissues, resulting in restricted expansion in tumour masses at primary sites and enhanced CRC metastasis in mouse models. Reduced ß-catenin activity by a chemical inhibitor MSAB promoted the CRC metastasis. Our findings demonstrate why reduced ß-catenin activity is needed for CRC progression and provide a mechanism by which interactions between CRC cells and stromal cells affect disease promotion.

Effects of aflatoxin and fumonisin on gene expression of growth factors and inflammation-related genes in a human hepatocyte cell line.

Aflatoxin B1 (AFB1) and fumonisin B1 (FB1) are mycotoxins widely distributed in maize and maized-based products, often occurring together. The implications of co-exposure to aflatoxin and fumonsin for human health are numerous, but a particular concern is the potential of FB1 to modulate AFB1 hepatotoxicity. This study evaluated the toxicity of these mycotoxins, alone or combined, in a human non-tumorigenic liver cell line, HHL-16 cells, and assessed the effects of AFB1 and FB1 on expression of genes involved in immune and growth factor pathways. The results demonstrated that in HHL-16 cells, both AFB1 and FB1 had dose-dependent and time-dependent toxicity, and the combination of them showed a synergistic toxicity in the cells. Moreover, AFB1 caused upregulation of IL6, CCL20, and BMP2, and downregulation of NDP. In combination of AFB1 with FB1, gene expression levels of IL6 and BMP2 were significantly higher compared to individual FB1 treatment, and had a tendency to be higher than individual AFB1 treatment. This study shows that FB1 may increase the hepatoxicity of AFB1 through increasing the inflammatory response and disrupting cell growth pathways.

Inhibition of BMP signaling pathway induced senescence and calcification in anaplastic meningioma.

PURPOSE: Meningiomas are the most common type of brain tumors and are generally benign, but malignant atypical meningiomas and anaplastic meningiomas frequently recur with poor prognosis. The metabolism of meningiomas is little known, so few effective treatment options other than surgery and radiation are available, and the targets for treatment of recurrence are not well defined. The Aim of this paper is to find the therapeutic target. METHODS: The effects of bone morphogenetic protein (BMP) signal inhibitor (K02288) and upstream regulator Gremlin2 (GREM2) on meningioma's growth and senescence were examined. In brief, we examined as follows: 1) Proliferation assay by inhibiting BMP signaling. 2) Comprehensive analysis of forced expression GREM2.3) Correlation between GREM2 mRNA expression and proliferation marker in 87 of our clinical samples. 4) Enrichment analysis between GREM2 high/low expressed groups using RNA-seq data (42 cases) from the public database GREIN. 5) Changes in metabolites and senescence markers associated with BMP signal suppression. RESULTS: Inhibitors of BMP receptor (BMPR1A) and forced expression of GREM2 shifted tryptophan metabolism from kynurenine/quinolinic acid production to serotonin production in malignant meningiomas, reduced NAD + /NADH production, decreased gene cluster expression involved in oxidative phosphorylation, and caused decrease in ATP. Finally, malignant meningiomas underwent cellular senescence, decreased proliferation, and eventually formed psammoma bodies. Reanalyzed RNA-seq data of clinical samples obtained from GREIN showed that increased expression of GREM2 decreased the expression of genes involved in oxidative phosphorylation, similar to our experimental results. CONCLUSIONS: The GREM2-BMPR1A-tryptophan metabolic pathway in meningiomas is a potential new therapeutic target.

Unravelling the role of HAS2, GREM1, and PTGS2 gene expression in cumulus cells: implications for human oocyte development competency - a systematic review and integrated bioinformatic analysis.

The leading indicator for successful outcomes in in-vitro fertilization (IVF) is the quality of gametes in oocytes and sperm. Thus, advanced research aims to highlight the parameter in assessing these qualities - DNA fragmentation in sperm and oocyte development capacity (ODC) via evaluation of microenvironments involving its maturation process. Regarding oocytes, most evidence reveals the role of cumulus cells as non-invasive methods in assessing their development competency, mainly via gene expression evaluation. Our review aims to consolidate the evidence of GDF-9 derivatives, the HAS2, GREM1, and PTGS2 gene expression in cumulus cells used as ODC markers in relevant publications and tailored to current IVF outcomes. In addition to that, we also added the bioinformatic analysis in our review to strengthen the evidence aiming for a better understanding of the pathways and cluster of the genes of interest - HAS2, GREM1, and PTGS2 in cumulus cell level. Otherwise, the current non-invasive method can be used in exploring various causes of infertility that may affect these gene expressions at the cumulus cell level. Nevertheless, this method can also be used in assessing the ODC in various cohorts of women or as an improvement of markers following targeted tools or procedures by evaluating the advancement of these gene expressions following the targeted intervention.

Transfection of hypoxia-inducible factor-1α mRNA upregulates the expression of genes encoding angiogenic growth factors.

Hypoxia-Inducible Factor-1α (HIF-1α) has presented a new direction for ischemic preconditioning of surgical flaps to promote their survival. In a previous study, we demonstrated the effectiveness of HIF-1a DNA plasmids in this application. In this study, to avoid complications associated with plasmid use, we sought to express HIF-1α through mRNA transfection and determine its biological activity by measuring the upregulation of downstream angiogenic genes. We transfected six different HIF-1a mRNAs-one predominant, three variant, and two novel mutant isoforms-into primary human dermal fibroblasts using Lipofectamine, and assessed mRNA levels using RT-qPCR. At all time points examined after transfection (3, 6, and 10 h), the levels of HIF-1α transcript were significantly higher in all HIF-1α transfected cells relative to the control (all p < 0.05, unpaired Student's T-test). Importantly, the expression of HIF-1α transcription response genes (VEGF, ANG-1, PGF, FLT1, and EDN1) was significantly higher in the cells transfected with all isoforms than with the control at six and/or ten hours post-transfection. All isoforms were transfected successfully into human fibroblast cells, resulting in the rapid upregulation of all five downstream angiogenic targets tested. These findings support the potential use of HIF-1α mRNA for protecting ischemic dermal flaps.

SFRP1 decreases WNT-Mediated M2 macrophage marker expression in breast tissue.

The Wnt family of secreted proteins are involved in mammary gland development and tumorigenesis. It has recently been shown that Wnt ligands promote M2 macrophage polarization and so we sought to determine the effects of a Wnt signaling antagonist, Secreted Frizzled Related Protein 1 (SFRP1), on M2 marker expression. We measured a murine M2 marker (Arg1) in mice with a targeted deletion of Sfrp1 during different stages of mammary gland development including puberty, pregnancy, and lactation, as well as in response to obesity. Next, to determine whether Wnt signaling/antagonism affects human M2 markers (CD209 and CCL18), we treated a human patient derived explant (PDE) breast tissue sample with exogenous Wnt3a in the presence and absence of rSFRP1. Finally, we expanded our PDE study to 13 patients and performed bulk RNAseq analysis following the treatment described above. We found that in loss of Sfrp1 in the murine mammary gland increased Arg1 expression. Moreover, we showed that Wnt3a increases CD209 and CCL18 mRNA and protein expression in breast PDEs and that their expression is decreased in response to rSFRP1. Our RNAseq analysis unveiled novel genes that were affected by Wnt3a treatment and subsequently reversed when rSFRP1 was added. Validation of these data exhibited that chemokines involved in promoting macrophage polarization and cancer metastasis, including CCL11 and CCL26, were stimulated by Wnt3a signaling and their expression was abrogated by treatment with rSFRP1. Our data suggest that SFRP1 may be an important mediator that tempers Wnt signaling in the tumor microenvironment.

PGRN inhibits CD8+T cell recruitment and promotes breast cancer progression by up-regulating ICAM-1 on TAM.

BACKGROUND: Tumor microenvironment actually reduces antitumor effect against the immune attack by exclusion of CD8+T cells. Progranulin (PGRN) is a multifunctional growth factor with significant pathological effects in multiple tumors; however, its role in immunity evasion of breast cancer (BCa) is not completely understood. METHODS: We depleted GRN (PGRN gene) genetically in mice or specifically in PY8119 murine BCa cell line, and mouse models of orthotopic or subcutaneous transplantation were used. Chimeric mice-deficient of PGRN (Grn-/-) in bone marrow (BM) compartment was also generated. Association of PGRN expression with chemokine production or BCa development was investigated by histological and immunological assays. RESULTS: We found PGRN was involved in exhaustion of cytotoxic CD8+T cell in BCa with the increasing expressions of M2 markers and intercellular cell adhesion molecule-1 (ICAM-1) on macrophages. Specifically, ablation of PGRN in PY8119 cells reduced tumor burden, accompanied by the infiltrating of cytotoxic CD8+T cells into tumor nests. Moreover, our result revealed that blockade of PD-1 in PGRN-depleted tumors exhibited better antitumor effect in vivo and significantly decreased tumor burden. CONCLUSION: These findings suggest that inhibition of PGRN may act as a potential immune-therapeutic strategy by recovering infiltration of CD8+T cell in BCa tissue and thereby enhancing the response to anti-PD-1 therapy.

Maternal RNA transcription in Dlk1-Dio3 domain is critical for proper development of the mouse placental vasculature.

The placenta is a unique organ for ensuring normal embryonic growth in the uterine. Here, we found that maternal RNA transcription in Dlk1-Dio3 imprinted domain is essential for placentation. PolyA signals were inserted into Gtl2 to establish a mouse model to prevent the expression of maternal RNAs in the domain. The maternal allele knock-in (MKI) and homozygous (HOMO) placentas showed an expanded junctional zone, reduced labyrinth and poor vasculature impacting both fetal and maternal blood spaces. The MKI and HOMO models displayed dysregulated gene expression in the Dlk1-Dio3 domain. In situ hybridization detected Dlk1, Gtl2, Rtl1, miR-127 and Rian dysregulated in the labyrinth vasculature. MKI and HOMO induced Dlk1 to lose imprinting, and DNA methylation changes of IG-DMR and Gtl2-DMR, leading to abnormal gene expression, while the above changes didn't occur in paternal allele knock-in placentas. These findings demonstrate that maternal RNAs in the Dlk1-Dio3 domain are involved in placental vasculature, regulating gene expression, imprinting status and DNA methylation.

A systematic review of progranulin concentrations in biofluids in over 7,000 people-assessing the pathogenicity of GRN mutations and other influencing factors.

BACKGROUND: Pathogenic heterozygous mutations in the progranulin gene (GRN) are a key cause of frontotemporal dementia (FTD), leading to significantly reduced biofluid concentrations of the progranulin protein (PGRN). This has led to a number of ongoing therapeutic trials aiming to treat this form of FTD by increasing PGRN levels in mutation carriers. However, we currently lack a complete understanding of factors that affect PGRN levels and potential variation in measurement methods. Here, we aimed to address this gap in knowledge by systematically reviewing published literature on biofluid PGRN concentrations. METHODS: Published data including biofluid PGRN concentration, age, sex, diagnosis and GRN mutation were collected for 7071 individuals from 75 publications. The majority of analyses (72%) had focused on plasma PGRN concentrations, with many of these (56%) measured with a single assay type (Adipogen) and so the influence of mutation type, age at onset, sex, and diagnosis were investigated in this subset of the data. RESULTS: We established a plasma PGRN concentration cut-off between pathogenic mutation carriers and non-carriers of 74.8 ng/mL using the Adipogen assay based on 3301 individuals, with a CSF concentration cut-off of 3.43 ng/mL. Plasma PGRN concentration varied by GRN mutation type as well as by clinical diagnosis in those without a GRN mutation. Plasma PGRN concentration was significantly higher in women than men in GRN mutation carriers (p = 0.007) with a trend in non-carriers (p = 0.062), and there was a significant but weak positive correlation with age in both GRN mutation carriers and non-carriers. No significant association was seen with weight or with TMEM106B rs1990622 genotype. However, higher plasma PGRN levels were seen in those with the GRN rs5848 CC genotype in both GRN mutation carriers and non-carriers. CONCLUSIONS: These results further support the usefulness of PGRN concentration for the identification of the large majority of pathogenic mutations in the GRN gene. Furthermore, these results highlight the importance of considering additional factors, such as mutation type, sex and age when interpreting PGRN concentrations. This will be particularly important as we enter the era of trials for progranulin-associated FTD.