Frizzled Class Receptor Research Articles

Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B.

Clostridium difficile infection (CDI) is a major nosocomial disease associated with significant morbidity and mortality. The pathology of CDI stems primarily from the 2 C. difficile–secreted exotoxins—toxin A (TcdA) and toxin B (TcdB)—that disrupt the tight junctions between epithelial cells leading to the loss of colonic epithelial barrier function. Here, we report the engineering of a series of monomeric and dimeric designed ankyrin repeat proteins (DARPins) for the neutralization of TcdB. The best dimeric DARPin, DLD-4, inhibited TcdB with a half maximal effective concentration (EC50) of 4 pM in vitro, representing an approximately 330-fold higher potency than the Food and Drug Administration (FDA)-approved anti-TcdB monoclonal antibody bezlotoxumab in the same assay. DLD-4 also protected mice from a toxin challenge in vivo. Cryo-electron microscopy (cryo-EM) studies revealed that the 2 constituent DARPins of DLD-4–1.4E and U3—bind the central and C-terminal regions of the delivery domain of TcdB. Competitive enzyme-linked immunosorbent assay (ELISA) studies showed that the DARPins 1.4E and U3 interfere with the interaction between TcdB and its receptors chondroitin sulfate proteoglycan 4 (CSPG4) and frizzled class receptor 2 (FZD2), respectively. Our cryo-EM studies revealed a new conformation of TcdB (both apo- and DARPin-bound at pH 7.4) in which the combined repetitive oligopeptides (CROPS) domain points away from the delivery domain. This conformation of the CROPS domain is in stark contrast to that seen in the negative-stain electron microscopy (EM) structure of TcdA and TcdB at the same pH, in which the CROPS domain bends toward and “kisses” the delivery domain. The ultrapotent anti-TcdB molecules from this study serve as candidate starting points for CDI drug development and provide new biological tools for studying the pathogenicity of C. difficile. The structural insights regarding both the “native” conformation of TcdB and the putative sites of TcdB interaction with the FZD2 receptor, in particular, should help accelerate the development of next-generation anti–C. difficile toxin therapeutics.

FZD6 (Frizzled class receptor 6)

FZD6 (Frizzled class receptor 6)

FZD5 (frizzled class receptor 5)

FZD5 (frizzled class receptor 5)

NPTX2 promotes colorectal cancer growth and liver metastasis by the activation of the canonical Wnt/\u03b2-catenin pathway via FZD6

Accumulating evidence from clinical and epidemiological studies has highlighted the close correlation between the individual risk of cancer and nervous system diseases. The expression of neuronal pentraxin 2 (NPTX2) is absent in Alzheimer’s disease, anxiety, and depression. Herein, we found that NPTX2 mRNA and protein expression was significantly upregulated in colorectal carcinoma (CRC). NPTX2 expression level gradually increased with CRC progression and was closely associated with poor prognosis. In vitro and in vivo studies demonstrated that NPTX2 promoted CRC proliferation and metastasis through the activation of the Wnt/β-catenin signaling pathway. As NPTX2 receptors are absent on CRC cells, NPTX2 was shown to physically interact with frizzled class receptor 6 (FZD6) to promote β-catenin translocation into the cell nucleus, resulting in an increase in the expression of MYC, cyclin D1, snail, and N-cadherin along with a decrease in the expression of E-cadherin. Knockdown of FZD6 expression with a small-interfering RNA almost completely reversed the proliferative effects of NPTX2 on CRC development. In conclusion, NPTX2, a molecule related to nervous system diseases, promotes CRC cell proliferation and metastasis through the activation of the Wnt/β-catenin pathway via direct interaction with FZD6.

Dynamic expression of ZNF382 and its tumor-suppressor role in hepatitis B virus-related hepatocellular carcinogenesis

Hepatitis B virus (HBV) infection is the primary cause of hepatocellular carcinoma (HCC). Zinc-finger protein 382 (ZNF382), which belongs to zinc-finger protein family, has been documented to be downregulated in certain types of cancer. However, its role in HCC remains largely unknown. In this study, we demonstrated that ZNF382 expression was significantly elevated in HBV-infected liver cirrhosis tissues relative to HBV-negative normal liver tissues at protein levels, but not at mRNA levels, and was positively correlated with the levels of HBV DNA and hepatitis B virus X protein (HBx). Further studies revealed that ZNF382 was a target of miR-6867, and HBx promoted the translation of ZNF382 during HBV chronic infection through Erk-mediated miR-6867 inhibition. In addition, our data showed that ZNF382 was frequently downregulated by promoter methylation in HBV-related HCCs relative to HBV-infected liver cirrhosis tissues, and decreased expression of ZNF382 was strongly correlated with poor survival in early-stage HCC patients. Functional studies demonstrated that ZNF382 was a potent tumor suppressor in HCC cells through inhibiting cell proliferation, colony formation, migration, invasion, and tumorigenic potential in nude mice, and inducing cell apoptosis. Mechanistically, ZNF382 exerted its tumor-suppressor functions in HCC through transcriptionally repressing its downstream targets such as Fos proto-oncogene (FOS), Jun proto-oncogene (JUN), disheveled segment polarity protein 2 (DVL2), and frizzled class receptor 1 (FZD1), thereby impairing the activities of activating protein 1 (AP-1) and Wnt/β-catenin pathways and activating p53 signaling. Altogether, our data show that ZNF382 acts as a tumor suppressor, and is co-regulated by HBx and epigenetic mechanism in HBV-related hepatocellular carcinogenesis.

Overexpression of FZD1 is Associated with a Good Prognosis and Resistance of Sunitinib in Clear Cell Renal Cell Carcinoma.

Frizzled class receptor 1 (FZD1), a receptor for Wnt signaling pathway. Overexpression of FZD1 has been detected in many cancer tissues and cells resulting in tumor development and drug resistance. However, its expression status and prognostic merit in renal cancer still remains unclear. We screened the FZD1 mRNA in clear cell renal cell carcinoma (ccRCC) and papillary renal cell carcinoma (pRCC) from TCGA database and Oncomine database. We then detected FZD1 mRNA expression in sunitinib-resistant cells and the corresponding parental cells by qRT-PCR. FZD1 level was significantly upregulated in renal cancer tissues, renal cancer cell lines and their corresponding sunitinib-resistant cells. FZD1 level was also associated with the clinicopathological characteristics of ccRCC patients that could discriminate metastasis, pathological stage, recurrence and prognosis in ccRCC patients. The Kaplan-Meier survival curve and the log-rank test revealed FZD1 was higher in lower clinical stage and grade that correlated with better overall survival (OS) and disease-free survival (DFS) in total and subgroups of ccRCC patients. Both univariate and multivariate cox regression analysis indicated that high FZD1 level was an independent predictor of good prognosis for OS (HR 0.569, P=0.001) and DFS (HR 0.559, P=0.036) in ccRCC patients. Using cBioportal program, less than 1% mutation in the patients with renal cancer was observed, the alterations in FZD1 were correlated with better OS (P=0.0404) in ccRCC patients. Finally, the result of KEGG pathway analysis predicted seven potential pathways that FZD1 and its related genes got involved in ccRCC, including Hippo signaling pathway. This indicated potential therapeutic targets of ccRCC. In conclusion, our results suggested that expression status of FZD1 had a diagnostic value and prognostic value in ccRCC patients, it also may serve as a potential drug target to relieve sunitinib resistance in renal cancer patients.

ACVR1-knockout promotes osteogenic differentiation by activating the Wnt signaling pathway in mice.

Osteogenic differentiation refers to the process of bone formation and remodeling, which is controlled by complex molecular mechanisms. Activin A receptor type I (ACVR1) is reported to be associated with osteogenic differentiation. However, the underlying molecular mechanism remains elusive. Therefore, this study evaluates the function of ACVR1 in osteogenic differentiation through the Wnt signaling pathway. The expression of osteocalcin (Oc) and osterix together with osteogenic differentiation and mineralization was examined in ACVR1-knockout (KO) mouse. Furthermore, the Wnt signaling pathway was inhibited in bone marrow stromal cells (BMSCs) of mice to explore the role of the Wnt signaling pathway in osteogenic differentiation by means of alkaline phosphatase (ALP) activity detection and evaluation of mineralized nodules and calcium content. Subsequently, the effect of ACVR1 on the Wnt signaling pathway was assessed by determining the expression of ACVR1, β-catenin, glycogen synthase kinase 3 β (GSK3β), dickkopf-related protein 1 (DKK1), and frizzled class receptor 1 (FZD1). Both their effects on osteogenic differentiation were further evaluated by determination of Oc, osterix, and Runx2 expression. AVCR1 KO mice exhibited increased Oc and osterix expression and promoted bone resorption and formation. ACVR1-knockout was observed to activate the Wnt signaling pathway with an increase of β-catenin and reductions in GSK3β, DKK1, and FZD1. With the inhibited Wnt signaling pathway expression of Oc, osterix, and Runx2 was decreased, and ALP activity, mineralized nodule, and calcium content in cellular matrix were decreased as well, indicating that inactivation of the Wnt signaling pathway reduced the differentiation of BMSCs into osteoclasts. These findings indicate that ACVR1-knockout promotes osteogenic differentiation by activating the Wnt signaling pathway in mice.

MicroRNA-101 inhibits cell migration and invasion in bladder cancer via targeting FZD4.

Dysfunction of microRNAs (miRs) has been implicated in the development and progression of various human cancers. Our previous study demonstrated that miR-101 inhibited bladder cancer cell proliferation and invasion through inhibition of c-FOS expression. As an miR generally has many targets, other targets of miR-101 may also serve important roles in bladder cancer progression. Reverse transcription-quantitative polymerase chain reaction and western blot analyses were used to examine mRNA and protein expression, respectively. Wound healing and Transwell assays were conducted to study cell migration and invasion, respectively. The luciferase reporter gene assay was performed to verify one of the targets of miR-101. The data in the present study indicate that the expression of miR-101 is significantly reduced in bladder cancer tissues compared with that in adjacent non-tumour tissues. In addition, miR-101 expression is also downregulated in bladder cancer cell lines compared with that in normal bladder epithelial cells. Furthermore, low expression of miR-101 was significantly associated with tumour metastasis, advanced clinical stage, and poor prognosis in bladder cancer. Frizzled class receptor 4 (FZD4) was identified as a novel target of miR-101 in bladder cancer cells. The expression of FZD4 was significantly upregulated in bladder cancer tissues and cell lines. Both miR-101 overexpression and FZD4 inhibition caused a significant reduction of the migration and invasion of bladder cancer cells, whereas overexpression of FZD4 reversed the suppressive effects of miR-101 on bladder cancer cell migration and invasion. In conclusion, it was demonstrated that miR-101 downregulation is associated with bladder cancer progression and that miR-101 can inhibit bladder cancer cell migration and invasion via directly targeting FZD4. The present study expands the understanding of the molecular mechanisms underlying bladder cancer progression.

MiR-212-5p regulates the proliferation and apoptosis of AML cells through targeting FZD5.

To explore the effects of microRNA-212-5p (miR-212-5p) on biological functions of acute myeloid leukemia (AML) and to find the potential molecular mechanism. We measured the expression level of miR-212-5p in 35 AML patients and 20 patients with idiopathic thrombocytopenic purpura (ITP) as control cases. Besides, the miR-212-5p expression at cellular level was checked as well. In order to screen the functional targets of miR-212-5p, online prediction software was used and gene frizzled class receptor 5 (FZD5) attracted our attention. The effects of miR-212-5p on biological functions of AML cell line (Kasumi-1) were analyzed by subsequent experiments. The mRNA and protein expressions of FZD5 were detected by quantitative Real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB) analysis, respectively. Cell proliferation was tested by cell counting kit-8 (CCK-8) assay. Cell cycle and apoptosis were measured by flow cytometry. Finally, protein expression of β-catenin was analyzed by WB assay. In AML cases and cells, miR-212-5p was found to be lowly expressed. The potential target of miR-212-5p was predicated in three public databases. Through a series of experiments including qRT-PCR, WB and luciferase assay, we identified FZD5 as a functional target of miR-212-5p. In further cellular functional experiments on Kasumi-1, we found overexpression of miR-212-5p in Kasumi-1 cells greatly inhibited the cell viability and proliferation. The ratio of cells in G0/G1 phase and the proportion of apoptotic cells increased after miR-212-5p overexpression. Furthermore, Wnt/β-catenin signal pathway was the most apparent pathway that was regulated by miR-212-5p according to WB results. However, the effects of miR-212-5p were suppressed after restoring the expression of FZD5. Expression of miR-212-5p was significantly lower in AML patients and cell lines, indicating that miR-212-5p served as a tumor-suppressor gene in AML. According to our in vitro experiments, miR-212-5p/FZD5 was likely to become a new therapeutic target for AML.

FZD4 (frizzled class receptor 4)

FZD4 (frizzled class receptor 4)

Hsa_circ_0000177-miR-638-FZD7-Wnt Signaling Cascade Contributes to the Malignant Behaviors in Glioma.

As a new member of the noncoding RNA family, circular RNAs (circRNAs) have been demonstrated as critical regulators in various physiological and pathological processes, such as tumorigenesis. However, the role of circRNAs has not been well understood until now. In our study, we found that circRNA hsa_circ_0000177 was upregulated in glioma tissues and cell lines. Also, hsa_circ_0000177 overexpression was associated with poor prognosis in glioma patients. Through functional experiments, we found that hsa_circ_0000177 knockdown dramatically inhibited glioma cell proliferation and invasion in vitro. Consistently, hsa_circ_0000177 knockdown significantly repressed glioma growth in vivo. In terms of mechanism, we used bioinformatics analysis and identified hsa_circ_0000177 as a miR-638 sponge. We showed that miR-638 inhibition could restore the proliferation and invasion of glioma cells transfected with hsa_circ_0000177 small interfering RNA. Furthermore, we demonstrated that frizzled class receptor 7 (FZD7) was targeted by miR-638 and upregulated by hsa_circ_0000177. Through upregulating FZD7 expression, hsa_circ_0000177 activated Wnt signaling and facilitated glioma growth. Taken together, our study revealed a novel signaling pathway involved in glioma progression.

Wnt3a-regulated TCF4/β-catenin complex directly activates the key Hedgehog signalling genes Smo and Gli1.

The Wnt and Hedgehog signalling pathways serve key roles in diverse developmental processes. However, the molecular associations between these two signalling pathways remains unclear. Previous transcriptome studies on human foreskin fibroblasts have indicated that Wnt signalling activation induces the expression of key Hedgehog signalling genes, including smoothened, frizzled class receptor (Smo) and GLI family zinc finger 1 (Gli1). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) results revealed that Wnt3a treatment induced the expression of the key Hedgehog signalling genes, including Smo, patched (PTCH), Gli1, Gli2 and Gli3. In addition, western blot analyses demonstrated that Wnt3a treatment resulted in the accumulation of cellular Smo and Gli proteins. Furthermore, promoter sequence analysis revealed that the putative β-catenin/T-cell factor (TCF)-4 complex binding motifs (T/AC/GAAAG) were located within 1.5 kb of the Smo and Gli1 promoters. Results of the chromatin immunoprecipitation experiments and yeast-one hybrid assays revealed that TCF4 directly binds to the Smo and Gli1 promoters, with two binding sites for Smo and a single binding site for Gli1. Further analysis showed that the β-catenin/TCF4 complex binds to the Smo and Gli1 promoters. To investigate the functions of TCF4 and β-catenin in transcriptional regulation of Smo and Gli1, TCF4 and β-catenin were transiently expressed in fibroblast cells. RT-qPCR results demonstrated that overexpression of TCF4 and β-catenin induced the expression of Smo and Gli1. In addition, small interfering RNA-mediated suppression of β-catenin resulted in the downregulation of Smo and Gli1 expression levels, even under Wnt3a treatment. Suppression of β-catenin and Gli1 expression inhibited cell proliferation. Taken together, the results of the present study suggested that the β-catenin/TCF4 complex directly activates Smo and Gli1 by binding to their promoters, which in turn controls cell proliferation in human fibroblasts.

Identification and validation of a Hedgehog pathway-based 3-gene prognostic signature for gastric cancers.

Developing prognostic factors for patients with gastric cancer (GC) is crucial for the accurate identification of subgroups with distinct clinical outcomes and the development of effective treatment strategies. The aim of this study was to determine novel gene expression signatures from the hedgehog (Hh) signaling pathway as predictors of risk with biological significance. The Cancer Genome Atlas (TCGA) GC (STAD) cohort was used as the training dataset to select for significant prognostic Hh genes. Three Hh genes, indian hedgehog (IHH), patched 1 (PTCH1) and smoothened frizzled class receptor (SMO), were identified to be significant prognostic factors. On this basis, a 3-Hh-gene set was constructed and the high-risk patients of the training cohort were distinguished against low-risk cases [hazard ratio (HR)=1.73, 95% confidence interval (CI)=1.26-2.39, P=0.00069]. Then the gene signature was externally validated in a combined dataset from Gene Expression Omnibus (n=631), and experimentally confirmed in an independent cohort of 126 clinical GC samples by immunohistochemistry (IHC). Validation in the combined GEO dataset yielded consistent results (HR=1.45, 95% CI=1.17-1.81, P=0.00068), and remained significant for stages I-III, HER2-positive and surgery alone subgroups. Subsequently, we further demonstrated that this mRNA-based gene set could be successfully transferred to an IHC-based signature in our local cohort (HR=2.04, 95% CI=1.09-3.82, P=0.02). In addition, this signature served as an independent prognostic indicator for overall survival in the multivariate Cox analysis (HR=2.133, 95% CI=1.110-4.099, P=0.02). In conclusion, we successfully generated a stable III-Hh-gene model with the ability to separate patients into prognostic subgroups, which may have notable biological importance and be easily utilized clinically.

Luteolin attenuates Wnt signaling via upregulation of FZD6 to suppress prostate cancer stemness revealed by comparative proteomics

The mechanisms underlying luteolin-induced inhibition of prostate cancer (PCa) stemness have remained elusive. Here, we report that luteolin suppresses PCa stemness through Wnt signaling by upregulation of FZD6 (frizzled class receptor 6). Luteolin inhibits PCa cell proliferation, migration, self-renewal as well as the expression of prostate cancer stem cell markers in vitro. Through iTRAQ-based quantitative proteomics study, we identified 208 differentially expressed proteins in luteolin-treated PC-3 cells. Subsequent mechanistic analysis revealed that luteolin inhibits Wnt signaling by transcriptional upregulation of FZD6, and thereby suppressing the stemness of PCa cells. Furthermore, we identified FZD6 as a tumor suppressor that can abolish PCa stemness. In summary, our findings demonstrate that suppression of Wnt signaling by upregulation of FZD6 is a mechanism underlying luteolin-induced inhibition of PCa stemness. Our work suggests a new therapeutic strategy against human prostate cancer caused by aberrant activation of Wnt signaling.

Dysregulated genes targeted by microRNAs and metabolic pathways in bladder cancer revealed by bioinformatics methods.

The present study aimed to identify bladder cancer-associated microRNAs (miRNAs) and target genes, and further analyze the potential molecular mechanisms involved in bladder cancer. The mRNA and miRNA expression profiling dataset GSE40355 was downloaded from the Gene Expression Omnibus database. The Limma package in R was used to identify differential expression levels. The Human microRNA Disease Database was used to identify bladder cancer-associated miRNAs and Target prediction programs were used to screen for miRNA target genes. Enrichment analysis was performed to identify biological functions. The Database for Annotation, Visualization and Integration Discovery was used to perform OMIM_DISEASE analysis, and then protein-protein interaction (PPI) analysis was performed to identify hubs with biological essentiality. ClusterONE plugins in cytoscape were used to screen modules and the InterPro database was used to perform protein domain enrichment analysis. A group of 573 disease dysregulated genes were identified in the present study. Enrichment analysis indicated that the muscle organ development and vascular smooth muscle contraction pathways were significantly enriched in terms of disease dysregulated genes. miRNAs targets (frizzled class receptor 8, EYA transcriptional coactivator and phosphatase 4, sacsin molecular chaperone, calcium voltage-gated channel auxiliary subunit β2, peptidase inhibitor 15 and catenin α2) were mostly associated with bladder cancer. PPI analysis revealed that calmodulin 1 (CALM1), Jun proto-oncogene, AP-1 transcription factor subunit (JUN) and insulin like growth factor 1 (IGF1) were the important hub nodes. Additionally, protein domain enrichment analysis indicated that the serine/threonine protein kinase active site was enriched in module 1 extracted from the PPI network. Overall, the results suggested that the IGF signaling pathway and RAS/MEK/extracellular signal-regulated kinase transduction signaling may exert vital molecular mechanisms in bladder cancer, and that CALM1, JUN and IGF1 may be used as novel potential therapeutic targets.

MicroRNA-216b actively modulates diabetic angiopathy through inverse regulation on FZD5

BackgroundIn this work, we examined the angiogenic function of microRNA-216b in an in vitro rat diabetic model of myocardial microvascular endothelial cells (MMECs). MethodsMMECs were extracted from Wistar rats (MMEC(WI)) or diabetic Goto-Kakizaki (GK) rats (MMEC(GK)) and cultured in vitro. QRT-PCR was applied to compare miR-216b between MMEC(WI) and MMEC(GK). MiR-216b was downregulated in MMEC(GK). Its effects on angiogenic development, including invasion and proliferation, were evaluated. In MMEC(GK), putative miR-216b downstream target gene, frizzled class receptor 5 (FZD5), was evaluated by dual-luciferase reporter, qRT-PCR and western blot assays, respectively. FZD5 was further downregulated in MMEC(GK) with stable miR-216b downregulation to evaluate its functional role in regulating diabetic angiogenesis. ResultsMiR-216b was markedly overexpressed in MMEC(GK). MiR-216b downregulation significantly enhanced angiogenesis in MMEC(GK) by promoting invasion and proliferation. FZD5 was inversely upregulated in miR-216b-downregulated MMEC(GK). Subsequently, FZD5 downregulation suppressed angiogenic development, by inhibiting invasion and proliferation in miR-216b-downregulated MMEC(GK). ConclusionMicroRNA-216b was overexposed in diabetic MMECs and its downregulation may actively enhance angiogenesis in diabetic angiopathy through inverse regulation on FZD5.

Nonsense mutations in FZD2 cause autosomal-dominant omodysplasia: Robinow syndrome-like phenotypes.

Omodysplasia-2 (OMOD2; OMIM%16475) is a rare autosomal dominant (AD) skeletal dysplasia characterized by shortened humeri, short first metacarpal, craniofacial dysmorphism (frontal bossing, depressed nasal bridge, bifid nasal tip, and long philtrum), and variable degrees of genitourinary anomalies. This clinical phenotype overlaps with that of AD type Robinow syndrome. Recently, a mutation in FZD2 encoding a Frizzled Class Receptor 2 has been identified in a family with AD omodysplasia (an affected girl and her affected mother). Here, we present the second report on a heterozygous novel nonsense FZD2 mutation in OMOD2 or Robinow syndrome-like phenotype. The proband was a 16-year-old boy, who has been followed from infancy to adolescence. He presented with rhizomelic short stature with elbow restriction, mild facial dysmorphism (depressed broad bridge, short nose, anteverted nostrils, long philtrum, and low-set ears), and genital hypoplasia. Radiological examination in infancy showed short, broad humeri with relatively narrow distal ends, mildly broad femora, thick proximal ulnae with hypoplastic, dislocated proximal radii, and short first metacarpals. The abnormal skeletal pattern was persistent in adolescence; however, the humeri and femora became less undermodeled, while the humeri and radii became mildly bowed. Molecular analysis identified a de novo, heterozygous, nonsense mutation (c.1640C>A, p.S547*) in FZD2. The affected codon was next to the previously reported mutation (p.Trp548*). The results indicate that OMOD2 or Robinow syndome-like phenotype can be caused by a heterozygous nonsense FZD2 mutation impairing Wnt signaling. Further molecular studies will permit better clarification of the phenotypic spectrum in patients with OMOD2.

A Novel Variant of the FZD4 Gene in a Chinese Family Causes Autosomal Dominant Familial Exudative Vitreoretinopathy

Background/Aims: Familial exudative vitreoretinopathy (FEVR) is a complex hereditary eye disorder characterized by incomplete development of the retinal vasculature, thereby affecting retinal angiogenesis. Methods: In this study, a Chinese autosomal dominant FEVR pedigree was recruited. Ophthalmic examinations were performed, targeted next-generation sequencing was used to identify the causative gene, and Sanger sequencing was conducted to verify the candidate mutation. Co-segregation analysis was performed to evaluate pathogenicity. Semi-quantitative reverse transcription-PCR was applied to investigate the spatial and temporal expression patterns of the frizzled class receptor 4 (FZD4) gene in the mouse. Results: A novel heterozygous, deleterious variant of the FZD4 gene, c.A749G (p.Y250C), was identified in this FEVR pedigree, which co-segregated with the clinical phenotype. The amino acid tyrosine (Y) is highly conserved both orthologously and paralogously. The FZD4 gene was highly expressed in the retina, sclera of the eye, ovary, kidney, and liver; ubiquitously expressed in other tissues; and highly expressed in 6 different developmental stages/times of retinal tissue. Conclusion: Our study is the first to identify that the novel heterozygous variant c.A749G (p.Y250C) in the FZD4 gene may be the disease-causing mutation in this FEVR family, extending its mutation spectrum. These findings further our understanding of the molecular pathogenesis of FEVR and will facilitate the development of methods for the diagnosis, prevention, and genetic counseling of this disease.

Preliminary identification of key miRNAs, signaling pathways, and genes associated with Hirschsprung's disease by analysis of tissue microRNA expression profiles.

Hirschsprung's disease (HSCR) is a congenital gut motility disorder of infants, and if left untreated, it is fatal to the affected infants. This study aimed to identify key microRNAs (miRNAs), signaling pathways and genes involved in the pathogenesis of HSCR. The miRNA microarray dataset GSE77296 was downloaded. Nine colon tissue samples were available: six from HSCR patients and three matched control samples. Differentially expressed miRNAs (DEMs) were identified after data preprocessing. Target genes of the selected upregulated and downregulated DEMs were predicted. In addition, functional enrichment analyses for the selected DEMs and target genes were conducted. Finally, interaction networks between the DEMs and target genes were constructed. A total of 162 DEMs (73 upregulated and 89 downregulated) were obtained. A total of 2511 DEM-target gene pairs for the 40 selected DEMs were identified, including 1645 pairs for the upregulated DEMs and 866 pairs for the downregulated DEMs. The upregulated DEM miR-141-3p and down-regulated DEM miR-30a-3p were identified as key miRNAs by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and network analyses. Besides, KEGG pathway enrichment analysis revealed that pathways in cancer and the mitogen-activated protein kinase (MAPK) signaling pathway were key pathways. The key genes frizzled class receptor 3 (FZD3) and docking protein 6 (DOK6) were obtained through the DEM-target gene interaction networks. Two key miRNAs (miR-141-3p and miR-30a-3p), the MAPK signaling pathway and two key genes (FZD3 and DOK6) were implicated in the pathogenesis of HSCR.

Non-canonical Wnt mediated neurogenic differentiation of human bone marrow-derived mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (BM-MSCs), which are characterized by multipotency and self-renewal, are responsible for tissue regeneration and repair. We have previously reported in adipose tissue-derived MSCs that only Wnt5a is enhanced at neurogenic differentiation, and the mechanism of differentiation is dependent on the Wnt5a/JNK pathway; however, the role of Wnt/MAPK pathway is yet to be investigated in neurogenic differentiation in BM-MSCs. We compared the transcriptional expression of Wnt in neurogenic induced-hBM-MSCs (NI-hBM-MSCs) with that in primary hBM-MSCs, using RT-PCR, qPCR, and western blotting. Although the expression of Wnt1 and Wnt2 was unchanged, the expression of Wnt4, Wnt5a, and Wnt11 increased after neurogenic differentiation. In addition, only the expression of frizzled class receptor (Fzd) 3 gene was increased, but not of most of the Fzds and Wnt ligands in NI-hBM-MSCs. Interestingly, Wnt4, Wnt5a, and Wnt11 gene expressions significantly increased in NI-hBM-MSCs by qPCR. In addition, the protein expression level of Wnt4 and Wnt5a, but not Wnt3, increased after neurogenic induction. Furthermore, the expressions of phosphorylated-GSK-3β, ERK1/2, and PKC decreased; however, JNK was activated after neurogenic differentiation. Thus, non-canonical Wnts, i.e., Wnt4, Wnt5a, and Wnt11, regulate neurogenic differentiation through Fzd3 activation and the increase in downstream targets of JNK, which is one of the non-canonical pathways, in hBM-MSCs.