BACKGROUND: 5-Fluorouracil (5FU) is a primary chemotherapeutic agent used to treat oral squamous cell carcinoma (OSCC). However, the development of drug resistance has significantly limited its clinical application. Therefore, there is an urgent need to determine the mechanisms underlying drug resistance and identify effective targets. In recent years, the Wingless and Int-1 (WNT) signaling pathway has been increasingly studied in cancer drug resistance; however, the role of WNT3, a ligand of the canonical WNT signaling pathway, in OSCC 5FU-resistance is not clear. This study delved into this potential connection.
METHODS: 5FU-resistant cell lines were established by gradually elevating the drug concentration in the culture medium. Differential gene expressions between parental and resistant cells underwent RNA sequencing analysis, which was then substantiated via Real-time quantitative PCR (RT-qPCR) and western blot tests. The influence of the WNT signaling on OSCC chemoresistance was ascertained through WNT3 knockdown or overexpression. The WNT inhibitor methyl 3-benzoate (MSAB) was probed for its capacity to boost 5FU efficacy.
RESULTS: In this study, the WNT/β-catenin signaling pathway was notably activated in 5FU-resistant OSCC cell lines, which was confirmed through transcriptome sequencing analysis, RT-qPCR, and western blot verification. Additionally, the key ligand responsible for pathway activation, WNT3, was identified. By knocking down WNT3 in resistant cells or overexpressing WNT3 in parental cells, we found that WNT3 promoted 5FU-resistance in OSCC. In addition, the WNT inhibitor MSAB reversed 5FU-resistance in OSCC cells.
CONCLUSIONS: These data underscored the activation of the WNT/β-catenin signaling pathway in resistant cells and identified the promoting effect of WNT3 upregulation on 5FU-resistance in oral squamous carcinoma. This may provide a new therapeutic strategy for reversing 5FU-resistance in OSCC cells.

OBJECTIVE: Preeclampsia (PE) is a vascular remodeling disorder cloesly linked to trophoblast dysfunction, involving defects in their proliferation, migration, and apoptosis. Maternal exosomal microRNAs (miRNAs) have been reported to play pivotal roles in the development of PE. However, the mechanism underlying the role of maternal exosomes in trophoblast dysfunction regarding the development of PE is poorly understood.
METHODS: Plasma exosomes from maternal peripheral blood were collected from pregnant women with PE and from those with normal pregnancy. Bioinformatics analysis was used to identify significantly differentially expressed miRNAs under these two conditions. The expression of the miR-3198 gene in plasma exosomes was detected using quantitative real-time polymerase chain reaction. Dual luciferase reporter assay was used to confirm binding of miR-3198 and 3\'UTR region of WNT3. Cell proliferation was examined using the Cell Count Kit-8 and EdU assays, and flow cytometry was performed to detect apoptosis and cell cycle. Changes in cell migration were examined using transwell and scratch assays.
RESULTS: Patients with PE showed decreased expression of plasma-derived exosomal miR-3198. The proliferation and migration abilities of HTR-8/SVneo and primary human trophoblast cells were both improved when cocultured with miR-3198-rich exosomes. Exposure to miR-3198-enriched exosomes facilitated cell cycle progression but reduced apoptosis in HTR-8/SVneo cells. Notably, overexpression of miR-3198 partially prevented the inhibitory effects of WNT3 on proliferation and migration in HTR-8/SVneo cells.
CONCLUSIONS: Exosomal miR-3198 in the maternal peripheral blood may regulate the biological functions of trophoblasts by targeting WNT3 and influence the development of diseases of placental origin.

BACKGROUND: Zinc finger protein X-linked (ZFX) has been shown to promote the growth of tumor cells, including leukemic cells. However, the role of ZFX in the growth and drug response of chronic myeloid leukemia (CML) stem/progenitor cells remains unclear.
METHODS: Real-time quantitative PCR (RT-qPCR) and immunofluorescence were used to analyze the expression of ZFX and WNT3 in CML CD34+ cells compared with normal control cells. Short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats/dead CRISPR-associated protein 9 (CRISPR/dCas9) technologies were used to study the role of ZFX in growth and drug response of CML cells. Microarray data were generated to compare ZFX-silenced CML CD34+ cells with their controls. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were performed to study the molecular mechanisms of ZFX to regulate WNT3 expression. RT-qPCR and western blotting were used to study the effect of ZFX on β-catenin signaling.
RESULTS: We showed that ZFX expression was significantly higher in CML CD34+ cells than in control cells. Overexpression and gene silencing experiments indicated that ZFX promoted the in vitro growth of CML cells, conferred imatinib mesylate (IM) resistance to these cells, and enhanced BCR/ABL-induced malignant transformation. Microarray data and subsequent validation revealed that WNT3 transcription was conservatively regulated by ZFX. WNT3 was highly expressed in CML CD34+ cells, and WNT3 regulated the growth and IM response of these cells similarly to ZFX. Moreover, WNT3 overexpression partially rescued ZFX silencing-induced growth inhibition and IM hypersensitivity. ZFX silencing decreased WNT3/β-catenin signaling, including c-MYC and CCND1 expression.
CONCLUSIONS: The present study identified a novel ZFX/WNT3 axis that modulates the growth and IM response of CML stem/progenitor cells.

BACKGROUND: This study evaluated if genetic variations in the WNT family members and RUNX2 are associated with craniofacial maturation, investigating dental and skeletal maturity in children and teenagers.
METHODS: Radiographs from pre-orthodontic treatment of Brazilian patients (7 to 17 years-old) were used to assess dental (panoramic radiographs) and skeletal maturity (cephalometric radiographs). The chronological age (CA) was calculated based on the date of birth and the time the radiographs were performed. For the dental maturity analysis, the Demirjian (1973) method was used and a delta [dental age - chronological age (DA-CA)] was calculated. For the skeletal maturity analysis, the Baccetti et al. (2005) method was used and the patients were classified as \"delayed skeletal maturation\", \"advanced skeletal maturation\" or \"normal skeletal maturation\". DNA isolated from buccal cells was used for genotyping of two genetic variations in WNT family genes: rs708111 (G > A) in WNT3A and rs1533767 (G > A) in WNT11; and two genetic variations in RUNX2: rs1200425 (G > A) and rs59983488 (G > T). A statistical analysis was performed and values of p < 0.05 indicated a significant difference.
RESULTS: There were no associations between dental maturity and genotypes (p > 0.05). In the skeletal maturity analysis, the allele A in the rs708111 (WNT3A) was statistically more frequent in patients with delayed skeletal maturation (Prevalence Ratio = 1.6; 95% Confidence Interval = 1.00 to 2.54; p-value = 0.042).
CONCLUSIONS: The rs708111 in the WNT3A gene impacts on skeletal maturation.

BACKGROUND: Achyranthes bidentata Blume (A. bidentata) is a common Chinese herb used to treat osteoarthritis (OA). Achyranthoside D (Ach-D) is a glucuronide saponin isolated from A. bidentata.
OBJECTIVE: To assess the mechanisms of action of Ach-D and its effects on OA.
METHODS: The effects of Ach-D were evaluated in rats underwent anterior cruciate ligament transection (ACLT) with medial meniscectomy (MMx) and in interleukin (IL)-1β-induced chondrocytes. Histological changes in rat cartilage tissues were detected using Safranin O-Fast green and haematoxylin-eosin staining. Immunohistochemical staining, qRT-PCR, ELISA, immunoblotting, and immunofluorescence were conducted to examine cartilage degeneration-related and inflammation-related factor expression. CCK-8, LDH assay, and EdU staining were performed to detect chondrocyte death.
RESULTS: Ach-D dose-dependently reduced the Osteoarthritis Research Society International (OARSI) scores, alleviated cartilage injury, and decreased the serum concentrations of CTX-II and COMP in ACLT-MMx models. Ach-D increased the expression levels of collagen II and aggrecan and decreased the levels of cartilage degeneration-related proteins, ADAMTS-5, MMP13, and MMP3, in rat cartilage tissues. Additionally, nod-like receptor protein 3 (NLRP3)-related inflammation was reduced by Ach-D, as shown by the significantly inhibited expression levels of NLRP3, ASC, GSDMD, IL-6, TNF-α, IL-1β, and IL-18 in rat cartilage tissues. In primary rat chondrocytes, Ach-D protected against IL-1β-induced viability loss and LDH release. Wnt3a is the target protein of Ach-D. Mechanistically, Ach-D alleviated OA by inhibiting Wnt signalling.
CONCLUSIONS: ACH-D may reduce inflammation and cartilage degeneration by inhibiting the Wnt signalling pathway, thereby reducing OA.

We previously found that (pro)renin receptor ((P)RR) augments Wnt3 protein without affecting Wnt3 gene transcription in colorectal cancer (CRC) cells, thus contributes to CRC initiation. The present study aims to investigate whether (P)RR further promotes CRC progression following oncogenesis and the related mechanisms. Notably, we deeply elaborate how (P)RR affects Wnt3 protein level and the key enzyme that mediates this process.
Immunohistochemistry, western blotting and immunofluorescence were performed to detect protein expression status. A kind of gastrointestinal epithelium-specific ATP6AP2 ((P)RR encoding gene) knock-in mice were generated using Crispr/Cas9 system.
We found that increased (P)RR expression in primary CRC lesions is positively associated with higher Wnt3 protein level and disease progression. Progressive CRC presents less colocalization of Wnt3 and an E3 ubiquitin ligase NEDD4L in primary lesions than non-progressive CRC. In colon cancer cells, (P)RR dramatically inhibits the NEDD4L-mediated Wnt3 protein ubiquitination. ATP6AP2 knock-in mice show more diminished Wnt3-NEDD4L colocalization in their gut epithelium in comparison to wildtype mice. They also have abnormal gut bacterial flora distribution. Especially, Lachnospiraceae_NK4A136 and Bacteroides genus, which are generally protective against CRC, are suppressed in guts of ATP6AP2 knock-in mice.
Collectively, (P)RR promotes CRC progression through inhibiting the NEDD4L-mediated Wnt3 ubiquitination and modulating gut microbiota. Video Abstract.

Hepatoblastoma is the most common type of hepatic tumors occurring in children between 0 and 5 years. And the exact pathophysiology of the disease is still mysterious. Accumulating studies on LncRNA have shown its pivotal role in the development and progression of distinct human cancers. However, the role of LINC01023 in hepatoblastoma is unknown. The relative expression of LINC01023, miR-378a-5p, and Wnt3 on hepatoblastoma tissue and cell lines was determined by quantitative polymerase chain reaction (qRT-PCR). The effect of LINC01023 downregulation and upregulation on cell proliferation, colony formation and apoptosis activities in HUH6 and HepG2 Cells was assessed by CKK8, clonogenic and flow cytometry analysis, respectively. Dual luciferase, RNA immunoprecipitation (RIP), and RNA pull-down were performed to confirm the interaction between LINC01023 and miR-378a-5p. Similarly, Dual luciferase assay was performed to confirmed the interaction between Wnt3 and miR-378a-5p. The xenograft tumorgenicity test was performed to elucidate the tumorgenicity potential of LINC01023. LINC01023 was significantly upregulated in hepatoblastoma tissue and cell lines rather than in adjacent normal hepatic tissue and QSG7701 cell lines. LINC01023 silencing attenuated cell proliferation, colony formation and increased cell apoptosis. Conversely, LINC01023 upregulation results in significant increase in cell proliferation, and colony formation activities however, a significant reduction in apoptosis activity was reported. Interaction between the LINC01023 and WNT3 was confirmed by dual luciferase assay. Xenograft animal tumorgenicity test confirmed the in-vivo tumorigenesis potential of LINC01203. To the best of our knowledge, this study is the first study demonstrating the role of LINC01023 in hepatoblastoma tumorigenesis through the LINC01023/miR-378a-5p/Wnt3 axis. It could be a potential therapeutic target and a prognostic biomarker in hepatoblastoma.

Menopausal women often face long-term estrogen treatment. G protein-coupled estrogen receptor (GPER) expressed in intestinal crypt was activated by estrogen therapy, but it was unclear whether chronic GPER activation during menopause had an effect on intestinal stem cells (ISCs). We tested the effect of chronic GPER activation on ISCs of ovariectomized (OVX) mice by injection of the selective GPER agonist G-1 for 28 days, or G-1 stimulation of organoids derived from crypts of OVX mice. G-1 up-regulated crypt depth, the number of Ki67+, bromodeoxyuridine+ cells and Olfm4+ ISCs, and the expression of ISCs marker genes (Lgr5, Olfm4 and Axin2). G-1 administration promoted organoid growth, increased the number of EdU+ cells per organoid and protein expression of Cyclin D1 and cyclin B1 in organoids. After G-1 treatment in vivo or in vitro, Paneth cell-derived Wnt3, Wnt3 effector β-catenin and Wnt target genes c-Myc and Cyclin D1 increased in ileum or organoids. Once blocking the secretion of Wnt3 from Paneth cells, the effects of G-1 on organoids growth, ISCs marker genes and Wnt/β-catenin signaling were abolished. G-1 did not affect the number of Paneth cells in ex vivo organoids, while activated Mmp7/cryptdin program in Paneth cells, promoted their maturation, and increased the expression of lysozyme protein. G-1 pretreatment in OVX mice inhibited radiation-induced ISCs proliferation injury and enhanced the resistance of mice to intestinal injury. In conclusion, chronic GPER activation prompted the Wnt3 synthesis in Paneth cells, thus increased the proliferation of ISCs via activation of Wnt3/β-catenin signaling in OVX mice.

The therapeutic application of neural stem cells (NSCs) in the central nerve system (CNS) injury is a promising strategy for combating irreversible neuronal loss. However, a variety of obvious inflammatory responses following nerve injury rapidly create an unfavorable microenvironment for survival and neuronal differentiation of NSCs in lesion area, limiting the efficacy of NSC-based therapy for CNS injury. It remained unknown how to effectively increase the neuronal differentiation efficiency of NSCs through transplantation. Here, we demonstrated that curcumin (CCM)-activated olfactory ensheathing cells (aOECs) effectively promoted neuronal differentiation of NSCs in the activated microglial inflammatory condition, and co-transplantation of aOECs and NSCs improved neurological recovery of rats after spinal cord injury (SCI), as evidenced by higher expression levels of neuronal markers and lower expression levels of glial markers in the differentiated cells, greater number of Tuj-1-positive cells as well as higher Basso, Beattie, and Bresnahan (BBB) locomotor scale, compared to the corresponding controls. Pathologically, hematoxylin and eosin (HE) staining and immunostaining also showed that aOECs remarkably enhanced the in vivo neuronal differentiation of NSCs and migration, and nerve repair. Further analysis revealed that the underlying mechanisms of aOECs potentiating the neuronal conversion of NSCs under inflammatory environment were tightly associated with up-regulation of anti-inflammatory cytokines and neurotrophic factors in OECs, and importantly, the activation of Wnt3/β-catenin pathway was likely involved in the mechanisms underlying the observed cellular events. Therefore, this study provides a promising strategy for SCI repair by co-transplantation of aOECs and NSCs.

Diabetic neuropathy is a chronic condition that affects a significant number of individuals with diabetes. Streptozotocin injection intraperitoneally to rodents produces pancreatic islet β-cell destruction causing hyperglycemia, which affect the brain leading to memory and cognition impairment. Dapagliflozin may be able to reverse beta-cell injury and alleviate this impairment. This effect may be via neuroprotective effect or possible involvement of the antioxidant, and anti-apoptotic properties. Forty rats were divided into four groups as follows: The normal control group, STZ-induced diabetes group, STZ-induced diabetic rats followed by treatment with oral dapagliflozin group and normal rats treated with oral dapagliflozin. Behavioral tests (Object location memory task and Morris water maze) were performed. Serum biomarkers (blood glucose and insulin) were measured and then the homeostatic model assessment for insulin resistance (HOMA-IR) was calculated. In the hippocampus the followings were determined; calmodulin, ca-calmodulin kinase Ⅳ (CaMKIV), protein kinase A (PKA) and cAMP-responsive element-binding protein to determine the transcription factor CREB and its signaling pathway also Wnt signaling pathway and related parameters (WnT, B-catenin, lymphoid enhancer binding factor LEF, glycogen synthase kinase 3β). Moreover, nuclear receptor-related protein-1, acetylcholine and its hydrolyzing enzyme acetylcholine esterase, oxidative stress parameter malondialdehyde (MDA) and apoptotic parameter caspase-3 were determined. STZ was able to cause destruction to pancreatic β-cells which was reflected on glucose levels causing diabetes. Diabetic neuropathy was clear in the rats performing the behavioral tests. Memory and cognition parameters in the hippocampus were negatively affected. Oxidative stress and apoptotic parameter were elevated while the electrical activity was declined. Dapagliflozin was able to reverse the previously mentioned parameters and behavior. Thus, to say dapagliflozin significantly showed neuroprotective action along with antioxidant, and anti-apoptotic properties.