Activation of the wnt/β-catenin/CYP1B1 pathway alleviates oxidative stress and protects the blood-brain barrier under cerebral ischemia/reperfusion conditions

Accumulating evidence suggests that oxidative stress and the Wnt/β-catenin pathway participate in stroke-induced disruption of the blood-brain barrier.However,the potential links between them following ischemic stroke remain largely unknown.The present study found that cerebral ischemia leads to oxidative stress and repression of the Wnt/β-catenin pathway.Meanwhile,Wnt/β-catenin pathway activation by the pharmacological inhibito r,TWS119,relieved oxidative stress,increased the levels of cytochrome P4501B1(CYP1B1)and tight junction-associated proteins(zonula occludens-1[ZO-1],occludin and claudin-5),as well as brain microvascular density in cerebral ischemia rats.Moreove r,rat brain microvascular endothelial cells that underwent oxygen glucose deprivation/reoxygenation displayed intense oxidative stress,suppression of the Wnt/β-catenin pathway,aggravated cell apoptosis,downregulated CYP1B1and tight junction protein levels,and inhibited cell prolife ration and migration.Overexpression ofβ-catenin or knockdown ofβ-catenin and CYP1B1 genes in rat brain mic rovascular endothelial cells at least partly ameliorated or exacerbated these effects,respectively.In addition,small interfering RNA-mediatedβ-catenin silencing decreased CYP1B1 expression,whereas CYP1B1 knoc kdown did not change the levels of glycogen synthase kinase 3β,Wnt-3a,andβ-catenin proteins in rat brain microvascular endothelial cells after oxygen glucose deprivatio n/reoxygenation.Thus,the data suggest that CYP1B1 can be regulated by Wnt/β-catenin signaling,and activation of the Wnt/β-catenin/CYP1B1 pathway contributes to alleviation of oxidative stress,increased tight junction levels,and protection of the blood-brain barrier against ischemia/hypoxia-induced injury.

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.

MicroRNA-329-3p inhibits the Wnt/β-catenin pathway and proliferation of osteosarcoma cells by targeting transcription factor 7-like 1

An important factor in the emergence and progre sion of osteosarcoma(OS)is the dysregulated expression of microRNAs(miRNAs).Transcription factor 7-like 1(TCF7LI),a member of the T cell factor/lymphoid enhancer factor(TCF/LEF)transcription factor family,interacts with the Wnt signaling pathway regulator β-catenin and acts as a DNA-specific binding protein.This study sought to elucidate the impact of the interaction between miR 3293p and TCF7L1 on.the growth and apoptosis of OS and analyze the regulatory expression relationship between miRNA and mRNA in osteosarcoma cells using a variety of approaches.MiR329-3p was significantly downregulated,while TCF7L1 was considerably up-regulated in all examined OS cell lines.Additionally,a clinical comparison study was performed using the TCGA database.Subsequently,the regulatory relationship between miR-329-3p and TCF7L1 on the proliferation and apoptosis of OS cells was verified through in vitro and in vivo experiments.When miR 329-3p was transfected into the OS cell line,the expression of TCF7L1 decreased,the proliferation of OS cells was inhibited,the cytoskeleton disintegrated,and the nucleus condensed to fom apoptotic bodies.The expression of proteins that indicate apoptosis increased simultaneously.The cell cycle was arrested in the G0/G1 phase,and the G1/S transition was blocked.The introduction of miR 3293p also inhibited downstream Cyclin D1 of the Wnt pathway.Xenograf experiments indicated that the overexpression of miR-329-3p signi ficanly inhibited the growth of OS xenografts in nude mice,and the expression of TCF7L1 and C-Myc in tumor tssues decreased.MiR 329-3p was significantly reduced in OS cells and played a suppressive role in tumorigenesis and proliferation by targeting TCF7L1 both in vitro and in vivo.Osteosarcoma cell cycle arrest and pathway inhibition were observed upon the regulation of TCF7LI by miR 3293p.Summarizing these results,it can be inferred that miR.3293p exerts anticancer efects in osteosarcoma by inhibiting TCF7L1.

LSM12 facilitates the progression of colorectal cancer by activating the WNT/CTNNB1 signaling pathway

Aberrant activation of the WNT signaling pathway is a joint event in colorectal cancer(CRC),but the molecular mechanism is still unclear.Recently,RNA-splicing factor LSM12(like-Sm protein 12)is highly expressed in CRC tissues.This study aimed to verify whether LSM12 is involved in regulating CRC progression via regulating the WNT signaling pathway.Here,we found that LSM12 is highly expressed in CRC patient-derived tissues and cells.LSM12 is involved in the proliferation,invasion,and apoptosis of CRC cells,similar to the function of WNT signaling in CRC.Furthermore,protein interaction simulation and biochemical experiments proved that LSM12 directly binds to CTNNB1(also known asβ-Catenin)and regulates its protein stability to affect the CTTNB1-LEF1-TCF1 transcriptional complex formation and the associated WNT downstream signaling pathway.LSM12 depletion in CRC cells decreased the in vivo tumor growth through repression of cancer cell growth and acceleration of cancer cell apoptosis.Taken together,we suggest that the high expression of LSM12 is a novel factor leading to aberrant WNT signaling activation,and that strategies targeting this molecular mechanism may contribute to developing a new therapeutic method for CRC.

沉默牛磺酸上调基因1通过Wnt/CTNNB1信号通路提高胃癌细胞对顺铂和5-氟尿嘧啶的敏感性

目的探讨牛磺酸上调基因1(TUG1)调控胃癌细胞对顺铂(CDDP)和5-氟尿嘧啶(5-FU)敏感性的作用效果及机制。方法实时定量PCR法检测TUG1在CDDP和5-FU耐药的胃癌组织和细胞系(SGC7901/R)中的表达。应用增强型CCK8法检测细胞的增殖活力,并计算CDDP和5-FU的半抑制浓度(IC50)。双荧光素酶实验检测Wnt/CTNNB1信号通路活性。Western boltting检测CTNNB1蛋白的表达。结果与对照组胃癌组织和细胞系(SGC7901)相比,TUG1在CDDP和5-FU耐药的胃癌组织和细胞系(SGC7901/R)中高表达。TUG1沉默上调了SGC7901/R细胞中CDDP和5-FU的IC50,提高了化疗敏感性。TUG1沉默降低了SGC7901/R细胞中Wnt/CTNNB1信号通路的活性,并抑制了CTNNB1蛋白的表达。CTNNB1过表达激活了SGC7901/R细胞中Wnt/CTNNB1信号通路,逆转了TUG1沉默对CDDP和5-FU化疗敏感性的促进作用。结论TUG1与胃癌的化疗不敏感相关,沉默TUG1通过Wnt/CTNNB1信号通路提高胃癌细胞对CDDP和5-FU的敏感性。

Up-regulated Wnt1-inducible signaling pathway protein 1 correlates with poor prognosis and drug resistance by reducing DNA repair in gastric cancer

BACKGROUND Wnt1-inducible signaling pathway protein 1(WISP1)is upregulated in several types of human cancer,and has been implicated in cancer progression.However,its clinical implications in gastric cancer(GC)remain unclear.AIM To explore the expression pattern and clinical significance of WISP1 in GC.METHODS Public data portals,including Oncomine,The Cancer Genome Atlas database,Coexpedia,and Kaplan-Meier plotter,were analyzed for the expression and clinical significance of WISP1 mRNA levels in GC.One hundred and fifty patients who underwent surgery for GC between February 2010 and October 2012 at the Affiliated Hospital of Jiangnan University were selected for validation study.WISP1 levels were measured at both the mRNA and protein levels by RTqPCR,Western blot analysis,and immunohistochemistry(IHC).In addition,the in situ expression of WISP1 in the GC tissues was determined by IHC,and the patients were accordingly classified into high-and low-expression groups.The correlation of WISP1 expression status with patient prognosis was then determined by univariate and multivariate Cox regression analyses.WISP1 was knocked down by RNA interference.The 50%inhibitory concentration of oxaliplatin was detected by CellTiter-Blue assay.RESULTS WISP1 levels at both the mRNA and protein levels were remarkably upregulated in GC tissues compared to normal tissues.Moreover,IHC revealed that WISP1 expression was associated with T stage and chemotherapy outcome,but not with lymph node metastasis,age,gender,histological grade,or histological type.GC patients with high WISP1 expression showed a poor overall survival.Multivariate survival analysis indicated that WISP1 was an important prognostic factor for GC patients.Mechanistically,knock-down of WISP1 expression enhanced sensitivity to oxaliplatin by reducing DNA repair and enhancing DNA damage.CONCLUSION Significantly upregulated WISP1 expression is associated with cancer progression,chemotherapy outcome,and prognosis in GC.Mechanistically,knock-down of WISP1 expression enhances oxaliplatin sensitivity by reducing DNA repair and enhancing DNA damage.WISP1 may be a potential therapeutic target for GC treatment or a potential biomarker for diagnosis and prognosis.

Characterization of Wnt1-inducible Signaling Pathway Protein-1 in Obese Children and Adolescents

Wnt1-inducible signaling pathway protein-1(WISP1),a member of the CCN family,is increasingly being recognized as a potential target for obesity and type 2 diabetes mellitus.Recent studies have shown that WISP1 can regulate low-grade inflammation in obese mice,and circulating WISP1 levels are associated with obesity and type 2 diabetes mellitus in adults.Herein,we measured serum WISP1 levels in obese youth and explored its relationships with pro-inflammatory cytokine interleukin 18(IL-18)and other metabolic indexes.Totally,44 normal-weight and 44 obese children and adolescents were enrolled.Physical and laboratory data were recorded,and then serum levels of WISP1 and IL-18 were determined by enzyme-linked immunosorbent assays.Results showed that serum levels of WISP1 were significantly higher in obese children and adolescents than in normal-weight healthy controls (1735.444-15.29 vs. 1364.084-18.69 pg/mL).WISP1 levels were significantly positively correlated with body mass index (BMI)and BMI z-score (r=0.392,P=0.008;r=0.474,P=0.001,respectively) in obese group;circulating IL-18 was increased in obese individuals (1229.064-29.42 vs. 295.874-13.30 pg/mL).Circulating WISP1 levels were significantly correlated with IL-18 (r=0.542,P<0.001),adiponectin (r=0.585,P<0.001)and leptin (r=0.592,P<0.001).The multivariate stepwise regression analysis showed that higher IL-18 levels represented the main determinant of increased WISP1 levels after adjusting for BMI,waist circumference, fasting insulin,homeostatic model assessment of insulin resistance (HOMA-IR)and HbAlc in obese individuals (β=0.542,P=0.000).WISP1 can be involved in glucose/lipid metabolism in obese youth,which may be modulated by IL-18.Increased WISP1 levels may be a risk factor of obesity and insulin resistance,and WISP1 has a potential therapeutic effect on insulin resistance in obese children and adolescents.

Tissue Inhibitor of Metalloprotease-1(TIMP-1)Regulates Adipogenesis of Adipose-derived Stem Cells(ASCs)via the Wnt Signaling Pathway in an MMP-independent Manner

Tissue inhibitor of m etalloprotease-1(TIM P-1)is a tissue inhibitor o f matrix metalloproteinases(MMPs).It however exerts multiple effects on biological processes,such as cell growth,proliferation,differentiation and apoptosis,in an MMP-independent manner.This study aimed to examine the role of TIMP-1 in adipogenesis of adipose-derived stem cells(ASCs)and the underlying mechanism.We knocked down the TIMP-1 gene in ASCs through lentiviral vectors encoding TIMP-1 small interfering RNA(siRNA),and then found that the knockdown of TIMP-1 in ASCs promoted the adipogenic differentiation of stem cells and inhibited the Wnt/β-catenin signaling pathway in ASCs.We also noted that mutant TIMP-1 without the inhibitory activity on MMPs promoted the activation of Wnt/β-catenin pathway as well as the recombinant wild type TIMP-1 did,which indicated that the effect of TIMP-1 on Wnt/β-catenin pathway was MMPindependent.Our study suggested that TIMP-1 negatively regulated the adipogenesis of ASCs via the Wnt/β-catenin signaling pathway in an MMP-independent manner.

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.

Tip60-siRNA Regulates ABCE1 Acetylation and Inhibits the Proliferation, Migration and Invasion of Esophageal Cancer via the Wnt Pathway

Objective: To investigate the effect of Tip60 gene silencing on the ABCE1 acetylation level and cell proliferation, migration and invasion in TE-1 cells of oesophageal cancer. Methods: The siRNA sequence of Tip60 was transfected with esophageal cancer TE-1 cells. Transfected siRNA vector cells were used as experimental group (si-T), siRNA no-loaded somatic cells were transfected as control group (si-NC), and untransfected TE-1 cells were used as blank group (Group N). ABCE1 mRNA was detected by qRT-PCR, the expression of ABCE1 protein, proliferation-related protein β catenin (β-catenin), GSK3β, and c-myc by Western blot, the protein acetylation level by immunoprecipitation, MTT assay for cell viability, scratch healing and Transwell compartment assay for migration and invasion ability. Results: After 48 h downregulation of the Tip60 gene, TE-1 cells showed no significant changes in the ABCE1 mRNA and protein expression. The acetylation level of ABCE1 decreased significantly, compared with the control group and the blank group. After Tip60 gene silencing, the expression of β-catenin and c-myc protein decreased, while the expression of GSK-3β protein increased. Cytofunctology experiments showed that the proliferative activity, migration and invasion ability of TE-1 cells in the experimental group were significantly inhibited. Conclusion: Down regulation of Tip60 gene can deacetylate ABCE1 protein and inhibit the proliferation activity, migration and invasion ability of esophageal cancer by blocking the conduction of Wnt signaling pathway.

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.

The effect of Wnt/β-catenin signaling on PD-1/PDL-1 axis in HPV-related cervical cancer

Infection with high-risk human papillomavirus(HPV),including HPV-16 and HPV-18,is the main cause of malignancies,such as cervical cancer.Viral oncoproteins encoded by HPV are expressed in HPV-positive cancers and associated with the early cancer stages and the transformation of normal cells.The signaling pathways involved in the transformation of normal cells to cancerous form and the subsequently expressed programmed cell death-ligand 1(PD-L1)on the surface of the transformed cells lead to a disruption in recognition of tumor cells by the immune cell system,including T lymphocytes and dendritic cells which lead to the development of cervical cancer malignancy.These cells also produce modest levels of cytokines during exhaustion,tumor-infiltrating T CD4+cells with high levels of PD-1 and CD39 release considerable quantities of cytokines.The Wnt/β-catenin signaling pathway,which controls the expression of genes involved in the tumor cells’markers,is demonstrated to be one of the most potent cancer stimulants.It leads to the evasion of the tumor cells from immune cell detection and ultimately avoids being recognized by dendritic cells or T-cells.PD-L1,as an inhibitory immune checkpoint,is essential for controlling immune system activity by inhibiting T-cells’inflammatory function.In the present review,we looked into how Wnt/β-catenin affects the expression of PD-L1 and related genes like c-MYC in cancer cells and its role in the development of HPV-induced malignancy.We hypothesized that blocking these pathways could be a potential immunotherapy and cancer prevention method.

Integrative analysis of aberrant Wnt signaling in hepatitis B virus-related hepatocellular carcinoma

AIM: To comprehensively understand the underlying molecular events accounting for aberrant Wnt signaling activation in hepatocellular carcinoma(HCC).METHODS: This study was retrospective. The HCC tissue specimens used in this research were obtained from patients who underwent liver surgery. The Catalogue of Somatic Mutations in Cancer(COSMIC) database was searched for the mutation statuses of CTNNB1, TP53, and protein degradation regulator genes of CTNNB1. Dual-luciferase reporter assay was performed with TOP/FOP reporters to detect whether TP53 gain-of-function(GOF) mutations could enhance the transcriptional activity of Wnt signaling. Methylation sensitive restriction enzyme-quantitative PCR was used to explore the methylation status of Cp G islands located in the promoters of APC, SFRP1, and SFRP5 in HCCs with different risk factors. Finally, nestedreverse transcription PCR was performed to examine the integration of HBx in front of LINE1 element and the existence of HBx-LINE1 chimeric transcript in Hepatitis B virus-related HCC. All results in this article were analyzed with the software SPSS version 19.0 for Windows, and different groups were compared by χ2 test as appropriate.RESULTS: Based on the data from COSMIC database, compared with other solid tumors, mutation frequency of CTNNB1 was significantly higher in HCC(P < 0.01). The rate of CTNNB1 mutation was significantly less frequent in Hepatitis B virus-related HCC than in other etiologies(P < 0.01). Dual-luciferase reporter system and TOP/FOP reporter assays confirmed that TP53 GOF mutants were able to enhance the transcriptional ability of Wnt signaling. An exclusive relationship between the status of TP53 and CTNNB1 mutations was observed. However, according to the COSMIC database, TP53 GOF mutation is rare in HCC, which indicates that TP53 GOF mutation is not a reason for the aberrant activation of Wnt signaling in HCC. APC and AXIN1 were mutated in HCC. By using methylation sensitive restriction enzyme-quantitative PCR, hypermethylation of APC was detected in HCC with different risk factors, whereas SFRP1 and SFRP5 were not hypermethylated in any of the HCC etiologies, which indicates thatthe mutation of APC and AXIN1, together with the methylation of APC could take part in the overactivation of Wnt signaling. Nested-reverse transcription PCR failed to detect the integration of HBx before the LINE1 element, or the existence of an HBx-LINE1 chimeric transcript, suggesting that integration could not play a role in the aberrant activation of Wnt signaling in HCC.CONCLUSION: In HCC, genetic/epigenetic aberration of CTNNB1 and its protein degradation regulators are the major cause of Wnt signaling overactivation.

miR-103-3p targets Ndel1 to regulate neural stem cell proliferation and differentiation

The regulation of adult neural stem cells(NSCs) is critical for lifelong neurogenesis. MicroRNAs(miRNAs) are a type of small, endogenous RNAs that regulate gene expression post-transcriptionally and influence signaling networks responsible for several cellular processes. In this study, mi R-103-3 p was transfected into neural stem cells derived from embryonic hippocampal neural stem cells. The results showed that mi R-103-3 p suppressed neural stem cell proliferation and differentiation, and promoted apoptosis. In addition, mi R-103-3 p negatively regulated Nud E neurodevelopment protein 1-like 1(Ndel1) expression by binding to the 3′ untranslated region of Ndel1. Transduction of neural stem cells with a lentiviral vector overexpressing Ndel1 significantly increased cell proliferation and differentiation, decreased neural stem cell apoptosis, and decreased protein expression levels of Wnt3 a, β-catenin, phosphor-GSK-3β, LEF1, c-myc, c-Jun, and cyclin D1, all members of the Wnt/β-catenin signaling pathway. These findings suggest that Ndel1 is a novel mi R-103-3 p target and that mi R-103-3 p acts by suppressing neural stem cell proliferation and promoting apoptosis and differentiation. This study was approved by the Animal Ethics Committee of Nantong University, China(approval No. 20200826-003) on August 26, 2020.

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.

靶向调控CTNNB1表达水平对胃癌细胞系SGC-7901增殖及侵袭能力的影响

目的探讨调控Wnt/CTNNB1通路对胃癌增殖和侵袭能力的影响。方法人胃癌细胞系SGC-7901,体外培养慢病毒转染的稳转SGC-7901,分成CTNNB1-shRNA组、shRNA组与对照组。qRT-PCR和免疫印迹检测稳转细胞系中AKT、CTNNB1、Wnt2、Cyp19A1的表达,CCK-8测定不同细胞系增殖能力,划痕试验测定胃癌细胞系的转移能力,Transwell小室测定胃癌细胞系侵袭能力,集落形成试验测定胃癌细胞系的集落形成能力。结果AKT、CTNNB1、Wnt2、Cyp19A1 mRNA和蛋白在CTNNB1-shRNA组表达最低(P<0.05),在shRNA组和对照组中表达差异无统计学意义(P>0.05);24、48、72 h细胞增殖水平在CTNNB1-shRNA组细胞最低(P<0.05),shRNA组和对照组细胞增殖差异无统计学意义(P>0.05);划痕后24 h CTNNB1-shRNA组24 h迁移细胞率最低(P<0.05),shRNA组与对照组迁移细胞率差异无统计意义(P>0.05);CTNNB1-shRNA组侵袭细胞数量和集落形成数量最低(P<0.05),shRNA组与对照组侵袭细胞数量差异无统计意义(P>0.05)。结论通过下调Wnt/CTNNB1通路能引起该通路相关下游因子在胃癌细胞中低表达,从而影响胃癌增殖和侵袭能力,有望成为胃癌治疗新靶点。

利拉鲁肽对高糖介导人牙周膜成纤维细胞的影响

目的研究利拉鲁肽(Lira)对高糖介导人牙周膜成纤维细胞(hPDLFs)凋亡及Wnt/连环蛋白β(CTNNB)1通路的影响及机制。方法体外复苏培养hPDLFs,取对数生长期的hPDLFs调整其密度为5×10^(8)个/L。对照组hPDLFs在常规培养基中培养〔含11 mmol/L葡萄糖(GLU)〕,甘露醇组hPDLFs在含有19.5 mmol/L甘露醇培养基中培养,高糖组在含40 mmol/L GLU的培养基中培养,Lira组在含有40 mmol/L GLU和100 nmol/ml Lira的培养基中培养。3 d后利用CCK-8法检测各组细胞活力,利用Hoechst染色检测细胞凋亡情况同时利用比色法检测各组hPDLFs凋亡相关指标含半胱氨酸的天冬氨酸蛋白水解酶(Caspase)3、6、9活性,检测各组hPDLFs内游离Ca^(2+)浓度,利用酶联免疫吸附试验(ELISA)检测测各组hPDLFs上清液中基质金属蛋白酶(MMP)-2、MMP-3和MMP-9表达,采用实时荧光定量PCR及Western印迹检测各组hPDLFs中Wnt3a、CTNNB1、Bcl2相关X蛋白(Bax)、重组人B细胞淋巴瘤因子2XL(Bcl-xl)的mRNA和蛋白表达。结果与对照组和甘露醇组比较,高糖组hPDLFs凋亡率及细胞内游离Ca^(2+)浓度显著增加,细胞活力显著降低(P<0.05),Caspase3、6、9活性和细胞上清液中MMP-2、MMP-3和MMP-9浓度显著升高(P<0.05),Wnt3a、CTNNB1及Bax mRNA和蛋白水平均表达显著升高,Bcl-xl mRNA和蛋白表达显著降低(P<0.05)。与高糖组比较,Lira组hPDLFs凋亡细胞数目显著减少,细胞内游离Ca^(2+)浓度显著降低,细胞活力显著升高(P<0.05),Caspase3、6、9活性显著降低,细胞上清液中MMP-2、MMP-3和MMP-9浓度显著降低(P<0.05),Wnt3a、CTNNB1及Bax mRNA和蛋白表达显著降低,Bcl-xl mRNA和蛋白表达显著升高(P<0.05)。结论Lira对高糖介导的hPDLFs损伤具有保护作用,降低细胞凋亡率,促进细胞增殖,其机制可能通过抑制Wnt/CTNNB1通路激活,抑制下游靶基因Bax,促进Bcl-xl的表达实现的,并且抑制细胞Caspase活性及上清液中MMP相关蛋白的释放。

Apoptosis of mesenchymal stem cells is regulated by Rspo1 via the Wnt/β-catenin signaling pathway

Objective: The aim of this study was to investigate the effect and possible mechanism of action of roof plate-specific spondin1 (Rspo1) in the apoptosis of rat bone marrow mesenchymal stem cells (BMSCs). Methods: Osteogenic and adipogenic differentiation of BMSCs was identified by Alizarin Red and Oil Red O staining, respectively. BMSC surface markers (cluster of differentiation 29 [CD29], CD90, and CD45) were detected using flow cytometry. BMSCs were transfected with an adenoviral vector encoding Rspo1 (BMSCs-Rspo1 group). The expression levels of Rspo1 gene and Rspo1 protein in the BMSCs-Rspo1 group and the two control groups (untransfected BMSCs group and BMSCs-green fluorescent protein [GFP] group) were analyzed and compared by quantitative polymerase chain reaction and Western blot. The occurrence of apoptosis in the three groups was detected by flow cytometry and acridine orange-ethidium bromide (AO-EB) double dyeing. The activity of the Wnt/β-catenin signaling pathway was evaluated by measuring the expression levels of the key proteins of the pathway (β-catenin, c-Jun N-terminal kinase [JNK], and phospho-JNK). Results: Osteogenic and adipogenic differentiation was confirmed in cultured BMSCs by the positive expression of CD29 and CD90 and the negative expression of CD45. Significantly increased expression levels of Rspo1 protein in the BMSCs-Rspo1 group compared to those in the BMSCs (0.60 ± 0.05 vs. 0.13 ± 0.02;t=95.007, P=0.001) and BMSCs-GFP groups (0.60 ± 0.05 vs. 0.10 ± 0.02;t=104.842, P=0.001) were observed. The apoptotic rate was significantly lower in the BMSCs-Rspo1 group compared with those in the BMSCs group ([24.06 ± 2.37]% vs.[40.87 ± 2.82]%;t =49.872, P =0.002) and the BMSCs-GFP group ([24.06 ± 2.37]% vs.[42.34 ± 0.26]%;t =62.358, P =0.001). In addition, compared to the BMSCs group, the protein expression levels of β-catenin (2.67 ± 0.19 vs. 1.14 ± 0.14;t =-9.217, P =0.000) and JNK (1.87 ± 0.17 vs. 0.61 ± 0.07;t =-22.289, P =0.000) were increased in the BMSCs-Rspo1 group. Compared to the BMSCs-GFP group, the protein expression levels of β-catenin (2.67 ± 0.19 vs. 1.44 ± 0.14;t =-5.692, P =0.000) and JNK (1.87 ± 0.17 vs. 0.53 ± 0.06;t =-10.589, P =0.000) were also upregulated in the BMSCs-Rspo1 group. Moreover, the protein expression levels of phospho-JNK were increased in the BMSCs-Rspo1 group compared to those in the BMSCs group (1.89 ± 0.10 vs. 0.63 ± 0.09;t =-8.975, P =0.001) and the BMSCs-GFP group (1.89 ± 0.10 vs. 0.69 ± 0.08;t =-9.483, P =0.001). Conclusion: The Wnt/β-catenin pathway could play a vital role in the Rspo1-mediated inhibition of apoptosis in BMSCs.

Strawberry Notch 1 (SBN01) promotes proliferation of spermatogonial stem cells via the noncanonical Wnt pathway in mice

While it is known that spermatogonial stem cells (SSCs) initiate the production of male germ cells, the mechanisms of SSC self-renewal, proliferation, and differentiation remain poorly understood. We have previously identified Strawberry Notch 1 (SBN01), a vertebrate strawberry notch family protein, in the proteome profile for mouse SSC maturation and differentiation, revealing SBN01 is associated with neonatal testicular development. To explore further the location and function of SBN01 in the testes, we performed Sbnol gene knockdown in mice to study the effects of SBN01 on neonatal testicular and SSC development. Our results revealed that SBN01 is required for neonatal testicular and SSC development in mice. Particularly, in vitro Sbnol gene knockdown with morpholino oligonucleotides caused a reduction of SSCs and inactivation of the noncanonical Wnt pathway, through Jun N-terminal kinases. Our study suggests SBN01 maintains SSCs by promoting the noncanonical Wnt pathway.

Role of Wnt signaling pathway hypofunction mediated by dephosphorylation of β-catenin in impaired wound healing of type 1 diabetic rats

Objective To investigate the role of Wnt signaling pathway hypofunction mediated by dephosphorylation ofβ-catenin in the impaired wound healing of type 1 diabetic rats.Methods The back skin defect wounds were produced in rats with type 1 diabetes.These rats were divided into control,diabetes,lithium chloride treatment,and epidermal growth factor(EGF)