MiR-340 regulates the growth and metabolism of renal cell carcinoma cells by targeting frizzled class receptor 3.

MicroRNA(miR)-340 is known as a multifunctional miRNA related to various types of cancer while its role in renal cell carcinoma (RCC) remains to be further investigated. In the present study, an apparent increase in miR-340 expression was observed in both clear cell RCC tissues and RCC cell line 786-O and Caki-1. Functionally, the overexpression of miR-340 promoted cell proliferation, migration, invasion, extracellular alanine (Ala) level, and glycolysis level in 786-O cells. Then, frizzled class receptor 3 (FZD3) was determined as the target gene of miR-340 and its expression level was negatively regulated by miR-340. The FZD3 silencing abrogated the inhibitory effect of miR-340 knockdown on cell proliferation, migration, invasion, Ala level, and glycolysis level in 786-O cells. In conclusion, miR-340 promotes proliferation, migration, and invasion of RCC cells via suppressing FZD3 expression, and the promotion effect of miR-340 on RCC progression may be due to its regulatory effect on glycolysis and Ala level.

Concurrent Wnt pathway component expression in breast and colorectal cancer.

Wnt signaling pathway regulates important cell functions such as proliferation and migration and is frequently deregulated in colorectal and breast cancer. Thus, it constitutes an attractive therapeutic target with many drugs being investigated in clinical trials. Eighty-two breast and 102 colorectal carcinomas were analyzed for: relative mRNA expression levels of Wnt pathway components namely Wnt3 ligand, Frizzled 7 receptor and LEF1 transcriptional factor, their concurrent expression patterns and their correlation with clinicopathological features. Regarding breast carcinomas, increased relative mRNA expression levels of WNT3 were found in 54 % of cases whereas decreased relative mRNA expression levels were observed in FZD7 and LEF1 in 82 % and 43 % of cases, respectively. Expression levels of WNT3 were significantly correlated with tumour grade (p = 0.021) in breast cancer. As far as colorectal carcinomas are concerned, increased relative mRNA expression levels of WNT3, FZD7 and LEF1 were found in 60 %, 37 % and 48 % of cases respectively. A statistically significant correlation emerged between LEF1expression levels and pT-category (p = 0.027), suggesting a possible association with tumour aggressiveness in colorectal carcinomas. Statistically significant linear correlations were observed between the expression of WNT3/LEF1 (R = 0.233, p = 0.035) and FZD7/LEF1 (R = 0.359, p = 0.001) in breast carcinomas as well as in colorectal carcinomas (R = 0.536, p < 0.01 and R = 0.210, p = 0.034) respectively. Our results demonstrate a possible clinical significance of Wnt pathway gene expression levels in both tumour types. The distinct expression patterns and simultaneous expression of the investigated genes underscore the complexity of this pathway in breast and colorectal carcinogenesis and highlights the necessity of patient selection with regard to the effectiveness of Wnt pathway inhibitors.

Inhibition of Frizzled-2 by small interfering RNA protects rat hepatic BRL-3A cells against cytotoxicity and apoptosis induced by Hypoxia/Reoxygenation.

Frizzled-2 plays an important role in maintaining normal hepatic cell functionality. This study aimed to investigate the role of inhibition of Frizzled-2 in protecting rat liver BRL-3A cells from Hypoxia/Reoxygenation (H/R). In vitro H/R hepatic cell model was established by culturing BRL-3A cells under H/R condition. Frizzled-2 siRNA was transfected into BRL-3A cells to inhibit Frizzled-2 signaling. Wnt5a and Frizzled-2 were significantly increased in BRL-3A cells upon H/R treatment. H/R treatment induced cell cytotoxicity, the early apoptosis rate and the intracellular Ca2+ level in BRL-3A cells while silencing frizzled-2 gene decreased the H/R induced cell cytotoxicity, apoptosis and intracellular Ca2+ level. In vivo mice study further showed the up-regulation of Frizzled-2/Wnt 5 pathway and cleaved Caspase-3 expression in liver tissues under ischemia and reperfusion injury (IRI). In summary, inhibition of Frizzled-2 by its siRNA may protects BRL-3A cells by attenuating the H/R induced cell cytotoxicity and apoptosis.

Effect of circ MTHFD2 on resistance to pemetrexed in gastric cancer through regulating expression of miR-124.

OBJECTIVE: The aim of this study was to explore the effect of circ MTHFD2 on resistance to pemetrexed (MTA) in gastric cancer by regulating the expression of micro ribonucleic acid (miR)-124. MATERIALS AND METHODS: Human gastric cancer MGC-803 cells were induced by MTA at an increasing concentration. MGC-803/MTA resistant cell model was successfully established after 5 months. The half-maximal inhibitory concentration (IC50) of MTA on the two kinds of cells was detected via cell counting kit-8 (CCK-8) assay. Differentially expressed circular RNAs (circRNAs) were screened using circRNA microarray analysis. Meanwhile, the target miRNAs of circRNAs were predicted using bioinformatics tool and verified via luciferase reporter assay, respectively. In MGC-803 cells, the effects of overexpression and knockdown of circ MTHFD2 on the expression of miR-124 were detected via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Finally, the effects of circ MTHFD2 on the protein expressions of FDZ5 and multidrug resistance gene-1 (MDR-1) were detected via Western blotting. RESULTS: MGC-803/MTA resistant cell lines were successfully constructed via persistent drug exposure to MTA at an increasing concentration for 5 months. Compared with parental cells, MGC-803/MTA resistant cells showed significantly enhanced drug resistance. Subsequently, differentially expressed circRNAs were screened using circRNA microarray analysis. A total of 673 circRNAs were screened out based on Fold Change >3 and adjusted p-value. The results of qRT-PCR showed that the expression levels of all differentially expressed circRNAs were significantly changed when compared with MGC-803/MTA resistant cells. Compared with MGC-803/MTA resistant cells, the drug resistance of cells increased significantly in circ MTHFD2 overexpression group. However, it markedly decreased in circ MTHFD2 knockdown group. According to the results of bioinformatics and luciferase reporter assay, circ MTHFD2 could target bind to miR-124. In MGC-803/MTA cells, miR-124 could remarkably increase the resistance of MGC-803/MTA cells to MTA. Western blotting results revealed that overexpression of circ MTHFD2 significantly increased the protein expressions of FDZ5 and MDR-1. However, miR-124 mimics reversed the inhibitory effect of circ MTHFD2 on FDZ5 and MDR-1. CONCLUSIONS: Circ MTHFD2 directly binds to miR-124 through the molecular sponge effect. This may induce increased protein expression of MDR-1, ultimately enhancing the drug resistance of MGC-803/MTA cells.

Abnormal axon guidance signals and reduced interhemispheric connection via anterior commissure in neonates of marmoset ASD model.

In autism spectrum disorder (ASD), disrupted functional and structural connectivity in the social brain has been suggested as the core biological mechanism underlying the social recognition deficits of this neurodevelopmental disorder. In this study, we aimed to identify genetic and neurostructural abnormalities at birth in a non-human primate model of ASD, the common marmoset with maternal exposure to valproic acid (VPA), which has been reported to display social recognition deficit in adulthood. Using a comprehensive gene expression analysis, we found that 20 genes were significantly downregulated in VPA-exposed neonates. Of these, Frizzled3 (FZD3) and PIK3CA were identified in an axon guidance signaling pathway. FZD3 is essential for the normal development of the anterior commissure (AC) and corpus callosum (CC); hence, we performed diffusion tensor magnetic resonance imaging with a 7-Tesla scanner to measure the midsagittal sizes of these structures. We found that the AC size in VPA-exposed neonates was significantly smaller than that in age-matched controls, while the CC size did not differ. These results suggest that downregulation of the genes related to axon guidance and decreased AC size in neonatal primates may be linked to social brain dysfunctions that can happen later in life.

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.

Retinoic acid-induced expression of Hnf1b and Fzd4 is required for pancreas development in Xenopus laevis.

Retinoic acid (RA) is required for pancreas specification in Xenopus and other vertebrates. However, the gene network that is directly induced by RA signalling in this context remains to be defined. By RNA sequencing of in vitro-generated pancreatic explants, we identified the genes encoding the transcription factor Hnf1ß and the Wnt-receptor Fzd4/Fzd4s as direct RA target genes. Functional analyses of Hnf1b and Fzd4/Fzd4s in programmed pancreatic explants and whole embryos revealed their requirement for pancreatic progenitor formation and differentiation. Thus, Hnf1ß and Fzd4/Fzd4s appear to be involved in pre-patterning events of the embryonic endoderm that allow pancreas formation in Xenopus.

A fusion-protein approach enabling mammalian cell production of tumor targeting protein domains for therapeutic development.

A single chain Fv fragment (scFv) is a fusion of the variable regions of heavy (VH ) and light (VL ) chains of immunoglobulins. They are important elements of chimeric antigen receptors for cancer therapy. We sought to produce a panel of 16 extracellular protein domains of tumor markers for use in scFv yeast library screenings. A series of vectors comprising various combinations of expression elements was made, but expression was unpredictable and more than half of the protein domains could not be produced using any of the constructs. Here we describe a novel fusion expression system based on mouse TEM7 (tumor endothelial marker 7), which could facilitate protein expression. With this approach we could produce all but one of the tumor marker domains that could not otherwise be expressed. In addition, we demonstrated that the tumor associated antigen hFZD10 produced as a fusion protein with mTEM7 could be used to enrich scFv antibodies from a yeast display library. Collectively our study demonstrates the potential of specific fusion proteins based on mTEM7 in enabling mammalian cell production of tumor targeting protein domains for therapeutic development.

Overexpression of FZD1 and CAIX are Associated with Invasion, Metastasis, and Poor-Prognosis of the Pancreatic Ductal Adenocarcinoma.

Approximately 80% of patients with pancreatic ductal adenocarcinoma (PDAC) have metastatic disease with poor prognosis, but clinically available biomarkers have not yet been identified. This study was to investigate the clinical significance of FZD1 and CAIX in PDACs. FZD1 and CAIX protein expression was measured using EnVision immunohistochemistry. Positive FZD1 or CAIX expression was significantly higher in PDAC than that in precursor lesions (p < 0.01). Positive FZD1 or CAIX expression was significantly lower in cases with well-differentiated adenocarcinoma, no-metastasis of the lymph node, no-invasion of regional tissues, and TNM I/II stage disease than in cases with poorly-differentiated adenocarcinoma, metastasis and invasion, and TNM stage III+ IV stage disease (p < 0.05 or p < 0.01). The expression of FZD1 positively correlated with CAIX in PDAC (P = 0.000). Univariate Kaplan-Meier analysis showed that FZD1 and/or CAIX expression (p < 0.001) was significantly associated with shorter overall survival (p < 0.05). Cox multivariate analysis showed that differentiation, tumor mass, lymph node metastasis, invasion, TNM stage, FZD1 and CAIX levels negatively correlated with overall survival. Positive FZD1 and CAIX expressions are poor prognostic factors in PDAC patients. FZD1 and CAIX might be important biological markers for the carcinogenesis, metastasis, invasion, and prognosis of PDAC.

Wnt and frizzled expression during regeneration of internal organs in the holothurian Eupentacta fraudatrix.

Several genes of the Wnt and Frizzled families in the holothurian Eupentacta fraudatrix are characterized, and the complete coding sequences of wntA, wnt4, wnt6, wnt16, frizzled1/2/7, frizzled4, and frizzled5/8 are obtained. The dynamics of expression of these genes during regeneration of internal organs after evisceration are studied. Evisceration and the associated damages supposedly induce the expression of wnt16 on third day after evisceration. Genes wntA, wnt4, wnt6, and frizzled1/2/7 up-regulate during the period of active morphogenesis (5-7 days after evisceration) and might participate in regulation of tissue and organ formation. The signaling induced via Frizzled5/8 is could be necessary for formation of the anterior (ectodermal) part of the digestive system and development of the calcareous ring on 10th day after evisceration. Our data suggest that the Wnt signaling pathway plays a significant role in the regulation of regeneration of internal organs in holothurians.

Circular Noncoding RNA HIPK3 Mediates Retinal Vascular Dysfunction in Diabetes Mellitus.

BACKGROUND: The vascular complications of diabetes mellitus are the major causes of morbidity and mortality among people with diabetes. Circular RNAs are a class of endogenous noncoding RNAs that regulate gene expression in eukaryotes. In this study, we investigated the role of circular RNA in retinal vascular dysfunction induced by diabetes mellitus. METHODS: Quantitative polymerase chain reactions, Sanger sequencing, and Northern blots were conducted to detect circular HIPK3 (circHIPK3) expression pattern on diabetes mellitus-related stresses. MTT (3-[4,5-dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assays, EdU (5-ethynyl-2'-deoxyuridine) incorporation assays, Transwell migration assays, and Matrigel assays were conducted to detect the role of circHIPK3 in retinal endothelial cell function in vitro. Retinal trypsin digestion, vascular permeability assays, and ELISA assays were conducted to detect the role of circHIPK3 in retinal vascular dysfunction in vivo. Bioinformatics analysis, luciferase activity assays, RNA pull-down assays, and in vitro studies were conducted to reveal the mechanism of circHIPK3-mediated retinal vascular dysfunction. RESULTS: circHIPK3 expression was significantly upregulated in diabetic retinas and retinal endothelial cells following stressors related to diabetes mellitus. circHIPK3 silencing or overexpressing circHIPK3 changed retinal endothelial cell viability, proliferation, migration, and tube formation in vitro. circHIPK3 silencing in vivo alleviated retinal vascular dysfunction, as shown by decreased retinal acellular capillaries, vascular leakage, and inflammation. circHIPK3 acted as an endogenous miR-30a-3p sponge to sequester and inhibit miR-30a-3p activity, which led to increased vascular endothelial growth factor-C, FZD4, and WNT2 expression. Ectopic expression of miR-30a-3p mimicked the effect of circHIPK3 silencing on vascular endothelial phenotypes in vivo and in vitro. CONCLUSIONS: The circular RNA circHIPK3 plays a role in diabetic retinopathy by blocking miR-30a function, leading to increased endothelial proliferation and vascular dysfunction. These data suggest that circular RNA is a potential target to control diabetic proliferative retinopathy.

Let7b modulates the Wnt/ß-catenin pathway in liver cancer cells via downregulated Frizzled4.

Let7 microRNA implicated in many cellular processes and participated in the progress of various tumors. Similarly, Wnt signaling pathway plays an important role in morphogenesis, differentiation, cell survival, and proliferation. However, there is little research focusing on the relevance between Let7b and Wnt/ß-catenin signaling pathway, especially in liver cancer cell. To study this, human liver cancer cells HUH7 and MHCC97H were cultured, enhanced, or inhibited the expression of Let7b in two cell lines. Western blotting was used to measure the expression of Wnt signaling-related protein ß-catenin and Frizzled family receptor. CD24+133+ was used as a cancer stem cell marker, and the proportion of CD24+133+ in liver cancer cell lines was observed by flow cytometry. The proliferation, invasiveness, and migration of liver cancer cells were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, transwell, and wound healing assays. The research revealed that enhanced expression of Let7b decreased the expression of Frizzled4, while inhibited Let7b expression increased Frizzled4 expression. Enhanced Let7b expression reduced the proportion of cancer stem cell in liver cancer cell; meanwhile, Let7b inhibition increased the proportion of cancer stem cell. Upregulated Let7b expression repressed the proliferation, invasion, and migration of liver cancer cell. This study showed that Let7b modulates the proliferation, invasiveness, and migration of liver cancer cell and reduces the proportion of cancer stem cells in liver cancer cell by inhibiting Wnt/ß-catenin signaling pathway via downregulated Frizzled4.

MicroRNA-505 predicts prognosis and acts as tumor inhibitor in cervical carcinoma with inverse association with FZD4.

PURPOSE: We investigated the expression and mechanisms of microRNA-505 (miR-505) and its downstream target gene Frizzled-4 (FZD4) in cervical cancer. METHODS: miR-505 expression was evaluated by qRT-PCR in cervical cancer cell lines and human carcinomas. Cancer patients' clinicopathological factors and survival were analyzed based on their tumorous miR-505 levels. Ca-Ski and HeLa cells were transduced with lentivirus to upregulate or downregulate miR-505. Their impacts on cervical cancer were evaluated by in vitro proliferation, invasion and in vivo tumorigenicity assays, respectively. Target gene of miR-505, FZD4, was evaluated by dual-luciferase reporter assay. Its expression in cervical cancer cell was evaluated by qRT-PCR. FZD4 was either upregulated or downregulated in cervical cancer cells to further assess its impact on modulating cervical cancer development in vitro. RESULTS: MiR-505 is lowly expressed in cervical cancer cell lines and human carcinomas. Low tumorous miR-505 expression was associated with patients' advanced tumor stage and short survival. In Ca-Ski and HeLa cells, lentivirus-mediated miR-505 upregulation suppressed cancer proliferation and invasion in vitro, and tumorigenicity in vivo, whereas miR-505 downregulation had no functional effects. FZD4 was confirmed to be a downstream target of miR-505, and found to be upregulated in cervical cancer. Genetic modification of FZD4 in cervical cancer cells yielded a significant change in cancer growth, as FZD4 upregulation suppressed whereas FZD4 downregulation promoted cervical cancer proliferation and invasion In vitro. CONCLUSION: MiR-505 may act as a cancer inhibitor and prognostic factor in cervical cancer. FDZ4 is reversely expressed as miR-505, and has dramatic regulatory function in cervical cancer.

Clinical implication of Frizzled 2 expression and its association with epithelial-to-mesenchymal transition in hepatocellular carcinoma.

The epithelial-to-mesenchymal transition (EMT) is an initial, critical step in hepatocellular carcinoma (HCC) tumor invasion and metastasis. Frizzled 2 (Fzd2) expression might drive EMT through the non-canonical Wnt pathway, one of the various EMT signaling pathways. The expression of epithelial (E-cadherin) and mesenchymal (vimentin) markers, as well as that of Wnt5b, Stat3, IL-6, Jak2 and Fzd2, were measured in 15 HCC cell lines. The EMT status (vimentin to E-cadherin mRNA expression ratio), Fzd2 mRNA expression, and pSTAT3 protein expression were assessed by immunostaining in 100 HCC patients, and correlations of their expression with clinicopathological factors and prognosis were analyzed. Cell proliferation, migration, and invasiveness were assessed after Fzd2 knockdown. Fzd2 expression was significantly correlated with a mesenchymal phenotype in the HCC cell lines. Treatment of the cell lines with Fzd2 siRNA resulted in significantly reduced migration and invasiveness but did not affect proliferation. A significant correlation was detected between the EMT status and Fzd2 expression in the HCC patients. Multivariate analysis revealed that Fzd2 expression was an independent predictor of recurrence (P=0.034). Patients with high Fzd2 expression had significantly poorer recurrence­free survival than those with low expression (P=0.03). Finally, pSTAT3 expression was significantly correlated with the EMT and Fzd2 status (P=0.0028, and P=0.0066, respectively). Fzd2 expression induced EMT and enhanced cell migration and invasiveness, and it might be a novel predictor of HCC recurrence. Furthermore, Stat3 might be controlled by both the Wnt5/Fzd2 and IL-6/Jak2 signaling pathways and play an important role in EMT.

High variability of expression profiles of homeologous genes for Wnt, Hh, Notch, and Hippo signaling pathways in Xenopus laevis.

Cell signaling pathways, such as Wnt, Hedgehog (Hh), Notch, and Hippo, are essential for embryogenesis, organogenesis, and tissue homeostasis. In this study, we analyzed 415 genes involved in these pathways in the allotetraploid frog, Xenopus laevis. Most genes are retained in two subgenomes called L and S (193 homeologous gene pairs and 29 singletons). This conservation rate of homeologs is much higher than that of all genes in the X. laevis genome (86.9% vs 60.2%). Among singletons, 24 genes are retained in the L subgenome, a rate similar to the average for all genes (82.8% vs 74.6%). In addition, as general components of signal transduction, we also analyzed 32 heparan sulfate proteoglycan (HSPG)-related genes and eight TLE/Groucho transcriptional corepressors-related genes. In these gene sets, all homeologous pairs have been retained. Transcriptome analysis using RNA-seq data from developmental stages and adult tissues demonstrated that most homeologous pairs of signaling components have variable expression patterns, in contrast to the conservative expression profiles of homeologs for transcription factors. Our results indicate that homeologous gene pairs for cell signaling regulation have tended to become subfunctionalized after allotetraploidization. Diversification of signaling pathways by subfunctionalization of homeologs may enhance environmental adaptability. These results provide insights into the evolution of signaling pathways after polyploidization.

Transcriptional Regulation of Frizzled-1 in Human Osteoblasts by Sp1.

The wingless pathway has a powerful influence on bone metabolism and is a therapeutic target in skeletal disorders. Wingless signaling is mediated in part through the Frizzled (FZD) receptor family. FZD transcriptional regulation is poorly understood. Herein we tested the hypothesis that Sp1 plays an important role in the transcriptional regulation of FZD1 expression in osteoblasts and osteoblast mineralization. To test this hypothesis, we conducted FZD1 promoter assays in Saos2 cells with and without Sp1 overexpression. We found that Sp1 significantly up-regulates FZD1 promoter activity in Saos2 cells. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift (EMSA) assays identified a novel and functional Sp1 binding site at -44 to -40 from the translation start site in the FZD1 promoter. The Sp1-dependent activation of the FZD1 promoter was abolished by mithramycin A (MMA), an antibiotic affecting both Sp1 binding and Sp1 protein levels in Saos2 cells. Similarly, down-regulation of Sp1 in hFOB cells resulted in less FZD1 expression and lower alkaline phosphatase activity. Moreover, over-expression of Sp1 increased FZD1 expression and Saos2 cell mineralization while MMA decreased Sp1 and FZD1 expression and Saos2 cell mineralization. Knockdown of FZD1 prior to Sp1 overexpression partially abolished Sp1 stimulation of osteoblast differentiation markers. Taken together, our results suggest that Sp1 plays a role in human osteoblast differentiation and mineralization, which is at least partially mediated by Sp1-dependent transactivation of FZD1.

miR-135b reverses chemoresistance of non-small cell lung cancer cells by downregulation of FZD1.

BACKGROUND: Non-small cell lung cancer (NSCLC) chemoresistance usually limits the clinical efficacy of chemotherapeutic approaches. However, few reports have revealed the regulation of miR-135b and Frizzled-1 (FZD1) involved in NSCLC chemoresistance. METHODS: To identify the mechanism of miR-135b and FZD1 in NSCLC chemoresistance and to observe their biological functions, we detected the expression levels of miR-135b and FZD1 by conducting quantitative real-time polymerase chain reaction (RT-qPCR) and modified the expressions of miR-135b and FZD1 by transiently transfecting cells with miR-135b mimics or FZD1-siRNA. The 3'-untranslated region (3'-UTR) of FZD1 combined with miR-135b was verified through dual-luciferase reporter assay. RESULTS: Compared with that in A549 parental cell lines, the miR-135b expression in drug-resistant lung cancer cell lines (A549/DDP) was decreased and their FZD1 expression was increased. The increased miR-135b expression and silenced FZD1 expression enhanced the sensitivity of resistant cells to cisplatin treatment. The high expression of miR-135b in A549/DDP cells remarkably decreased the mRNA levels of FZD1. FZD1 was further identified as the functional downstream target of miR-135b by directly targeting the 3'-UTR of FZD1. CONCLUSION: The amplification of miR-135b suppressed NSCLC chemoresistance by directly mediating the FZD1 downregulation.

Wnt, Frizzled, and sFRP gene expression patterns during gastrulation in the starfish Patiria (Asterina) pectinifera.

By the initial phase of gastrulation, Wnt pathway regulation mediates endomesoderm specification and establishes the animal-vegetal axis, thereby leading to proper gastrulation in starfish. To provide insight into the ancestral mechanism regulating deuterostome gastrulation, we identified the gene expression patterns of Wnt, Frizzled (Fz), and secreted frizzled-related protein (sFRP) family genes, which play a role in the initial stage of the Wnt pathway, in starfish Patiria (Asterina) pectinifera embryos using whole mount in situ hybridization. We identified ten Wnt, four Fz, and two sFRP paralogues. From the hatching blastula to the late gastrula stage, the majority of the Wnt genes and both Fz5/8 and sFRP1/5 were expressed in the posterior and anterior half of the embryo, respectively. Wnt8, Fz1, and Fz4 showed restricted expression in the lateral ectoderm. On the other hand, several genes were expressed de novo in the restricted domain of the archenteron at the late gastrula stage. These results suggest that the canonical and/or non-canonical Wnt pathway might implicate endomesoderm specification, anterior-posterior axis establishment, anterior-posterior patterning, and archenteron morphogenesis in the developmental context of starfish embryos. From comparison with the expression patterns observed in Patria miniata, we consider that the Wnt pathway is conserved among starfishes.

The expression and function of Frizzled-7 in human renal cell carcinoma.

PURPOSE: Wnt/ß-catenin has emerged as an important signal pathway in renal cell carcinoma (RCC) pathogenesis. Frizzled 7 (Fzd7) is a member of Frizzled (Fzd) receptor family which binds with Wnt ligands and transduces canonical and non-canonical pathways. However, the expression of Fzd7 in human RCC is poorly investigated. METHODS: 53 RCC tissues and peri-tumor tissues were collected from the patients treated with radical nephrectomy. The expression of Fzd7 was investigated by immunohistochemical staining. Three RCC cells were transfected with Fzd7shRNA and GFPshRNA to investigate the function of Fzd7 in RCC cells. RESULTS: The immunohistochemical analysis showed that Fzd7 protein expression level was significantly increased in RCC tissues when compared with peri-tumor tissues, which suggested that Fzd7 might be involved in the formation of tumors. However, the Fzd7 expression was not correlated with clinicopathological parameters. Three RCC cell lines: 786-O, Caki-1, and OS-RC-2 also expressed Fzd7. With Fzd7 expression being interfered by shRNA, the RCC cell proliferation was mildly decreased. Wnt3a could stimulate the RCC cells proliferation, but the stimulation was decreased when Fzd7 expression was interfered. Restoring the Fzd7 expression led to the proliferation stimulation effect of Wnt3a being restored. CONCLUSIONS: This paper suggests that Fzd7 may act as one of the molecules that take part in the course of RCC formation. Fzd7 can be activated by Wnt3a to stimulate cell proliferation.

Frizzled2 mediates the migration and invasion of human oral squamous cell carcinoma cells through the regulation of the signal transducer and activator of transcription-3 signaling pathway.

Frizzled2 (Fzd2) is a receptor for wingless-type MMTV integration site family members (Wnts), the aberrant overexpression of which has been noted to contribute to cancer metastasis. The present study was performed to characterize the role of Fzd2 in the migration and invasion of oral squamous cell carcinomas (OSCC) in vitro. Using TSCCa cells (a tongue SCC cell line) for loss- or gain-of-function of Fzd2, we found that a forced overexpression of Fzd2 promoted TSCCa cell migration and invasion, decreased the expression of epithelial­cadherin (E-cadherin, an epithelial marker) and increased that of vimentin, Snail Slug, matrix metalloproteinases (MMPs)-2/-9/-13 and a-disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS5). By contrast, RNA interference (RNAi)-mediated knockdown of Fzd2 had opposite effects on OSCC cells. In addition, we found that the phosphorylation of signal transducer and activator of transcription-3 (STAT3) was enhanced by Fzd2 overexpression, but suppressed by Fzd2 depletion, and that STAT3­specific shRNA attenuated Fzd2 overexpression­induced cell invasion. In summary, the present study demonstrated that Fzd2 contributes to the migration and invasion of OSCC cells, at least partly through regulation of the STAT3 pathway. These results suggest Fzd2 as a novel therapeutic target for OSCC.