OBJECTIVES: Frizzled 7 (FZD7) is abnormally expressed and activated in a variety of cancers. In ovarian cancer, overexpression of FZD7 reduces the sensitivity of platinum-resistant ovarian cancer cells to ferroptosis, thereby allowing cancer cells to survive. However, whether FZD7 inhibits ferroptosis in ovarian cancer cells and its mechanisms are remain unclear. This study aims to explore the effects of FZD7 and its upstream regulator miR-1-3p on ferroptosis in ovarian cancer cells are evaluated to clarify the molecular mechanism for miR-1-3p and FZD7's involvement in ferroptosis in ovarian cancer cells. METHODS: Human ovarian cancer cell lines HO8910 and SKOV3 were used as the research subjects. In the first part of the experiment, human ovarian cancer cells were transfected with blank plasmid and FZD7 overexpression plasmid, respectively; in the second and third parts, human ovarian cancer cells were transfected with miR-1-3p mimics negative control, miR-1-3p mimics, miR-1-3p inhibitors negative control, and miR-1-3p inhibitors, respectively; in the fourth part of the experiment, human ovarian cancer cells were transfected with miR-1-3p mimics and miR-1-3p mimics+FZD7 overexpression plasmid, respectively, and normal cultured cells were set as the control group. The human ovarian cancer cell ferroptosis model was established by incubating human ovarian cancer cells with different treatments with ferroptosis inducer Erastin or RSL3. Real-time RT-PCR was used to detect the mRNA expression levels of FZD7 and miR-1-3p; Western blotting was used to detect the protein expression levels of FZD7; CCK-8 assay was used to detect the cell viability; lipid peroxidation colorimetric assay kit was used to detect the level of intracellular MDA; and iron assay kit was used to detect the level of intracellular Fe2+. Dual-luciferase assay was used to detect the targeting relationship between miR-1-3p and FZD7. RESULTS: Overexpression of FZD7 increased the cell viability of human ovarian cancer cell lines HO8910 or SKOV3 (P<0.05, P<0.01, or P<0.001) and decreased the intracellular MDA levels (P<0.01) in Erastin-treated or RSL3-treated ovarian cancer cells. FZD7 was a direct target of miR-1-3p, which inhibited the expression of FZD7 (P<0.01) by binding to the 3'-untranslated region (3'UTR) site of FZD7. MiR-1-3p mimics decreased the cell viability of human ovarian cancer cell lines HO8910 or SKOV3 (P<0.05, P<0.01, or P<0.001) and increased the intracellular MDA levels (P<0.01) in Erastin-treated or RSL3-treated ovarian cancer cells; while miR-1-3p inhibitors significantly increased the cell viability of human ovarian cancer cell lines HO8910 or SKOV3 (P<0.05, P<0.01, or P<0.001) and decreased the intracellular MDA levels (P<0.01) in Erastin-treated or RSL3-treated ovarian cancer cells. The effect of miR-1-3p mimics on enhancing the sensitivity of human ovarian cancer cells to Erastin-induced or RSL3-induced ferroptosis was abrogated by overexpression of FZD7(P<0.05 or P<0.01). CONCLUSIONS MiR-1-3p enhances the sensitivity of ovarian cancer cells to ferroptosis by targeting FZD7.
Female ; Humans ; Frizzled Receptors/genetics* ; MicroRNAs/genetics* ; Ovarian Neoplasms/genetics* ; Ferroptosis

OBJECTIVE: To explore the regulatory function of RNA binding motif protein 38 (RBM38) in human acute myeloid leukemia cells HL-60 and its mechanism. METHODS: The lentivirus carriers of overexpressed and knockdown RBM38 were constructed. After HL-60 cells were transfected, Western blot was used to analyze the expression level of RBM38 in HL-60 cells. The cell proliferation and cycle of HL-60 were detected by CCK-8 assay and flow cytometry assay, respectively. RNA immunoprecipitation coupled real-time PCR (RIP-qPCR) was used to detect the combination of RBM38 with mRNAs. Actinomycin D treatment followed by real-time PCR (AcD-qPCR) was used to detect the effect of RBM38 on the stability of target mRNAs. RESULTS: RBM38 in HL-60 cells was overexpressed or inhibited by lentivirus transduction. Overexpressed RBM38 promoted the cell cycle and proliferation of HL-60, while RBM38 knockdown repressed the two processes. RBM38 showed an interaction with FZD1 mRNA and enhancement of its stability. CONCLUSION RBM38 can regulate cell proliferation of HL-60 by improving the stability of FZD1 mRNA.
Cell Proliferation ; Frizzled Receptors ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; RNA Stability ; RNA-Binding Proteins/metabolism*

Astrocytoma ; genetics ; metabolism ; Brain Neoplasms ; genetics ; metabolism ; Frizzled Receptors ; genetics ; metabolism ; Glioblastoma ; genetics ; metabolism ; Glioma ; genetics ; metabolism ; Humans ; Paraffin Embedding ; RNA, Messenger ; metabolism ; Receptors, G-Protein-Coupled ; genetics ; metabolism ; Signal Transduction ; Wnt2 Protein ; genetics ; metabolism ; beta Catenin ; genetics ; metabolism

Insulin like growth factor (IGF)-1 signaling through type 1 IGF receptor (IGF1R) is essential to the normal growth and development of the central nervous system. IGF-1 stimulates proliferation of neural stem cells and neural progenitors. IGF-1 also promotes survival and differentiation of neurons and oligodendrocytes, including neuritic outgrowth, synaptogenesis, and myelin production. The phosphatidylinositol 3 kinase (PI3)-Akt pathway and mitogen-activated protein (MAP) kinase pathway are two predominant mediators of IGF1-IGF1R signaling in neural cells. beta-catenin, a key molecule of the canonical Wnt signaling pathway, is also a downstream target of PI3-Akt-glycogen synthase kinase 3beta (GSK3beta) pathway. IGF-1 signaling through IGF1R interacts with canonical Wnt pathway at the levels of GSK3beta and beta-catenin rather than at the levels of Wnt ligands and Frizzled receptors to promote neural proliferation in developing central nervous system.
beta Catenin ; Brain ; Central Nervous System ; Frizzled Receptors ; Glycogen Synthase Kinase 3 ; Growth and Development ; Insulin ; Insulin-Like Growth Factor I ; Ligands ; Myelin Sheath ; Neural Stem Cells ; Neurons ; Oligodendroglia ; Phosphatidylinositol 3-Kinase ; Phosphotransferases ; Wnt Proteins ; Wnt Signaling Pathway

OBJECTIVE: To investigate the mechanism by which fibroblasts with high WNT2b expression causes intestinal mucosa barrier disruption and promote the progression of inflammatory bowel disease (IBD). METHODS: Caco-2 cells were treated with 20% fibroblast conditioned medium or co-cultured with fibroblasts highly expressing WNT2b, with the cells without treatment with the conditioned medium and cells co-cultured with wild-type fibroblasts as the control groups. The changes in barrier permeability of Caco-2 cells were assessed by measuring transmembrane resistance and Lucifer Yellow permeability. In Caco-2 cells co-cultured with WNT2b-overexpressing or control intestinal fibroblasts, nuclear entry of β-catenin was detected with immunofluorescence assay, and the expressions of tight junction proteins ZO-1 and E-cadherin were detected with Western blotting. In a C57 mouse model of dextran sulfate sodium (DSS)-induced IBD-like enteritis, the therapeutic effect of intraperitoneal injection of salinomycin (5 mg/kg, an inhibitor of WNT/β-catenin signaling pathway) was evaluated by observing the changes in intestinal inflammation and detecting the expressions of tight junction proteins. RESULTS: In the coculture system, WNT2b overexpression in the fibroblasts significantly promoted nuclear entry of β-catenin (P < 0.01) and decreased the expressions of tight junction proteins in Caco-2 cells; knockdown of FZD4 expression in Caco-2 cells obviously reversed this effect. In DSS-treated mice, salinomycin treatment significantly reduced intestinal inflammation and increased the expressions of tight junction proteins in the intestinal mucosa. CONCLUSION Intestinal fibroblasts overexpressing WNT2b causes impairment of intestinal mucosal barrier function and can be a potential target for treatment of IBD.
Humans ; Mice ; Animals ; Caco-2 Cells ; beta Catenin/metabolism* ; Culture Media, Conditioned/pharmacology* ; Tight Junctions/metabolism* ; Intestinal Mucosa ; Inflammatory Bowel Diseases ; Tight Junction Proteins/metabolism* ; Inflammation/metabolism* ; Fibroblasts/metabolism* ; Mice, Inbred C57BL ; Glycoproteins/metabolism* ; Wnt Proteins/pharmacology* ; Frizzled Receptors/metabolism*

Wnt signaling are critical pathway involved in organ development, tumorigenesis, and cancer progression. WNT7A, a member of the Wnt family, remains poorly understood in terms of its role and the underlying molecular mechanisms it entails in head and neck squamous cell carcinoma (HNSCC). According to the Cancer Genome Atlas (TCGA), transcriptome sequencing data of HNSCC, the expression level of WNT7A in tumors was found to be higher than in adjacent normal tissues, which was validated using Real-time RT-PCR and immunohistochemistry. Unexpectedly, overexpression of WNT7A did not activate the canonical Wnt-β-catenin pathway in HNSCC. Instead, our findings suggested that WNT7A potentially activated the FZD7/JAK1/STAT3 signaling pathway, leading to enhanced cell proliferation, self-renewal, and resistance to apoptosis. Furthermore, in a patient-derived xenograft (PDX) tumor model, high expression of WNT7A and phosphorylated STAT3 was observed, which positively correlated with tumor progression. These findings underscore the significance of WNT7A in HNSCC progression and propose the targeting of key molecules within the FZD7/JAK1/STAT3 pathway as a promising strategy for precise treatment of HNSCC.
Animals ; Humans ; Squamous Cell Carcinoma of Head and Neck ; Carcinogenesis/genetics* ; Cell Transformation, Neoplastic ; Wnt Signaling Pathway ; Disease Models, Animal ; Head and Neck Neoplasms/genetics* ; Wnt Proteins ; Frizzled Receptors/genetics* ; Janus Kinase 1 ; STAT3 Transcription Factor

OBJECTIVETo investigate the role of Wnt signaling pathway on paraquat (PQ)induced PC12 cells damage.

METHODSUsing PC12 cells, in this study CCK8 assay was used to detect the effect of cell viability. The cell apoptosis and cell cycle was detected by flow cytometry. The real-time polymerase chain reaction (RT-PCR) was used to measure the mRNA expression of Wnt pathway key genes including Fzd1, Dvl2 and β-catenin and downstream genes including Bax, Bcl2, Survivin, Cyclin D1 and C-myc.

RESULTCompared with the control, PC12 cells viability in 50.00 and 100.00 µmol/L PQ treatment groups were obviously decreased, the cell cycle S phase arrest, and cell apoptosis increased (P<0.05). The 25.00, 50.00 and 100.00 µmol/L PQ treatment groups mRNA expression of Wnt pathway key genes including Fzd1, Dvl2 and β-catenin and downstream genes including apoptosis suppressor genes (Bcl-2 and survivin)and cyclin gene (Cyclin D1) were downregulated (P<0.05). The mRNA expression of pro-apoptosis gene (Bax) and cyclin gene (C-myc) were upregulated (P<0.05).

CONCLUSIONIt suggested that PQ can activate Wnt pathway to regulate downsteam genes expression, resulting in PC12 cell cycle arrest and apoptosis.

Adaptor Proteins, Signal Transducing ; metabolism ; Animals ; Apoptosis ; Apoptosis Regulatory Proteins ; metabolism ; Cell Cycle ; Cell Survival ; Dishevelled Proteins ; Down-Regulation ; Flow Cytometry ; Frizzled Receptors ; metabolism ; Gene Expression ; drug effects ; PC12 Cells ; Paraquat ; toxicity ; Phosphoproteins ; metabolism ; Rats ; Receptors, Neurotransmitter ; metabolism ; Wnt Signaling Pathway

BACKGROUND/AIMS: This study aimed to better understand gene expression profiles of human hepatic stellate cell (HSC) activation and the relationship with the Wnt signaling pathway. METHODS: The global transcript levels in platelet derived growth factor-BB (PDGF-BB)-stimulated hTERT HSCs were analyzed using oligonucleotide microarrays. Oligonucleotide microarrays with 19K human oligo chips were performed to obtain gene expression profiles associated with proliferation in human hTERT HSCs. The microarray data was verified by real time quantitative PCR and expression of the components of Wnt signaling was analyzed by Western blot. RESULTS: Microarray data showed 243 up-regulated and 265 down-regulated genes in PDGF-BB-treated HSCs. The changes in expression of glypican3 and BH3 interacting domain death agonist (BID) mRNA in real time quantitative PCR, especially among the highly up- or down-regulated genes, were statistically consistent with the microarray data. The Wnt signaling pathway components, frizzled10 (FZD10) and calcium/calmodulin-dependent protein kinase II alpha (CAMK2A), showed increased expression in the short time course microarray and the up-regulation of FZD10 also occurred at the protein level. Our data showed various gene expression profiles during activation of human HSC. CONCLUSIONS: The up-regulated expression of FZD10 and CAMK2A suggests that the Wnt/Ca2+ signaling pathway is active in hTERT HSCs and may participate in HSC activation and proliferation
Angiogenesis Inducing Agents/*pharmacology ; Blotting, Western ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics/metabolism ; Cell Line ; Cell Proliferation ; Frizzled Receptors/genetics/metabolism ; Gene Expression Profiling ; Hepatic Stellate Cells/cytology/*metabolism ; Humans ; Oligonucleotide Array Sequence Analysis ; Platelet-Derived Growth Factor/*pharmacology ; Polymerase Chain Reaction ; Receptors, G-Protein-Coupled/genetics/metabolism ; *Signal Transduction ; Up-Regulation ; Wnt Proteins/genetics/*metabolism

Wnt signaling is known to be important for diverse embryonic and post-natal cellular events and be regulated by the proteins Dishevelled and Axin. Although Dishevelled is activated by Wnt and involved in signal transduction, it is not clear how Dishevelled-mediated signaling is turned off. We report that guanine nucleotide binding protein beta 2 (Gnb2; Gbeta2) bound to Axin and Gbeta2 inhibited Wnt mediated reporter activity. The inhibition involved reduction of the level of Dishevelled, and the Gbeta2gamma2 mediated reduction of Dishevelled was countered by increased expression of Axin. Consistent with these effects in HEK293T cells, injection of Gbeta2gamma2 into Xenopus embryos inhibited the formation of secondary axes induced either by XWnt8 or Dishevelled, but not by beta-catenin. The DEP domain of Dishevelled is necessary for both interaction with Gbeta2gamma2 and subsequent degradation of Dishevelled via the lysosomal pathway. Signaling induced by Gbeta2gamma2 is required because a mutant of Gbeta2, Gbeta2 (W332A) with lower signaling activity, had reduced ability to downregulate the level of Dishevelled. Activation of Wnt signaling by either of two methods, increased Frizzled signaling or transient transfection of Wnt, also led to increased degradation of Dishevelled and the induced Dishevelled loss is dependent on Gbeta1 and Gbeta2. Other studies with agents that interfere with PLC action and calcium signaling suggested that loss of Dishevelled is mediated through the following pathway: Wnt/Frizzled-->Gbetagamma-->PLC-->Ca+2/PKC signaling. Together the evidence suggests a novel negative feedback mechanism in which Gbeta2gamma2 inhibits Wnt signaling by degradation of Dishevelled.
Adaptor Proteins, Signal Transducing/genetics/*metabolism ; Animals ; Blastomeres/cytology/*metabolism ; Cell Line ; Embryonic Development/genetics ; *Feedback, Physiological ; Frizzled Receptors/genetics/metabolism ; GTP-Binding Proteins/genetics/*metabolism ; Gene Expression Regulation, Developmental ; Humans ; Mutation ; Phosphoproteins/genetics/*metabolism ; Protein Binding ; RNA, Small Interfering/genetics ; Repressor Proteins/genetics/metabolism ; Transfection ; Wnt Proteins/*genetics/metabolism ; Xenopus ; Xenopus Proteins/*genetics/metabolism

OBJECTIVETo investigate the action mechanisms of the FZD5 gene in prostate cancer bone metastasis and search for some new treatments for this disease.

METHODSWe determined the expression level of the FZD5 gene in prostate cancer PC3 cells and, after transfection of siRNA into the PC3 cells and silence of the FZD5 gene, observed the changes in the migration and proliferation of the cells. We established the model of prostate cancer bone metastasis by tibial injection of prostate cancer cells in the nude mice. Then we injected control siRNA and FZD5-silenced siRNA into the tibia of the mice followed by evaluation of tumor-induced bone destruction by X-ray imaging at 0, 1, and 3 weeks and by HE staining at 3 weeks after injection.

RESULTSAfter transfection of FZD5-silenced siRNA into the prostate cancer PC3 cells, the expression of the FZD5 gene was decreased about 70%. The rate of cell proliferation was significantly lower in the gene silencing group than in the control (P < 0.05), and that of cell migration dropped by 30% in the former as compared with the latter group at 48 hours after FZD5 silencing (P < 0.05). At 3 weeks after injection of control siRNA or FZD5-silenced siRNA into the tibia of the mice, osteolytic damage was observed in both groups, though less in the FZD5 silencing group, with only a few remaining bone trabeculae visible.

CONCLUSIONSilencing the FZD5 gene can reduce the migration and proliferation of prostate cancer cells, help to suppress bone metastasis and destruction, and thereby improve the survival rate and quality of life of the patients.

Animals ; Bone Neoplasms ; genetics ; prevention & control ; secondary ; Cell Line, Tumor ; Cell Movement ; genetics ; Cell Proliferation ; genetics ; Frizzled Receptors ; genetics ; physiology ; Gene Expression ; Gene Silencing ; Male ; Mice ; Mice, Nude ; Osteolysis ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; Quality of Life ; RNA, Small Interfering ; administration & dosage ; genetics ; Transfection