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.

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.

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.

Preeclampsia is a hypertensive disorder of pregnancy. Many studies have shown that epigenetic mechanisms may play a role in preeclampsia. Moreover, our previous study indicated that the differentially methylated genes in preeclampsia were enriched in the Wnt/β-catenin signaling pathway. This study aimed to identify differentially methylated Wnt/β-catenin signaling pathway genes in the preeclamptic placenta and to study the roles of these genes in trophoblast cells in vitro. Using an Illumina Infinium HumanMethylation 850 K BeadChip, we found that the Wnt signaling pathway was globally hypermethylated in the preeclamptic group compared with the term birth group, but hypomethylated in the preeclamptic group compared with the preterm birth group. Among all Wnt/β-catenin signaling pathway factors, WNT3 was the most significantly differentially expressed gene and was hypomethylated in the preeclamptic group compared to the nonhypertensive groups, namely, the preterm birth group and term birth group. This result was confirmed by pyrosequencing. Through quantitative real-time PCR and western blot analysis, the WNT3 gene was found to be highly expressed in preeclamptic placental tissues, in contrast to other WNT factors, which were previously reported to be expressed at low levels in placental tissues. Additionally, in the HTR8/SVneo cell line, knockdown of WNT3 suppressed the Wnt/β-catenin signaling pathway, consistent with the findings for other WNT factors. These results prompted us to speculate that the WNT3 gene counteracts the low activation state of the Wnt signaling pathway in the preeclamptic placenta through methylation modification.

The aim of the study is to examine the mechanism of Aralia armata (Wall.) Seem (AAS) in improving intimal hyperplasia after vascular injury in rats. Rats with femoral artery injury were randomly divided into three groups: the model group, AAS low-dose group (40 mg/kg), and AAS high-dose group (80 mg/kg). The sham operation group was used as a control group. HE staining was used to observe the changes in femoral artery vessels. Immunohistochemistry was adopted to detect α-SMA, PCNA, GSK-3β, and β-catenin proteins in femoral artery tissue. The CCK-8 test and wound healing assay were employed to analyze the effect of AAS on proliferation and migration of vascular smooth muscle cells (VSMCs) cultured in vitro. Western blotting (WB) and polymerase chain reaction (PCR) assays were used to evaluate the molecular mechanism. AAS reduced the stenosis of blood vessels and the protein expressions of α-SMA, PCNA, GSK-3β, and β-catenin compared to the model group. In addition, AAS (0-15 μg/mL) effectively inhibited the proliferation and migration of VSMCs. Moreover, the results of WB and PCR showed that AAS could inhibit the activation of β-catenin induced by 15% FBS and significantly decrease the expression levels of Wnt3α, Dvl-1, GSK-3β, β-catenin, and cyclin D1 in the upstream and downstream of the pathway. AAS could effectively inhibit the proliferation and migration of neointima after vascular injury in rats by regulating the Wnt/β-catenin signaling pathway.

OBJECTIVE: To compare the expression of the Wnt signaling-associated proteins (Wnt3, β-catenin, MMP-7) in gastric cancer and precancerous lesions with positive and negative Helicobacter pylori (H.pylori, Hp) infection, and to further explore the mechanisms underlying the Wnt signaling pathway involving in the formation of gastric cancer and its relationship with Hp infection.
METHODS: The complete paraffin samples with pathologically confirmed diagnosis, who came from the First Hospital of Changsha from January 2018 to April 2020, were collected. All samples were randomly divided into a gastric cancer group (n=57), a precancerous lesion group (n=84), and a chronic superficial gastritis group (n=25). Improved Giemsa staining was used to detect Hp infection, and according the results of Hp infection the above groups were divided into a Hp positive subgroup and a negative subgroup. The expressions of Wnt3, β-catenin and MMP-7 were examined with immunohistochemistry.
RESULTS: The Wnt3, β-catenin, and MMP-7 were highly expressed in the gastric cancer group and the gastric precancerous lesion group. The Wnt3 and MMP-7 were highly expressed in cytoplasm, and β-catenin showed a tendency of cell membrane transferring to cytoplasm and nucleus, which was characterized by \"nuclear translocation\". The positive rates of the Wnt3, β-catenin, and MMP-7 expressions in the gastric cancer group were higher than those in the precancerous lesion group and the chronic superficial gastritis group (all P<0.05), which showed a gradually increasing trend with the deterioration of differentiation degree. In addition, the expressions of Wnt3, β-catenin, and MMP-7 in the Hp positive subgroup in the gastric cancer group and the precancerous lesion group were higher than those in the Hp negative subgroup (all P<0.05).
CONCLUSIONS: Aberrant activation of Wnt signaling pathway is involved in the occurrence and development of precancerous lesions and gastric cancer, and which is related with Hp infection. Meanwhile, the Wnt3, β-catenin and MMP-7 may be used as molecular markers for early diagnosis of gastric cancer and indicators to judge the degree of differentiation and malignancy of gastric cancer.
目的: 对比幽门螺杆菌(Helicobacter pylori，Hp)阳性及阴性胃癌和癌前病变组织中Wnt信号相关蛋白[Wnt3、β-连环蛋白(β-catenin)、基质金属蛋白酶-7(matrix metalloproteinase-7，MMP-7)]的表达，探讨Wnt信号通路参与胃癌形成的机制及其与Hp之间的关系。方法: 收集长沙市第一医院病理科2018年1月至2020年4月保存完整的已经病理确诊的石蜡标本，分为胃癌组(n=57)、癌前病变组(n=84)和慢性浅表性胃炎组(n=25)，各组标本均用改良Giemsa染色检测Hp感染后再分成Hp阳性亚组和Hp阴性亚组，采用免疫组织化学方法检测各组织标本Wnt3、β-catenin、MMP-7的表达。结果: Wnt3、β-catenin、MMP-7在胃癌组和癌前病变组均呈高表达，Wnt3、MMP-7高表达于细胞质，β-catenin呈细胞膜向细胞质及细胞核转移的趋势，表现为“核易位”特点。胃癌组Wnt3、β-catenin、MMP-7表达的阳性率高于癌前病变组和慢性浅表性胃炎组，且随分化程度的恶化，呈逐步升高趋势，差异均有统计学意义(均 P<0.05)；同时，Wnt3、β-catenin、MMP-7在胃癌组和癌前病变组的Hp阳性亚组中的表达均明显高于Hp阴性亚组，差异均有统计学意义(均P<0.05)。结论: Wnt信号通路的异常激活参与了胃癌前病变以及胃癌的发生、发展，并与Hp感染密切相关。同时，Wnt3、β-catenin和MMP-7有可能成为胃癌早期诊断的分子标志物及判断胃癌恶性程度的指标。.

Stem cells (SCs) play a key role in homeostasis and repair. While many studies have focused on SC self-renewal and differentiation, little is known regarding the molecular mechanism regulating SC elimination and compensation upon loss. Here, we report that Caspase-9 deletion in hair follicle SCs (HFSCs) attenuates the apoptotic cascade, resulting in significant temporal delays. Surprisingly, Casp9-deficient HFSCs accumulate high levels of cleaved caspase-3 and are improperly cleared due to an essential caspase-3/caspase-9 feedforward loop. These SCs are retained in an apoptotic-engaged state, serving as mitogenic signaling centers by continuously releasing Wnt3 and instructing proliferation. Investigating the underlying mechanism, we reveal a caspase-3/Dusp8/p38 module responsible for Wnt3 induction, which operates in both normal and Casp9-deleted HFSCs. Notably, Casp9-deleted mice display accelerated wound repair and de novo hair follicle regeneration. Taken together, we demonstrate that apoptotic cells represent a dynamic SC niche, from which emanating signals drive SC proliferation and tissue regeneration.