Cinobufotalin prevents bone loss induced by ovariectomy in mice through the BMPs/SMAD and Wnt/β-catenin signaling pathways

Background:Osteoporosis is a chronic bone disease characterized by bone loss and decreased bone strength.However,current anti-resorptive drugs carry a risk of various complications.The deep learning-based efficacy prediction system(DLEPS)is a forecasting tool that can effectively compete in drug screening and prediction based on gene expression changes.This study aimed to explore the protective effect and potential mechanisms of cinobufotalin(CB),a traditional Chinese medicine(TCM),on bone loss.Methods:DLEPS was employed for screening anti-osteoporotic agents according to gene profile changes in primary osteoporosis.Micro-CT,histological and morphological analysis were applied for the bone protective detection of CB,and the osteogenic differentiation/function in human bone marrow mesenchymal stem cells(hBMMSCs)were also investigated.The underlying mechanism was verified using qRT-PCR,Western blot(WB),immunofluorescence(IF),etc.Results:A safe concentration(0.25mg/kg in vivo,0.05μM in vitro)of CB could effectively preserve bone mass in estrogen deficiency-induced bone loss and promote osteogenic differentiation/function of hBMMSCs.Both BMPs/SMAD and Wnt/β-catenin signaling pathways participated in CB-induced osteogenic differentiation,further regulating the expression of osteogenesis-associated factors,and ultimately promoting osteogenesis.Conclusion:Our study demonstrated that CB could significantly reverse estrogen deficiency-induced bone loss,further promoting osteogenic differentiation/function of hBMMSCs,with BMPs/SMAD and Wnt/β-catenin signaling pathways involved.

Pachymic acid exerts antitumor activities by modulating the Wnt/β-catenin signaling pathway via targeting PTP1B

Objective:To determine the inhibitory effects of pachymic acid on lung adenocarcinoma(LUAD)cells and elucidate its underlying mechanism.Methods:CCK-8,wound healing,Transwell,Western blot,tube formation,and immunofluorescence assays were carried out to measure the effects of various concentrations of pachymic acid on LUAD cell proliferation,metastasis,angiogenesis as well as autophagy.Subsequently,molecular docking technology was used to detect the potential targeted binding association between pachymic acid and protein tyrosine phosphatase 1B(PTP1B).Moreover,PTP1B was overexpressed in A549 cells to detect the specific mechanisms of pachymic acid.Results:Pachymic acid suppressed LUAD cell viability,metastasis as well as angiogenesis while inducing cell autophagy.It also targeted PTP1B and lowered PTP1B expression.However,PTP1B overexpression reversed the effects of pachymic acid on metastasis,angiogenesis,and autophagy as well as the expression of Wnt3a andβ-catenin in LUAD cells.Conclusions:Pachymic acid inhibits metastasis and angiogenesis,and promotes autophagy in LUAD cells by modulating the Wnt/β-catenin signaling pathway via targeting PTP1B.

ZNF554 Inhibits Endometrial Cancer Progression via Regulating RBM5 and Inactivating WNT/β-Catenin Signaling Pathway

Objective:Uterine corpus endometrial carcinoma(UCEC),a kind of gynecologic malignancy,poses a significant risk to women’s health.The precise mechanism underlying the development of UCEC remains elusive.Zinc finger protein 554(ZNF554),a member of the Krüppel-associated box domain zinc finger protein superfamily,was reported to be dysregulated in various illnesses,including malignant tumors.This study aimed to examine the involvement of ZNF554 in the development of UCEC.Methods:The expression of ZNF554 in UCEC tissues and cell lines were examined by qRT-PCR and Western blot assay.Cells with stably overexpressed or knocked-down ZNF554 were established through lentivirus infection.CCK-8,wound healing,and Transwell invasion assays were employed to assess cell proliferation,migration,and invasion.Propidium iodide(PI)staining combined with fluorescence-activated cell sorting(FACS)flow cytometer was utilized to detect cell cycle distribution.qRT-PCR and Western blotting were conducted to examine relative mRNA and protein levels.Chromatin immunoprecipitation assay and luciferase reporter assay were used to explore the regulatory role of ZNF554 in RNA binding motif 5(RBM5).Results:The expression of ZNF554 was found to be reduced in both UCEC samples and cell lines.Decreased expression of ZNF554 was associated with higher tumor stage,decreased overall survival,and reduced disease-free survival in UCEC.ZNF554 overexpression suppressed cell proliferation,migration,and invasion,while also inducing cell cycle arrest.In contrast,a decrease in ZNF554 expression resulted in the opposite effect.Mechanistically,ZNF554 transcriptionally regulated RBM5,leading to the deactivation of the Wingless(WNT)/β-catenin signaling pathway.Moreover,the findings from rescue studies demonstrated that the inhibition of RBM5 negated the impact of ZNF554 overexpression onβ-catenin and p-glycogen synthase kinase-3β(p-GSK-3β).Similarly,the deliberate activation of RBM5 reduced the increase inβ-catenin and p-GSK-3βcaused by the suppression of ZNF554.In vitro experiments showed that ZNF554 overexpression-induced decreases in cell proliferation and migration were counteracted by RBM5 knockdown.Additionally,when RBM5 was overexpressed,it hindered the improvements in cell proliferation and migration caused by reducing the ZNF554 levels.Conclusion:ZNF554 functions as a tumor suppressor in UCEC.Furthermore,ZNF554 regulates UCEC progression through the RBM5/WNT/β-catenin signaling pathway.ZNF554 shows a promise as both a prognostic biomarker and a therapeutic target for UCEC.

Effects of Helicobacter pylori and Moluodan on the Wnt/β-catenin signaling pathway in mice with precancerous gastric cancer lesions

BACKGROUND Helicobacter pylori(H.pylori)is the primary risk factor for gastric cancer(GC),the Wnt/β-Catenin signaling pathway is closely linked to tumourigenesis.GC has a high mortality rate and treatment cost,and there are no drugs to prevent the progression of gastric precancerous lesions to GC.Therefore,it is necessary to find a novel drug that is inexpensive and preventive to against GC.AIM To explore the effects of H.pylori and Moluodan on the Wnt/β-Catenin signaling pathway and precancerous lesions of GC(PLGC).METHODS Mice were divided into the control,N-methyl-N-nitrosourea(MNU),H.pylori+MNU,and Moluodan groups.We first created an H.pylori infection model in the H.pylori+MNU and Moluodan groups.A PLGC model was created in the remaining three groups except for the control group.Moluodan was fed to mice in the Moloudan group ad libitum.The general condition of mice were observed during the whole experiment period.Gastric tissues of mice were grossly and microscopically examined.Through quantitative real-time PCR(qRT-PCR)and Western blotting analysis,the expression of relevant genes were detected.RESULTS Mice in the H.pylori+MNU group showed the worst performance in general condition,gastric tissue visual and microscopic observation,followed by the MNU group,Moluodan group and the control group.QRT-PCR and Western blotting analysis were used to detect the expression of relevant genes,the results showed that the H.pylori+MNU group had the highest expression,followed by the MNU group,Moluodan group and the control group.CONCLUSION H.pylori can activate the Wnt/β-catenin signaling pathway,thereby facilitating the development and progression of PLGC.Moluodan suppressed the activation of the Wnt/β-catenin signaling pathway,thereby decreasing the progression of PLGC.

Effects of Qigongwan on Wnt/β-catenin Signaling Pathway in Rats with Polycystic Ovary syndrome

[Objectives] To explore the therapeutic effect and mechanism of Qigongwan on PCOS model rats by detecting the changes in sex hormone levels in rats with polycystic ovary syndrome (PCOS), and observing the effects of ovarian pathological morphological changes, apoptosis and expression of Wnt/β-β catenin signaling pathway protein. [Methods] Ten of 40 female SD rats were randomly selected as the normal group, and the other 30 rats were treated with letrozole combined with high-fat diet to establish the PCOS rat model. After successful modeling, the model group was randomly divided into Qigongwan group, positive Daying-35 (Ethinylestradiol and Cyproterone Acetate Tablets) group and model group, with 10 rats in each group. Qigongwan group was given 14.7 g/(kg·d) by gavage, Daying-35 group was given 0.21 mg/(kg·d) by oral gavage, and normal group and model group were given the same amount of distilled water, and the intervention lasted for 21 d. ELISA method was used to detect the levels of hormones such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), estradiol (E 2) and progesterone (P) in serum. HE staining was used to observe the pathological morphological changes of ovarian tissues;TUNEL staining was used to observe apoptosis of ovarian tissue granule cells;the expression of Wnt, β-catenin protein in rat ovarian tissue was detected by immunohistochemistry. [Results] (i) Compared with the model group, Qigongwan group and Daying-35 group could significantly increase serum E 2 and P levels, significantly reduce serum T levels ( P <0.01), significantly reduce serum LH levels and LH/FSH ratio ( P <0.01), and increase serum FSH levels ( P <0.05) in different degrees. (ii)The results of HE staining showed that compared with the model group, Qigongwan and Daying-35 groups could improve follicular development and reduce atretic follicles in different degrees. Compared with Daying-35 group, the number of GC layers in Qigongwan group was significantly increased. (iii) The results of TUNEL staining showed that compared with the model group, the rate of TUNEL-positive cells in the Qigongwan group and Daying-35 group decreased significantly ( P <0.01). (iv) The immunohistochemical results showed that compared with the model group, the expression levels of wnt and β-catenin in the Qigongwan group and the Daying-35 group increased in different degrees ( P <0.05), and the expression range increased. [Conclusions] Qigongwan can regulate the secretion level of sex hormones such as FSH and LH, improve the pathological damage of ovarian tissue, and inhibit apoptosis of ovarian granule cells, and its mechanism may be related to the activation of Wnt/β-catenin signaling pathway.

TCM Intervention on Wnt/β-Catenin signaling pathway in the treatment of diabetic nephropathy

Diabetic nephropathy(DN)is the most serious microvascular complication of diabetes mellitus,which is highly prevalent worldwide.Abnormal activation of Wnt/β-catenin signaling pathway is an important mechanism of renal damage induced by hyperglycemia.Many studies have shown that TCM has the advantages of high efficiency and safety in the prevention and treatment of DN.Some TCM monomers and compounds repair podocyte function and inhibit transdifferentiation process by inhibiting the activation of Wnt/β-catenin signaling pathway,thus playing a protective role in kidney.Based on this,this paper will review the existing research results and related mechanisms of TCM intervention in Wnt/β-catenin signaling pathway in the treatment of DN,in order to promote the more effective and reasonable application of TCM in clinical practice.

Inhibitory effect of caffeic acid phenethyl ester on the growth of SW480 colorectal tumor cells involvesβ-catenin associated signaling pathway down-regulation

AIM： To study the anti-tumor effect of caffeic acid phenethyl ester （CAPE） and the influence of CAPE on β-catenin associated signaling pathway in SW480 colorectal cancer （CRC） cells. METHODS： SW480 cells were treated with CAPE at serial concentrations. The proliferative status of cells was measured by methabenzthiazuron （MTT） assay. Cell cycle and cell apoptosis were analyzed using flow cytometry （FCM）. Western blotting assay was used to evaluate the protein level of β-catenin, c-myc and cyclinD1. β-catenin localization was determined by indirect immunofluorescence. RESULTS： CAPE displayed a strong inhibitory effect in a significant dose- and time-dependent manner on SW480 cell growth. FCM analysis showed that the ratio of G0/G1 phase cells increased, S phase ratio decreased and apoptosis rate increased after SW480 cells were exposed to CAPE for 24 h. Pretreatment of SW480 cells with CAPE significantly suppressed β-catenin, c-myc and cyclinD1 protein expression. CAPE treatment was associated with decreased accumulation of β-catenin protein in nucleus and cytoplasm, and concurrently increased its accumulation on the surface of cell membrane. CONCLUSION： CAPE can inhibit SW480 cell proliferation by inducing cell cycle arrest and apoptosis. Decreased β-catenin and the associated signaling pathway target gene expression may mediate the anti-tumor effects of CAPE.

Neuroprotective effect of rapamycin on spinal cord injury via activation of the Wnt/β-catenin signaling pathway

The Wnt/β-catenin signaling pathway plays a crucial role in neural development, axonal guid- ance, neuropathic pain remission and neuronal survival. In this study, we initially examined the effect of rapamycin on the Wnt/β-catenin signaling pathway after spinal cord iniury, by intraperitoneally injecting spinal cord injured rats with rapamycin over 2 days. Western blot analysis and immunofluorescence staining were used to detect the expression levels of β-catenin protein, caspase-3 protein and brain-derived neurotrophic factor protein, components of the Wnt/β-catenin signaling pathway. Rapamycin increased the levels of β-catenin and brain-derived neurotrophic factor in the injured spinal cord, improved the pathological morphology at the injury site, reduced the loss of motor neurons, and promoted motor functional recovery in rats after spinal cord injury. Our experimental fndings suggest that the neuroprotective effect of rapamycin intervention is mediated through activation of the Wnt/β-catenin signaling pathway after spinal cord injury.

Mechanism of miR-21 via Wnt/β-catenin signaling pathway in human A549 lung cancer cells and Lewis lung carcinoma in mice

Objective:To study the mechanism of effect of miR-21 via Wnt/ β-catenin signaling pathway in human A549 lung cancer cells and Lewis lung carcinoma in mice.Methods:The effect of miR-21 on A549 cells were detected by MTT method.MiR-21 expression levels were overexpressed or inhibited in A549 cells by transfecting with miR-21 mimics or inhibitors.Correlation among key molecules(Wnt1,β-catenin.CyclinD1 and miR-21) of mRNA and protein levels in Wnt/β-catenin signaling pathway were studied by Real-time PCR and Western blot hybridization assay.Invasive ability of A549 cells was determined via Transwell chamber cell invasion assay;the role of miR-21 in A549 cells was explored via the Wnt/β-catenin signaling pathway.A Lewis lung carcinoma animal model was established to detect miR-21 expressions in tumor animals and controlled animal tissues,and verify expression changes of the above moleculesin the Wnt / β-catenin signaling pathway was determined in the animal level.Results:MTT assay results showed that miR-21 overexpression could markedly enhance cell absorbance value;that is,miR-21 could increase the ability proliferation of A549 cells.β-catenin and CyclinD1 expression levels were significantly higher in miR-21 mimic transfected cells(P<0.05),and Wnt 1 gene had no significant change.Wnt 1,β-catenin and CyclinD1 gene expression showed no significant change when miR-21 expression was suppressed,compared with controls.After cells were transfected with miR-21 mimics,cell invasion assay revealed that the perforated cells was significantly higher than the perforated cells in the control group(P<0.01).Lewis lung assay revealed that miR-21 expression levels in the Lewis lung carcinoma were significantly higher;and at the same time.Wnt1,β-catenin and CyclinD1 gene expression levels were significantly increased,compared to controls.Conclusions:In A549 human lung cancer cells and Lewis lung carcinoma in mice,key molecules β-catenin and CyclinD1 of miR-21 expressions and the Wnt/ β-catenin signaling pathway are positively correlated.

MiR-19a-3p regulates the Forkhead box F2-mediated Wnt/β-catenin signaling pathway and affects the biological functions of colorectal cancer cells

BACKGROUND Colorectal cancer(CRC)is one of the most common malignancies worldwide.AIM To explore the expression of microRNA miR-19a-3p and Forkhead box F2(FOXF2)in patients with CRC and the relevant mechanisms.METHODS Sixty-two CRC patients admitted to the hospital were enrolled into the study group,and sixty healthy people from the same period were assigned to the control group.Elbow venous blood was sampled from the patients and healthy individuals,and blood serum was saved for later analysis.MiR-19a-3p mimics,miR-19a-3p inhibitor,miR-negative control,small interfering-FOXF2,and short hairpin-FOXF2 were transfected into HT29 and HCT116 cells.Then quantitative polymerase chain reaction was performed to quantify the expression of miR-19a-3p and FOXF2 in HT29 and HCT116 cells,and western blot(WB)analysis was conducted to evaluate the levels of FOXF2,glycogen synthase kinase 3 beta(GSK-3β),phosphorylated GSK-3β(p-GSK-3β),β-catenin,p-β-catenin,α-catenin,Ncadherin,E-cadherin,and vimentin.The MTT,Transwell,and wound healing assays were applied to analyze cell proliferation,invasion,and migration,respectively,and the dual luciferase reporter assay was used to determine the correlation of miR-19a-3p with FOXF2.RESULTS The patients showed high serum levels of miR-19a-3p and low levels of FOXF2,and the area under the curves of miR-19a-3p and FOXF2 were larger than 0.8.MiR-19a-3p and FOXF2 were related to sex,tumor size,age,tumor-nodemetastasis staging,lymph node metastasis,and differentiation of CRC patients.Silencing of miR-19a-3p and overexpression of FOXF2 suppressed the epithelialmesenchymal transition,invasion,migration,and proliferation of cells.WB analysis revealed that silencing of miR-19a-3p and FOXF2 overexpression significantly suppressed the expression of p-GSK-3β,β-catenin,N-cadherin,and vimentin;and increased the levels of GSK-3β,p-β-catenin,α-catenin,and Ecadherin.The dual luciferase reporter assay confirmed that there was a targeted correlation of miR-19a-3p with FOXF2.In addition,a rescue experiment revealed that there were no differences in cell proliferation,invasion,and migration in HT29 and HCT116 cells co-transfected with miR-19a-3p-mimics+sh-FOXF2 and miR-19a-3p-inhibitor+si-FOXF2 compared to the miR-negative control group.CONCLUSION Inhibiting miR-19a-3p expression can upregulate the FOXF2-mediated Wnt/β-catenin signaling pathway,thereby affecting the epithelial-mesenchymal transition,proliferation,invasion,and migration of cells.Thus,miR-19a-3p is likely to be a therapeutic target in CRC.

Expression analysis of eight amphioxus genes involved in the Wnt/β-catenin signaling pathway

The Wnt/β-catenin signaling pathway plays a crucial role in the embryonic development of metazoans. Although the pathway has been studied extensively in many model animals, its function in amphioxus, the most primitive chordate, remains largely uncharacterized. To obtain basic data for functional analysis, we identified and isolated seven genes （Lrp5/6, Dvl, APC, Ckla, CklS, Gsk3β, and Gro） of the Wnt/β-catenin signaling pathway from the amphioxus （Branchiostoma floridae） genome. Phylogenetic analysis revealed that amphioxus had fewer members of each gene family than that found in vertebrates. Whole-mount in situ hybridization showed that the genes were maternally expressed and broadly distributed throughout the whole embryo at the cleavage and blastula stages. Among them, Dvl was expressed asymmetrically towards the animal pole, while the others were evenly distributed in all blastomeres. At the mid-gastrula stage, the genes were specifically expressed in the primitive endomesoderm, but displayed different patterns. When the embryo developed into the neurula stage, the gene expressions were mainly detected in either paraxial somites or the tail bud. With the development of the embryo, the expression levels further decreased gradually and remained only in some pharyngeal regions or the tail bud at the larva stage. Our results suggest that the Wnt/β-catenin pathway might be involved in amphioxus somite formation and posterior growth, but not in endomesoderm specification.

Role of Wnt/β-catenin Signaling Pathway in the Mechanism of Calcification of Aortic Valve

Aortic valve calcification is a common disease in the elderly, but its cellular and molecular mechanisms are not clear. In order to verify the hypothesis that Wnt/β-catenin signaling pathway is involved in the process of calcification of aortic valve, porcine aortic valve interstitial cells（VICs） were isolated, cultured and stimulated with oxidized low density lipoprotein（ox-LDL） for 48 h to induce the differentiation of VICs into osteoblast-like cells. The key proteins and genes of Wnt/β-catenin signaling pathway, such as glycogen synthase kinase 3β（GSK-3β） and β-catenin, were detected by using Western blotting and real-time polymerase chain reaction（PCR）. The results showed that the VICs managed to differentiate into osteoblast-like cells after the stimulation with ox-LDL and the levels of proteins and genes of GSK-3β and β-catenin were increased significantly in VICs after stimulation for 48 h（P0.05）. It is suggested that Wnt/β-catenin signaling pathway may play a key role in the differentiation of VICs into osteoblast-like cells and make great contribution to aortic valve calcification.

Effects of Wnt/β-catenin signaling pathway on the proliferation and invasive growth of cells in mantle cell lymphoma

Objective:To study the effects of Wnt/β-catenin signaling pathway on the proliferation and invasive growth of cells in mantle cell lymphoma.Methods: Patients who were diagnosed with mantle cell lymphoma by lymph node biopsy in Tongji Hospital? Tongji Medical College Huazhong University of Science & Technology between March 2015 and May 2017 were selected as lymphoma group of the research, and patients who were diagnosed with reactive hyperplasia by lymph node biopsy during the same period were selected as the control group. The protein expression of Wnt/β-catenin signaling pathway molecules in lymph node tissues were determined by enzyme-linked immunosorbent assay kit, and the mRNA expression of proliferation genes and invasion genes were determined by fluorescence quantitative PCR kit.Results:β-catenin, p-GSK-3β and Tcf/Lef protein levels as well as C-myc, CyclinD1, Est-1, MMP9 and MMP26 mRNA expression in lymph node tissues of lymphoma group were significantly higher than those of control group whereas DKK1 and WIF-1 protein levels as well as Bax, Apaf1, Caspase-3, TNFAIP3, TIMP2 and TIMP4 mRNA expression were significantly lower than those of control group. C-myc, CyclinD1, Est-1, MMP9 and MMP26 mRNA expression in lymphoma tissues were positively correlated withβ-catenin, p-GSK-3β and Tcf/Lef protein levels, and negatively correlated with DKK1 and WIF-1 protein levels;Bax, Apaf1, Caspase-3, TNFAIP3, TIMP2 and TIMP4 mRNA expression were negatively correlated withβ-catenin, p-GSK-3β and Tcf/Lef protein levels, and positively correlated with DKK1 and WIF-1 protein levels.Conclusion: The activation of Wnt/β-catenin signaling pathway can promote the proliferation and invasive growth of cells in mantle cell lymphoma.

Mutations in components of the Wnt signaling pathway in gastric cancer

AIM: To explore the contribution of AXIN1, AXIN2 and beta-catenin, components of Wnt signaling pathway, to the carcinogenesis of gastric cancer (GC), we examined AXIN1, AXIN2 exon7 and CTNNB1 (encoding beta- catenin) exon3 mutations in 70 GCs. METHODS: The presence of mutations was identified by polymerase chain reaction (PCR)-based denaturing high-performance liquid chromatography and direct DNA sequencing. Beta-catenin expression was detected by immunohistochemical analysis. RESULTS: Among the 70 GCs, 5 (7.1%) had mutations in one or two of these three components. A frameshift mutation (1 bp deletion) in exon7 of AXIN2 was found in one case. Four cases, including the case with a mutation in AXIN2, had frameshift mutations and missense mutations in AXIN1. Five single nucleotide polymorphisms (SNPs), 334 C>T, 874 C>T, 1396 G>A, 1690 C>T and 1942 T>G, were identified in AXIN1. A frameshift mutation (27 bp deletion) spanning exon3 of CTNNB1 was observed in one case. All four cases with mutations in AXIN1 and AXIN2 showed nuclear beta- catenin expression. CONCLUSION: These data indicate that the mutationsin AXIN1 and AXIN2 may contribute to gastric carcino- genesis.

Involvement of the Wnt signaling pathway and cell apoptosis in the rat hippocampus following cerebral ischemia/reperfusion injury

We investigated the role of the Wnt signaling pathway in cerebral ischemia/reperfusion injury by examining β-catenin and glycogen synthase kinase-3β protein expression in the rat hippocampal CA1 region following acute cerebral ischemia/reperfusion. Our results demonstrate that cell apoptosis increases in the CA1 region following ischemia/reperfusion. In addition, β-catenin and glycogen synthase kinase-3β protein expression gradually increases, peaking at 48 hours following reperfusion. Dickkopf-1 administration, after cerebral ischemia/reperfusion injury, results in decreased cell apoptosis, and β-catenin and glycogen synthase kinase-3β expression, in the CA1 region. This suggests that β-catenin and glycogen synthase kinase-3β, both components of the Wnt signaling pathway, participate in cell apoptosis following cerebral ischemia/reperfusion injury.

Ubiquitin-like modifier activating enzyme 2 promotes cell migration and invasion through Wnt/β-catenin signaling in gastric cancer

AIM To investigate the function and mechanism of ubiquitinlike modifier activating enzyme 2(Uba2) in progression of gastric cancer(GC) cells.METHODS Uba2 level in patients with GC was analyzed by Western blotting and immunohistochemistry. MTT and colony formation assays were performed to examine cell proliferation.Flow cytometry was used for cell cycle analysis.Wound healing and Transwell assays were conducted to examine the effects of Uba2 on migration and invasion.Expression levels of cell cycle-related proteins, epithelial-mesenchymal transition(EMT) biomarkers, and involvement of the Wnt/β-catenin pathway was assessed by Western blotting. Activation of the Wnt/β-catenin pathway was confirmed by luciferase assay.RESULTS Uba2 expression was higher in GC than in normal tissues.Increased Uba2 expression was correlated with tissue differentiation, Lauren's classification, vascular invasion,and TNM stage, as determined by the analysis of 100 GC cases(P < 0.05). Knock-down of Uba2 inhibited GC cell proliferation, induced cell cycle arrest, and altered expression of cyclin D1, P21, P27, and Bcl-2, while upregulation of Uba2 showed the opposite effects. The wound healing and Transwell assays showed that Uba2 promoted GC cell migration and invasion. Western blotting revealed alterations in EMT biomarkers, suggesting the role of Uba2 in EMT. Furthermore, the luciferase reporter assay indicated the involvement of the Wnt/β-catenin signaling pathway as a possible modulator of Uba2 oncogenic functions.CONCLUSION Uba2 plays a vital role in GC cell migration and invasion,possibly by regulating the Wnt/β-catenin signaling pathway and EMT.

CYP24A1 Involvement in Inflammatory Factor Regulation Occurs via the Wnt Signaling Pathway

Objective While the upregulation of cytochrome P450 family 24 subfamily A member 1(CYP24A1)gene expression has been reported in colon cancer,its role in tumorigenesis remains largely unknown.In this study,we aimed to investigate the involvement of CYP24A1 in Wnt pathway regulation via the nuclear factor kappa B(NF-κB)pathway.Methods The human colon cancer cell lines HCT-116 and Caco-2 were subjected to stimulation with interleukin-6(IL-6)as well as tumor necrosis factor alpha(TNF-α),with subsequent treatment using the NF-κB pathway-specific inhibitor ammonium pyrrolidinedithiocarbamate(PDTC).Furthermore,CYP24A1 expression was subjected to knockdown via the use of small interfering RNA(siRNA).Subsequently,NF-κB pathway activation was determined by an electrophoretic mobility shift assay,and the transcriptional activity ofβ-catenin was determined by a dual-luciferase reporter assay.A mouse ulcerative colitis(UC)-associated carcinogenesis model was established,wherein TNF-αand the NF-κB pathway were blocked by anti-TNF-αmonoclonal antibody and NF-κB antisense oligonucleotides,respectively.Then the tumor size and protein level of CYP24A1 were determined.Results IL-6 and TNF-αupregulated CYP24A1 expression and activated the NF-κB pathway in colon cancer cells.PDTC significantly inhibited this increase in CYP24A1 expression.Additionally,knockdown of CYP24A1 expression by siRNA could partially antagonize Wnt pathway activation.Upregulated CYP24A1 expression was observed in the colonic epithelial cells of UC-associated carcinoma mouse models.Anti-TNF-αmonoclonal antibody and NF-κB antisense oligonucleotides decreased the tumor size and suppressed CYP24A1 expression.Conclusion Taken together,this study suggests that inflammatory factors may increase CYP24A1 expression via NF-κB pathway activation,which in turn stimulates Wnt signaling.

RING finger and WD repeat domain 3 regulates proliferation and metastasis through the Wnt/β-catenin signalling pathways in hepatocellular carcinoma

BACKGROUND Hepatocellular carcinoma(HCC)exhibits high invasiveness and mortality rates,and the molecular mechanisms of HCC have gained increasing research interest.The abnormal DNA damage response has long been recognized as one of the important factors for tumor occurrence and development.Recent studies have shown the potential of the protein RING finger and WD repeat domain 3(RFWD3)that positively regulates p53 stability in response to DNA damage as a therapeutic target in cancers.AIM To investigate the relationship between HCC and RFWD3 in vitro and in vivo and explored the underlying molecular signalling transduction pathways.METHODS RFWD3 gene expression was analyzed in HCC tissues and adjacent normal tissues.Lentivirus was used to stably knockdown RFWD3 expression in HCC cell lines.After verifying the silencing efficiency,Celigo/cell cycle/apoptosis and MTT assays were used to evaluate cell proliferation and apoptosis.Subsequently,cell migration and invasion were assessed by wound healing and transwell assays.In addition,transduced cells were implanted subcutaneously and injected into the tail vein of nude mice to observe tumor growth and metastasis.Next,we used lentiviral-mediated rescue of RFWD3 shRNA to verify the phenotype.Finally,the microarray,ingenuity pathway analysis,and western blot analysis were used to analyze the regulatory network underlying HCC.RESULTS Compared with adjacent tissues,RFWD3 expression levels were significantly higher in clinical HCC tissues and correlated with tumor size and TNM stage(P<0.05),which indicated a poor prognosis state.RFWD3 silencing in BEL-7404 and HCC-LM3 cells increased apoptosis,decreased growth,and inhibited the migration in shRNAi cells compared with those in shCtrl cells(P<0.05).Furthermore,the in vitro results were supported by the findings of the in vivo experiments with the reduction of tumor cell invasion and migration.Moreover,the rescue of RFWD3 shRNAi resulted in the resumption of invasion and metastasis in HCC cell lines.Finally,gene expression profiling and subsequent experimental verification revealed that RFWD3 might influence the proliferation and metastasis of HCC via the Wnt/β-catenin signalling pathway.CONCLUSION We provide evidence for the expression and function of RFWD3 in HCC.RFWD3 affects the prognosis,proliferation,invasion,and metastasis of HCC by regulating the Wnt/β-catenin signalling pathway.

Angelica sinensis polysaccharides ameliorate 5-flourouracil-induced bone marrow stromal cell proliferation inhibition via regulating Wnt/β-catenin signaling

Chemotherapy may cause cellular oxidative stress to bone marrow.Oxidative damage of bone marrow hematopoietic microenvironment is closely related to chronic myelosuppression after chemotherapeutic treatment.Angelica sinensis polysaccharides(ASP)are major effective ingredients of traditional Chinese medicine Angelica with multi-target anti-oxidative stress features.In the current study,we investigated the protective roles and mechanisms of ASP on chemotherapy-induced bone marrow stromal cell(BMSC)damage.The human bone marrow stromal cell line HS-5 cells were divided into control group,5-FU group,5-FU+ASP group,and 5-FU+LiCl group to investigate the mechanism of ASP to alleviate 5-FU-induced BMSC proliferation inhibition.The results showed that 5-FU inhibits the growth of HS-5 cells in a time and dose-dependent manner;however,ASP partially counteracted the 5-FU-induced decrease in cell viability,whereas Wnt signaling inhibitor Dkk1 antagonized the effect of ASP on HS-5 cells.ASP reversed the decrease in total cytoplasmicβ-catenin,p-GSK-3β,and CyclinD1 following 5-FU treatment and modulated nuclear expression ofβ-catenin,Lef-1,and C-myc proteins.Furthermore,ASP also enhanced the antioxidant capacity of cells and reduced 5-FU-induced oxidative stress,attenuated FoxO1 expression,thus weakened its downstream apoptosis-related proteins and G0/G1 checkpoint-associated p27^(Kip1) expression to alleviate 5-FU-induced apoptosis and to promote cell cycle progression.All the results above suggest that the protective role of ASP in 5-FU-treated BMSCs proliferation for the chemotherapy may be related to its activating Wnt/β-catenin signaling and keeping homeostasis betweenβ-catenin and FoxO1 under oxidative stress.The study provides a potential therapeutic strategy for alleviating chemotherapeutic damage on BMSCs.

Effect of miR-124 Overexpression under Wnt/β-catenin Signaling Pathway on Inflammatory Factor and Epidermal Growth Factor in Rats with Precancerous Gastric Lesions

Objective:To investigate the effects of miR-124 overexpression on inflammatory factors and epidermal growth factor in rats with gastric precancerous lesions by modulating the Wnt/β-catenin signaling pathway.Methods:Eighty SPF-grade Wistar rats were selected as the research subjects.After adaptive feeding,they were divided into 4 groups using a random number table method.The control group was fed a regular diet,while the model group,low expression group,and overexpression group of rats were treated with N-methyl-N’-nitro-N-nitrosoguanidine to establish a model of gastric cancer precancerous lesions.After successful modeling,the control group and model group rats were injected intraperitoneally with 0.9%sodium chloride solution,the low-expression group rats were injected intraperitoneally with miR-124-5p inhibitor,and the over-expression group rats were injected intraperitoneally with miR-124-5p mimic.After 8 weeks of continuous injection,the levels of miR-124,Wnt/β-catenin signaling pathway-related proteins,inflammatory factors,and epidermal growth factor were observed and compared among the four groups of rats.Results:The differences in the relative expression of miR-124 among the four groups of rats were statistically significant(P<0.05);the differences in the expression of Wnt protein andβ-catenin protein among the four groups of rats were statistically significant(P<0.05),and the expression of Wnt protein andβ-catenin protein in the overexpression group was significantly higher than that of the control group,and significantly lower than that in the model group and the low-expression group;the levels of IL-1β,IL-6,and TNF-αof rats in the four groups of rats IL-1β,IL-6 and TNF-αlevels were statistically different(P<0.05),and the levels of IL-1β,IL-6 and TNF-αin rats in the overexpression group were higher than those in the model group and significantly lower than those in the model group and the low-expression group(P<0.05);the differences in the levels of EGF,HER1 and HER2 among the four groups of rats were statistically different(P<0.05),and the levels of EGF levels were higher than those of the control group,and EGF and HER1 were significantly lower than those of the model and low expression groups(P<0.05).Conclusion:Mi R-124 overexpression helps to reduce gastric mucosal damage and inflammatory response in rats with gastric precancerous lesions,and its mechanism of action may be linked to the inhibition of the Wnt/β-catenin signaling pathway.