sFRP4 mRNA Research Articles

Upregulation of sFRP1 Is More Profound in Female than Male 5xFAD Mice and Positively Associated with Amyloid Pathology.

The prevalence of Alzheimer's disease is greater in women, but the underlying mechanisms remain to be elucidated. We herein demonstrated that α-secretase ADAM10 was downregulated and ADAM10 inhibitor sFRP1 was upregulated in 5xFAD mice. While there were no sex effects on ADAM10 protein and sFRP1 mRNA levels, female 5xFAD and age-matched non-transgenic mice exhibited higher levels of sFRP1 protein than corresponding male mice. Importantly, female 5xFAD mice accumulated more Aβ than males, and sFRP1 protein levels were positively associated with Aβ42 levels in 5xFAD mice. Our study suggests that sFRP1 is associated with amyloid pathology in a sex-dependent manner.

Bta-miR-484 Targets SFRP1 and Affects Preadipocytes Proliferation, Differentiation, and Apoptosis.

MicroRNAs (miRNAs) are essential regulators of numerous biological processes in animals, including adipogenesis. Despite the abundance of miRNAs associated with adipogenesis, their exact mechanisms of action remain largely unknown. Our study highlights the role of bta-miR-484 as a major regulator of adipocyte proliferation, apoptosis, and differentiation. Here, we demonstrated that the expression of bta-miR-484 initially increased during adipogenesis before decreasing. Overexpression of bta-miR-484 in adipocytes ultimately inhibited cell proliferation and differentiation, reduced the number of EdU fluorescence-stained cells, increased the number of G1 phase cells, reduced the number of G2 and S phase cells, and downregulated the expression of proliferation markers (CDK2 and PCNA) and differentiation markers (CEBPA, FABP4, and LPL). Additionally, overexpression of bta-miR-484 promoted the expression of apoptosis-related genes (Caspase 3, Caspase 9, and BAX), and increased the number of apoptotic cells observed via flow cytometry. In contrast, bta-miR-484 inhibition in adipocytes yielded opposite effects to those observed during bta-miR-484 overexpression. Moreover, luciferase reporter assays confirmed SFRP1 as a target gene of bta-miR-484, and revealed that bta-miR-484 downregulates SFRP1 mRNA expression. These findings offer compelling evidence that bta-miR-484 targets SFRP1, inhibits proliferation and differentiation, and promotes apoptosis. Therefore, these results offer novel insights into the bta-miR-484 regulation of adipocyte growth and development.

Clinical Significance of SFRP1 Gene Methylation in Patients with Childhood Acute Lymphoblastic Leukemia

To investigate the clinical significance of SFRP1 gene and its methylation in childhood acute lymphoblastic leukemia (ALL) . Methylation-specific PCR (MSP) was used to detect the methylation status of SFRP1 gene in bone marrow mononuclear cells of 43 children with newly diagnosed ALL before chemotherapy (primary group) and when the bone marrow reached complete remission d 46 after induction of remission chemotherapy (remission group), the expression of SFRP1 mRNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR), the expression of SFRP1 protein was detected by Western blot, and clinical data of children were collected, the clinical significance of SFRP1 gene methylation in children with ALL was analyze. The positive rate of SFRP1 gene promoter methylation in the primary group (44.19%) was significantly higher than that in the remission group (11.63%) (χ2=11.328, P<0.05). The relative expression levels of SFRP1 mRNA and protein in bone marrow mononuclear cells of children in the primary group were significantly lower than those in the remission group (P<0.05). Promoter methylation of SFRP1 gene was associated with risk level (χ2=15.613, P=0.000) and survival of children (χ2=6.561, P=0.010) in the primary group, children with SFRP1 hypermethylation had significantly increased risk and shortened event-free survival time, but no significant difference in other clinical data. Hypermethylation of SFRP1 gene promoter may be involved in the development of childhood ALL, and its hypermethylation may be associated with poor prognosis.

Identification and Validation of Potentially Clinically Relevant CpG Regions within the Class 2 Tumor Suppressor Gene SFRP1 in Pancreatic Cancer.

In pancreatic cancer treatment, tumor stage-dependent chemotherapies are used to prolong overall survival. By measuring DNA promoter hypermethylation in the plasma of patients with stage IV pancreatic cancer, it was recently shown that promoter DNA methylation of the tumor suppressor gene SFRP1 has a high value for predicting failure of drug treatment with gemcitabine. In this study, we therefore aimed to identify as precisely as possible the region in the SFRP1 promoter that is frequently hypermethylated in pancreatic cancer tissue. First, we used the TCGA data set to define CpG-rich regions flanking the SFRP1 transcription start site that were significantly more methylated in pancreatic cancer compared to normal pancreatic acinar tissue. A core CpG island was identified that exhibited abundant tumor DNA methylation and anti-correlation of SFRP1 mRNA expression. To validate our in silico results, we performed bisulfide conversion followed by DNA pyrosequencing of 28 matched formalin-fixed, paraffin-embedded (FFPE) pancreatic cancer cases and six pancreatic cancer cell lines. A defined block of seven CpG sites within the core CpG island was identified, which confirmed our in silico results by showing significantly higher SFRP1 methylation in pancreatic cancer specimens than in normal pancreatic tissue. By selecting this core CpG island, we were able to determine a median overall survival benefit for the low SFRP1 methylation group compared to the high SFRP1 methylation group (702 versus 517 days, p = 0.01) in the TCGA pancreatic cancer cohort. We propose a compact pyrosequencing assay that can be used in the future to further investigate the prognostic value of SFRP1 promoter hypermethylation in predicting pancreatic cancer chemoresistance. Therefore, instead of DNA analysis from blood (liquid biopsy), DNA easily extractable from cancer tissue blocks (FFPE material) could be used.

Sfrp4 expression in thyroxine treated calvarial cells

AimsEvidence suggests alterations of thyroid hormone levels can disrupt normal bone development. Most data suggest the major targets of thyroid hormones to be the Htra1/Igf1 pathway. Recent discovery by our group suggests involvement of targets WNT pathway, specifically overexpression of antagonist Sfrp4 in the presence of exogenous thyroid hormone. Main methodsHere we aimed to model these interactions in vitro using primary and isotype cell lines to determine if thyroid hormone drives increased Sfrp4 expression in cells relevant to craniofacial development. Transcriptional profiling, bioinformatics interrogation, protein and function analyses were used. Key findingsAffymetrix transcriptional profiling found Sfrp4 overexpression in primary cranial suture derived cells stimulated with thyroxine in vitro. Interrogation of the SFRP4 promoter identified multiple putative binding sites for thyroid hormone receptors. Experimentation with several cell lines demonstrated that thyroxine treatment induced Sfrp4 expression, demonstrating that Sfrp4 mRNA and protein levels are not tightly coupled. Transcriptional and protein analyses demonstrate thyroid hormone receptor binding to the proximal promoter of the target gene Sfrp4 in murine calvarial pre-osteoblasts. Functional analysis after thyroxine hormone stimulation for alkaline phosphatase activity shows that pre-osteoblasts increase alkaline phosphatase activity compared to other cell types, suggesting cell type susceptibility. Finally, we added recombinant SFRP4 to pre-osteoblasts in combination with thyroxine treatment and observed a significant decrease in alkaline phosphatase positivity. SignificanceTaken together, these results suggest SFRP4 may be a key regulatory molecule that prevents thyroxine driven osteogenesis. These data corroborate clinical findings indicating a potential for SFRP4 as a diagnostic or therapeutic target for hyperostotic craniofacial disorders.

OR21-4 Comparison of the Effects of PTH (1-34), PTHrP (1-36) and Abaloparatide (ABL) on the Murine Osteoblast Transcriptome

Abstract Osteoporosis is a prevalent disease with substantial morbidity/mortality among the aging population. Due to gaps in knowledge, current therapeutics are limited in their ability to prevent degeneration of bone while also stimulating formation of new bone. Teriparatide (PTH (1-34)) and its analogs PTHrP (1-36) and abaloparatide (ABL), have been utilized for treatment of osteoporosis but have significant limitations in efficacy over long-term use. Research from our laboratory has shown PTH (1-34), PTHrP (1-36), and ABL exert time and dose-dependent differential responses in the osteoblast, leading us to hypothesize they may also differentially modulate the osteoblast transcriptome. In this study we show that treatment of mouse calvarial osteoblasts with 1 nM of these peptides for 4 h results in differing effects on the osteoblast transcriptome by performing gene enrichment analysis of RNA-Seq data. Genes were selected with a Log2 fold change &amp;gt;1 and a false discovery rate &amp;lt;0.05. These data were analyzed and compiled into heat maps for each peptide and smear/volcano plots. RNA-Sequencing revealed that PTH (1-34) regulated 367 genes, 194 were unique; PTHrP (1-36) regulated 117 genes, 15 were unique; ABL regulated 179 genes, 20 were unique. There were 74 genes shared only among PTH(1-34) and ABL; 16 genes shared only among PTH (1-34) and PTHrP; and 83 genes shared among all three peptides. Data collected show pathway-specific differences including, 1) cAMP/PKA, 2) Wnt/β-catenin, 3) Transcriptional regulation 4) Inflammatory response 5) Transmembrane transport, 6) Metabolism, and 7) NF-kB. Further analysis of the data illuminated that the three peptides increased Vitamin D receptor (VDR) and Cbp/p300-interacting transactivator 1 (CITED1) mRNAs similar to RankL expression. These findings were confirmed via qRT-PCR of additional cultured samples of mouse calvarial osteoblasts, treated with 1 nM of PTH (1-34), PTHrP (1-36), and ABL for 4 h prior to harvest. RankL mRNA, VDR mRNA, and CITED1 mRNA were measured for each sample and statistical differences were analyzed via Kruskal Wallis p&amp;lt;0.05. Additionally, we analyzed mRNA levels of several genes of interest, including WNT7b, WNT11, TCF7, SFRP4, FZD5, PP2R2A, and DVL3 mRNA. Pathway analysis and subsequent qRT-PCR confirmation has shown that PTH (1-34) and ABL lead to a significant increase in Wnt11 mRNA, while PTHrP (1-36) does not. Our findings highlight the complexity of the genetic and functional events triggered by PTH (1-34) and its analogs. Many studies have examined PTH signaling in the osteoblast/osteocyte; ours is the first to examine global effects of these peptides on the osteoblast transcriptome. Further delineation of which signaling events are attributable to PTH (1-34), PTHrP (1-36), and ABL exclusively and which are shared among all three will further our understanding of the effects these peptides have on the osteoblast and lead to refinement of PTH-derived treatments for osteoporosis. Presentation: Monday, June 13, 2022 11:45 a.m. - 12:00 p.m.

Total Saponins of Radix Clematis Regulate Fibroblast-Like Synoviocyte Proliferation in Rheumatoid Arthritis via the LncRNA OIP5-AS1/MiR-410-3p/Wnt7b Signaling Pathway

Background Rheumatoid arthritis (RA) is the most common autoimmune disease and affects multiple joints. Previous studies have shown that total saponins of Radix clematidis (TSC) have a clear therapeutic effect on RA, but the specific mechanism has not yet been clarified. Literature screening and previous research suggest that the lncRNA OIP5-AS1/miR-410-3p/Wnt7b signaling pathway exerts a regulatory effect on the pathogenesis of RA. In this study, we examined whether the TSC treatment of RA affects the lncRNA OIP5-AS1/miR-410-3p/Wnt7b pathway. Materials and Methods Freund's complete adjuvant was used to create an adjuvant arthritis (AA) rat model with rat synovial cells being harvested and cultured. The experiment comprises a normal group, model group, TSC optimal-dose group, TSC optimal-dose group + lncRNA OIP5-AS1siRNA group, lncRNA OIP5-AS1 siRNA group, and lncRNA OIP5-AS1 siRNA + NC group. MMT was used to screen the optimal concentration of TSC. The level of lncRNA OIP5-AS1, miR-410-3p, Wnt7b, β-catenin, c-Myc, cyclin D1, GSK-3β, and SFRP4 mRNA were detected by real-time-qPCR, the expression of Wnt7b, β-catenin, c-Myc, cyclin D1, GSK-3β, and p-GSK-3β (Ser9) protein were detected by immunofluorescence and Western blot. Results We found that TSC inhibits the proliferation of RA FLS, TSC significantly reduced lncRNA OIP5-AS1, Wnt7b, β-catenin, c-Myc, cyclin D1, and p-GSK-3β/GSK-3β mRNA/protein expression, whereas the miR-410-3p and SFRP4 mRNA/protein expression levels were significantly upregulated. Our data suggest that TSC can inhibit the excessive proliferation of FLS to treat RA, the mechanism of which may be closely related to regulation of the lncRNA OIP5-AS1/miR-410-3p /Wnt7b signaling axis and the Wnt signaling pathway.

Silencing of MBD2 and EZH2 inhibits the proliferation of colorectal carcinoma cells by rescuing the expression of SFRP.

The secreted frizzled related proteins (SFRPs) are extracellular inhibitors of WNT pathway signaling. Methyl-CpG binding domain protein 2 (MBD2) and enhancer of zeste homolog 2 (EZH2) are core members of the methylated DNA binding domain (MBD) and polycomb group (PcG) protein families for epigenetic regulation, respectively. This study aimed to ascertain the potential role of MBD2 and EZH2 proteins in colorectal cancer (CRC) and its effects on the expression of SFRP. Bioinformatics, real-time quantitative polymerase chain reaction (qPCR) and western blot analysis were used to detect the expression of MBD2, EZH2, and SFRP in CRC cell lines and tissues. The functions of MBD2 and EZH2 in regards to cell proliferation, cell cycle distribution, apoptosis and invasion were examined in CRC cell lines. Methylation-specific PCR (MSP) was used to detect the methylation status of the SFRP promoter. The results revealed that the mRNA expression levels of SFRP were significantly decreased in CRC tissues and cell lines compared to these levels in the adjacent tissues and NCM460, respectively. However, the mRNA levels of EZH2 and MBD2 genes were highly expressed in CRC cell lines. We found that reducing MBD2 and EZH2 expression together remarkably inhibited and decreased the proliferation, migration and invasion abilities of the CRC cell lines compared to reducing one of each. Flow cytometric analysis showed that knockdown of MBD2 and EZH2 together in CRC affected cell apoptosis and the cell cycle progression more effectively than knockdown of one of each. The mRNA expression of SFRP1 was reactivated by silencing of MBD2 but not EZH2 in SW480 and HCT116 cells. SFRP4 and SFRP5 mRNA expression was reactivated by silencing of EZH2 but not MBD2 only in SW480 cells. However, depletion of both MBD2 and EZH2 restored SFRP1, SFRP2, SFRP4, and SFRP5 mRNA expression more effectively in CRC cells. Interestingly, there was no significant change in the methylation status of SFRP1, SFRP2, SFRP4, and SFRP5 gene promoter between before and after interference with MBD2, EZH2, and both. In conclusion, our results suggest that silencing of MBD2 and EZH2 simultaneously was able to rescue the expression of SFRP and inhibit the proliferation of CRC cells more effectively. However, the underlying regulatory mechanism system of MBD2 and EZH2 for SFRP in CRC requires further research.

Sfrp4 Expression in Thyroxine Treated Calvarial Cells: A Novel Target

Evidence suggests alterations of thyroid hormone levels can disrupt normal craniofacial development. Most data suggest the major targets of thyroid hormones to be the Htra1/Igf1 pathway. Recent discovery by our group suggests involvement of targets of the WNT pathway, specifically overexpression of antagonist Sfrp4 in the presence of excess thyroid hormone. Here we aimed to model these interactions in vitro to determine if thyroid hormone directly drives Sfrp4 expression in cells relevant to craniofacial development. Affymetrix transcriptional profiling found Sfrp4 to be overexpressed in cranial suture derived cells stimulated with thyroxine in vitro. Bioinformatics interrogation of the SFRP4 promoter identified multiple putative binding sites for thyroid hormone receptors. Experimentation with several cell lines demonstrated that thyroxine treatment induced Sfrp4 expression. However, protein levels did not correlate with induction of gene expression, demonstrating that Sfrp4 mRNA and protein levels are not tightly coupled. Transcriptional and protein analyses demonstrate thyroid hormone receptor binding to the proximal promoter of the target gene Sfrp4. Interestingly functional analysis of stem cells, fibroblasts, or primary culture cells after thyroxine stimulation for alkaline phosphatase activity shows only pre‐osteoblasts are pushed to become positive after thyroid hormone stimulation, suggesting cell type susceptibility. Finally, we added recombinant SFRP4 to pre‐osteoblasts in combination with thyroxine treatment and observed a significant decrease in alkaline phosphatase positivity, suggesting SFRP4 may be a key regulatory molecule that prevents thyroxine driven osteogenesis. The implications for this relationship indicate SFRP4 as a potential diagnostic or therapeutic target for hyperostotic craniofacial disorders.Support or Funding InformationThe authors would like to thank Xing Ming Shi for isolation of the bone marrow mesenchymal cells. The following funding sources were used for this study a grant from the Cleft Palate Foundation, NIH/NIDCR R03DE023350A, 5T32DE017551, and F31DE026684, NIH/NCATS UL1 TR000062, NIH/NIGM P30GM103331, GM103342 and GM103499, and MUSC’s Office of the Vice President for Research.

Contribution of adipocyte precursors in the phenotypic specificity of intra-articular adipose tissues in knee osteoarthritis patients

BackgroundIntra-articular adipose tissues (IAATs) are involved in osteoarthritis (OA) pathophysiology. We hypothesize that mesenchymal cells residing in IAATs may account for the specific inflammatory and metabolic patterns in OA patients.MethodsAdipocyte precursors (preadipocytes and dedifferentiated fat cells (DFATc)) from IAATs (infrapatellar and suprapatellar fat pads) and autologous subcutaneous adipose tissues (SCATs) were isolated from knee OA patients. The ability of these precursors to differentiate into adipocytes was assessed by oil red O staining after 14 days of culture in adipogenic medium. The gene expression of adipocyte-related transcription factors (C/EBP-α and PPAR-γ) and development-related factors (EN1 and SFRP2) were analyzed. The inflammatory pattern was assessed by RT-qPCR and ELISA (interleukin 6 (IL-6), IL-8, Cox2, and prostaglandin E2 (PGE2)) after a 24-h stimulation by IL-1β (1 ng/mL) and by conditioned medium from OA synovium.ResultsIAAT preadipocytes displayed a significantly higher ability to differentiate into adipocytes and expressed significantly more C/EBP-α mRNA than SCAT preadipocytes. IAAT preadipocytes expressed significantly less EN-1 and SFRP2 mRNA than SCAT preadipocytes. Unstimulated IAAT preadipocytes displayed a less inflammatory pattern (IL-6, IL-8, and Cox2/PGE2) than SCAT preadipocytes. In contrast, the response of IAAT preadipocytes to an inflammatory stimulus (IL-1β and conditioned media of OA synovium) was exacerbated compared to that of SCAT preadipocytes. Similar results were obtained with DFATc.ConclusionIAAT adipocyte precursors from OA patients have a specific phenotype, which may account for the unique phenotype of OA IAATs. The exacerbated response of IAAT preadipocytes to inflammatory stimulation may contribute to OA pathophysiology.

Circulating miR-103 family as potential biomarkers for type 2 diabetes through targeting CAV-1 and SFRP4.

MicroRNA-103 (miR-103) family plays important roles in regulating glucose homeostasis in type 2 diabetes mellitus (DM2). However, the underlying mechanisms remain poorly characterized. The objective of this study was to test the hypothesis that circulating miR-103a and miR-103b, which regulate CAV-1 and SFRP4, respectively, are novel biomarkers for diagnosis of DM2. We determined the predictive potential of circulating miR-103a and miR-103b in pre-DM subjects (pre-DM), noncomplicated diabetic subjects, and normal glucose-tolerance individuals (control) using bioinformatic analysis, qRT-PCR, luciferase assays, and ELISA assays. We found that both miR-103a and miR-103b had high complementarity and conservation, modulated reporter gene expression through seed sequences in the 3'UTRs of CAV-1 and SFRP4 mRNA, and negatively regulated their mRNA and protein levels, respectively. We also found that increased miR-103a and decreased miR-103a in plasma were significantly and negatively correlated with reduced CAV-1 levels and elevated SFRP4 levels in pre-DM and DM2, respectively, and were significantly associated with glucose metabolism, HbA1c levels, and other DM2 risk factors for progression from a normal individual to one with pre-DM. Furthermore, we demonstrated that the reciprocal changes in circulating miR-103a and miR-103b not only provided high sensitivity and specificity to differentiate the pre-DM population but also acted as biomarkers for predicting DM2 with high diagnostic value. These findings suggest that circulating miR-103a and miR-103b may serve as novel biomarkers for diagnosis of DM2, providing novel insight into the mechanisms underlying pre-DM.

Investigation of Key Genes and Pathways in Inhibition of Oxycodone on Vincristine-Induced Microglia Activation by Using Bioinformatics Analysis.

Introduction The neurobiological mechanisms underlying the chemotherapy-induced neuropathic pain are only partially understood. Among them, microglia activation was identified as the key component of neuropathic pain. The aim of this study was to identify differentially expressed genes (DEGs) and pathways associated with vincristine-induced neuropathic pain by using bioinformatics analysis and observe the effects of oxycodone on these DEG expressions in a vincristine-induced microglia activation model. Methods Based on microarray profile GSE53897, we identified DEGs between vincristine-induced neuropathic pain rats and the control group. Using the ToppGene database, the prioritization DEGs were screened and performed by gene ontology (GO) and signaling pathway enrichment. A protein-protein interaction (PPI) network was used to explore the relationship among DEGs. Then, we built the vincristine-induced microglia activation model and detected several DEG expressions by real-time polymerase chain reaction (PCR) and western blotting. Meanwhile, the effects of different concentrations of oxycodone on inflammatory response in primary microglia induced by vincristine were observed. Results A total of 38 genes were differentially expressed between normal and vincristine-treated rats. GO and pathway enrichment analysis showed that prioritization DEGs are involved in cAMP metabolic process, inflammatory response, regulation of cell proliferation, and chemokine pathway. The in vitro studies showed that vincristine had dose-dependent cytotoxic effects in microglia. Compared to the control group, vincristine (0.001 μg/ml) could lead to inflammation in primary microglia induced by vincristine and upregulated the CXCL10, CXCL9, SFRP2, and PF4 mRNA and made an obvious reduction in IRF7 mRNA. At protein levels, oxycodone (50, 100 ng/ml) decreased the expression of CXCL10 and CXCL9 in activated microglia. Conclusion Our study obtained several DEG expressions and signaling pathways in the vincristine-induced neuropathic pain rat model by bioinformatics analysis. Oxycodone could alleviate the vincristine-induced inflammatory signaling in primary microglia and downregulate some DEGs. Further molecular mechanisms need to be explored in the future.

LncRNA-Safe contributes to cardiac fibrosis through Safe-Sfrp2-HuR complex in mouse myocardial infarction.

Rationale: As a hallmark of various heart diseases, cardiac fibrosis ultimately leads to end-stage heart failure. Anti-fibrosis is a potential therapeutic strategy for heart failure. Long noncoding RNAs (lncRNAs) have emerged as critical regulators of heart diseases that promise to serve as therapeutic targets. However, few lncRNAs have been directly implicated in cardiac fibrosis.Methods: The lncRNA expression profiles were assessed by microarray in cardiac fibrotic and remote ventricular tissues in mice with myocardial infarction. The mechanisms and functional significance of lncRNA-AK137033 in cardiac fibrosis were further investigated with both in vitro and in vivo models.Results: We identified 389 differentially expressed lncRNAs in cardiac fibrotic and remote ventricular tissues in mice with myocardial infarction. Among them, a lncRNA (AK137033) we named Safe was enriched in the nuclei of fibroblasts, and elevated in both myocardial infarction and TGF-β-induced cardiac fibrosis. Knockdown of Safe prevented TGF-β-induced fibroblast-myofibroblast transition, aberrant cell proliferation and secretion of extracellular matrix proteins in vitro, and mended the impaired cardiac function in mice suffering myocardial infarction. In vitro studies indicated that knockdown of Safe significantly inhibited the expression of its neighboring gene Sfrp2, and vice versa. The Sfrp2 overexpression obviously disturbed the regulatory effects of Safe shRNAs in both the in vitro cultured cardiac fibroblasts and myocardial infarction-induced fibrosis. Dual-Luciferase assay demonstrated that Safe and Sfrp2 mRNA stabilized each other via their complementary binding at the 3'-end. RNA electrophoretic mobility shift assay and RNA immunoprecipitation assay indicated that RNA binding protein HuR could bind to Safe-Sfrp2 RNA duplex, whereas the knockdown of HuR dramatically reduced the stabilization of Safe and Sfrp2 mRNAs, down-regulated their expression in cardiac fibroblasts, and thus inhibited TGF-β-induced fibrosis. The Safe overexpression partially restrained the phenotype change of cardiac fibroblasts induced by Sfrp2 shRNAs, but not that induced by HuR shRNAs.Conclusions: Our study identifies Safe as a critical regulator of cardiac fibrosis, and demonstrates Safe-Sfrp2-HuR complex-mediated Sfrp2 mRNA stability is the underlying mechanism of Safe-regulated cardiac fibrosis. Fibroblast-enriched Safe could represent a novel target for anti-fibrotic therapy in heart diseases.

MiR-509-5p improves the proliferative and invasive abilities of papillary thyroid carcinoma cells by inhibiting SFRP1.

IntroductionOur study was conducted to prove that miR-509-5p improved the proliferative and invasive abilities of papillary thyroid carcinoma (PTC) cells through inhibiting SFRP1 expression.Material and methodsQRT-PCR was conducted in order to detect the miR-509-5p expression levels in PTC and normal tissues. The miR-509-5p and SFRP1 mRNA expression levels in PTC cell lines K1, TPC-1, BCPAP and the human normal thyroid cell line HT-ori3 were also detected by qRT-PCR. The transfection was performed using Lipofectamine and lentiviral vectors. Pgcsil-008 was used as the SFRP1 gene vector. Western blot and dual luciferase reporter gene assay were conducted to investigate miR-509-5p’s direct regulation on SFRP1. MTT, clone formation, and Transwell assays were adopted to investigate the biological behaviors of PTC cells. TCF/LEF luciferase assays were used to prove that miR-509-5p influenced the Wnt/β-catenin signaling pathway by regulating SFRP1.ResultsMiR-509-5p was overexpressed in PTC cells and tissues in which SFRP1 was down-regulated. MiR-509-5p bound to the 3′-UTR of SFRP1 and therefore partially weakened the proliferative, migrating and invasive activities of PTC cells. MiR-509-5p promoted activation of the Wnt/β-catenin signaling pathway through down-regulating SFRP1.ConclusionsMiR-509-5p improved the proliferative, migrating and invasive abilities of PTC cells through inhibiting SFRP1 expression.

Sulfur dioxide attenuates hypoxia-induced pulmonary arteriolar remodeling via Dkk1/Wnt signaling pathway

ObjectiveThis study investigated the impact of SO2 on rats with hypoxic pulmonary vascular structural remodeling and its possible mechanisms. Materials and methodsPulmonary vascular morphological change was examined by HE staining. RNA-based high-throughput sequencing (RNA-seq) was performed to detect differential expression of mRNAs in Normal and Hypoxia–induced Pulmonary hypertension (HPH) rats. The Real-time quantitative RT-PCR (q RT-PCR) was used for validation of wnt7b, sfrp2, cacna1f, DKK1, CaSR and vimentin mRNA expression levels. Protein levels of CaSR, Vimentin, Caspase3, E-cadherin and P-Akt1/2/3 were detected by Western blot and immunohistochemistry. ResultsThis study showed that SO2 significantly attenuated the interstitial thickening and prominent media hypertrophy of pulmonary arteries. SO2 downregulated p-Akt1/2/3 protein level and upregulated E-cadherin protein level in lung tissues, which inhibited the proliferation and epithelial-to-mesenchymal transition (EMT) in HPH rats. RNA-seq and PCR validation results showed that levels of Wnt7b, Sfrp2 and Cacna1f mRNAs decreased and Dkk1 mRNA level increased obviously in HPH rats. Moreover, SO2 attenuated the mRNA and protein level of CaSR, which was activated in HPH rats and resulted in the proliferation of PASMCs. Besides, the mRNA and protein expression of vimentin in PASMCs significantly reduced after SO2 treatment. ConclusionTogether, these findings indicate that SO2 could attenuate hypoxia-induced pulmonary arteriolar remodeling and may suppress the proliferation and migration of PASMCs at least in part through the Dkk1/Wnt signaling pathway.

MiR-542-3p prevents ovariectomy-induced osteoporosis in rats via targeting SFRP1.

Secreted frizzled‐related protein‐1 (SFRP1) is a negative regulatory molecule of the WNT signaling pathway and serves as a therapeutic target for bone formation in osteoporosis. In this study, we first established an ovariectomized (OVX) rat model to simulate postmenopausal osteoporosis and found significant changes in miR‐542‐3p and sFRP1 expression by RNA sequencing and qRT‐PCR. In addition, there was a significant negative correlation between miR‐542‐3p and sFRP1 mRNA levels in postmenopausal women with osteoporosis. We found that miR‐542‐3p inhibited the expression of sFRP1 mRNA by luciferase reporter assay. When the miR‐542‐3p binding site in sFRP1 3'UTR was deleted, it did not affect its expression. Western blot results showed that miR‐542‐3p inhibited the expression of SFRP1 protein. The expression of SFRP1 was significantly increased in osteoblast‐induced mesenchymal stem cells (MSC), whereas the expression of miR‐542‐3p was significantly decreased. And miR‐542‐3p transfected MSCs showed a significant increase in osteoblast‐specific marker expression, indicating that miR‐542‐3p is necessary for MSC differentiation. Inhibition of miR‐542‐3p reduced bone formation, confirmed miR‐542‐3p play a role in bone formation in vivo. In general, these data suggest that miR‐542‐3p play an important role in bone formation via inhibiting SFRP1 expression and inducing osteoblast differentiation.

Epigenetic loss of putative tumor suppressor SFRP3 correlates with poor prognosis of lung adenocarcinoma patients

ABSTRACTSecreted frizzled related protein 3 (SFRP3) contains a cysteine-rich domain (CRD) that shares homology with Frizzled CRD and regulates WNT signaling. Independent studies showed epigenetic silencing of SFRP3 in melanoma and hepatocellular carcinoma. Moreover, a tumor suppressive function of SFRP3 was shown in androgen-independent prostate and gastric cancer cells. The current study is the first to investigate SFRP3 expression and its potential clinical impact on non-small cell lung carcinoma (NSCLC). WNT signaling components present on NSCLC subtypes were preliminary elucidated by expression data of The Cancer Genome Atlas (TCGA). We identified a distinct expression signature of relevant WNT signaling components that differ between adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Of interest, canonical WNT signaling is predominant in LUAD samples and non-canonical WNT signaling is predominant in LUSC. In line, high SFRP3 expression resulted in beneficial clinical outcome for LUAD but not for LUSC patients. Furthermore, SFRP3 mRNA expression was significantly decreased in NSCLC tissue compared to normal lung samples. TCGA data verified the reduction of SFRP3 in LUAD and LUSC patients. Moreover, DNA hypermethylation of SFRP3 was evaluated in the TCGA methylation dataset resulting in epigenetic inactivation of SFRP3 expression in LUAD, but not in LUSC, and was validated by pyrosequencing of our NSCLC tissue cohort and in vitro demethylation experiments. Immunohistochemistry confirmed SFRP3 protein downregulation in primary NSCLC and indicated abundant expression in normal lung tissue. Two adenocarcinoma gain-of-function models were used to analyze the functional impact of SFRP3 on cell proliferation and regulation of CyclinD1 expression in vitro. Our results indicate that SFRP3 acts as a novel putative tumor suppressor gene in adenocarcinoma of the lung possibly regulating canonical WNT signaling.

Neonatal exposure to a glyphosate-based herbicide alters uterine decidualization in rats

We investigated whether defective modulation of uterine signaling may cause decidualization failure in rats neonatally exposed to a glyphosate-based herbicide (GBH). Female pups received vehicle or 2mg/kg of GBH from postnatal day (PND) 1 to PND7. On PND8 and PND21, Wnt5a and β-catenin expression was evaluated in uterine samples. On gestational day (GD) 9, Wnt5a, Wnt7a and β-catenin expression and Dkk1 and sFRP4 mRNA were evaluated on implantation sites. On PND8, GBH-exposed rats showed increased Wnt5a and β-catenin expression in luminal epithelium (LE), whereas on PND21, they showed increased Wnt5a and β-catenin expression in subepithelial stroma but decreased β-catenin expression in glandular epithelium. On GD9, GBH-exposed rats showed decreased Wnt5a and Wnt7a expression in the antimesometrial zone and LE respectively, without changes in β-catenin expression, while Dkk1 and sFRP4 were up- and down-regulated respectively. We concluded that neonatal GBH exposure may lead to embryo losses by disturbing uterine signaling.

MicroRNA-500a promotes migration and invasion in hepatocellular carcinoma by activating the Wnt/β-catenin signaling pathway.

Increased expression of microRNA-500a (miR-500a) has been reported in the serum of hepatocellular carcinoma (HCC) patients. However, the biological effects and mechanisms of miR-500a in hepatoma cells remain unclear. In this study, we found that miR-500a expression was up-regulated in HCC cell lines and tissues, and that high levels of miR-500a was associated with poor prognosis. We found that miR-500a upregulation promoted migration and invasion in two hepatoma cell lines, HCCLM3 and SMMC7721, while miR-500a downregulation had the opposite effect. We demonstrated that miR-500a activates the Wnt/β-catenin signaling pathway by directly binding to the 3'-untranslated region (UTR) of SFRP2 and GSK-3β mRNA. In conclusion, our results indicate miR-500a promotes HCC migration and invasion through activating Wnt/β-catenin signaling by directly binding to SFPR2 and GSK-3β.

Reduced skeletal muscle secreted frizzled‐related protein 3 is associated with inflammation and insulin resistance

To investigate the role of secreted frizzled-related protein 3 (Sfrp3) in insulin sensitivity (ISi) and β-cell function in humans across a spectrum of glucose homeostasis. Subjects included those with normal glucose homeostasis (NGT; n = 18), prediabetes (PD; n = 11), or type 2 diabetes (T2D; n=12). Serum and skeletal muscle (SkM) Sfrp3 levels were measured by ELISA and qPCR, respectively, and insulin signaling pathway was assessed by Western blot. IS and β-cell function were assessed by indices derived from frequently sampled intravenous glucose tolerance test. SkM Sfrp3 mRNA levels were significantly reduced in PD and T2D versus NGT. Similarly, serum Sfrp3 levels tended to be decreased in PD and T2D versus NGT. SkM Sfrp3 mRNA levels correlated negatively with circulating proinflammatory cytokines (IL-6, IFN-γ) and positively with IS. In vitro-differentiated myotubes from lean insulin-sensitive subjects treated with either lipopolysaccharide (LPS) or recombinant IL-6 demonstrated a dose-dependent reduction in Sfrp3 gene expression. Treatment of myotubes with recombinant Sfrp3 restored LPS- and IL-6-induced attenuation of insulin-stimulated Akt phosphorylation. Inflammation-induced reduction in SkM Sfrp3 expression may contribute to insulin resistance, and this effect may be prevented by addition of exogenous Sfrp3. Thus, Sfrp3 may be a novel target for insulin sensitization.