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.

下调METTL5通过Wnt/β-catenin信号通路抑制三阴乳腺癌细胞增殖、迁移与侵袭

目的探讨甲基转移酶5(methyltransferase-like 5,METTL5)在三阴乳腺癌(triple-negative breast cancer,TNBC)中的作用和潜在机制。方法采用免疫组织化学方法和Western blot检测TNBC肿瘤组织和细胞系中METTL5的表达情况。用靶向METTL5的shRNA(shRNA-METTL5)转染TNBC细胞后,用CCK-8、集落形成、伤口愈合以及Transwell实验分别检测细胞增殖活性、迁移与侵袭,Western blot检测Wnt/β-catenin信号关键蛋白的表达。构建异种移植瘤模型,验证敲降METTL5对TNBC细胞在体内生长以及Wnt/β-catenin信号活性的影响。结果METTL5在TNBC肿瘤组织和细胞系中表达上调(P<0.01)。敲降METTL5可抑制TNBC细胞的增殖、迁移和侵袭并降低了Wnt/β-catenin信号分子β-catenin、细胞周期蛋白(Cyclin)D1、基质金属蛋白酶(MMP)-2和MMP-7的表达(均P<0.01)。体内实验显示,敲降METTL5减缓了移植瘤生长和Wnt/β-catenin信号活性。结论敲降METTL5能抑制TNBC细胞的增殖、迁移与侵袭,其作用可能与抑制Wnt/β-catenin信号通路有关。

基于Wnt 5a/β-catenin信号通路探讨杜仲健骨方对骨质疏松症大鼠的治疗作用

目的探究不同剂量杜仲健骨方对骨质疏松症模型大鼠骨力学参数的影响及对Wnt5a/β-catenin信号通路的干预作用。方法60只SD大鼠分为对照组、模型组、杜仲健骨方低剂量组、杜仲健骨方中剂量组和杜仲健骨方高剂量组,采用去势(摘除大鼠双侧卵巢)制备骨质疏松症大鼠模型,对照组仅翻动两侧卵巢,不做其他处理。造模成功后杜仲健骨方各处理组分别给予低、中、高计量的杜仲健骨方灌胃90 d,实验终点检测大鼠骨密度、骨生物力学指标,骨形态学指标;通过Elisa法检测各组大鼠血清中抗酒石酸碱性磷酸酶(TRAP)、尿I型胶原交联N末端肽(U-NTX)、骨碱性磷酸酶(BALP)、雌二醇(E2)、尿型胶原交联C末端肽(U-CTX)、骨钙蛋白(BGP)等骨转化指标;通过qPCR和Western blot法检测骨组织中Wnt5a/β-catenin通路分子的mRNA和蛋白表达水平。结果杜仲健骨方能显著增加骨质疏松大鼠股骨、脊柱骨的骨密度(P<0.01),提高股骨生物力学参数水平(P<0.01);增加皮质骨面积百分比、骨小梁平均厚度、骨小梁面积、骨小梁体积(P<0.01),增加血清中的BALP、E2水平(P<0.01),降低血清中的BGP、TRAP、U-NTX、U-CTX水平(P<0.01);显著增加Wnt5a/β-catenin通路分子的mRNA和蛋白表达水平(P<0.01)。结论杜仲健骨方可以提高骨质疏松大鼠骨力学参数,并且调控Wnt5aβ-catenin信号通路,有利于骨质疏松症的治疗。

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.

Calculus bovis inhibits M2 tumor-associated macrophage polarization via Wnt/β-catenin pathway modulation to suppress liver cancer

BACKGROUND Calculus bovis(CB),used in traditional Chinese medicine,exhibits anti-tumor effects in various cancer models.It also constitutes an integral component of a compound formulation known as Pien Tze Huang,which is indicated for the treatment of liver cancer.However,its impact on the liver cancer tumor microenvironment,particularly on tumor-associated macrophages(TAMs),is not well understood.AIM To elucidate the anti-liver cancer effect of CB by inhibiting M2-TAM polarization via Wnt/β-catenin pathway modulation.METHODS This study identified the active components of CB using UPLC-Q-TOF-MS,evaluated its anti-neoplastic effects in a nude mouse model,and elucidated the underlying mechanisms via network pharmacology,transcriptomics,and molecular docking.In vitro assays were used to investigate the effects of CB-containing serum on HepG2 cells and M2-TAMs,and Wnt pathway modulation was validated by real-time reverse transcriptase-polymerase chain reaction and Western blot analysis.RESULTS This study identified 22 active components in CB,11 of which were detected in the bloodstream.Preclinical investigations have demonstrated the ability of CB to effectively inhibit liver tumor growth.An integrated approach employing network pharmacology,transcriptomics,and molecular docking implicated the Wnt signaling pathway as a target of the antineoplastic activity of CB by suppressing M2-TAM polarization.In vitro and in vivo experiments further confirmed that CB significantly hinders M2-TAM polarization and suppresses Wnt/β-catenin pathway activation.The inhibitory effect of CB on M2-TAMs was reversed when treated with the Wnt agonist SKL2001,confirming its pathway specificity.CONCLUSION This study demonstrated that CB mediates inhibition of M2-TAM polarization through the Wnt/β-catenin pathway,contributing to the suppression of liver cancer growth.

WNT/β-catenin-M2 macrophage interplay as a target for therapy against hepatocellular carcinoma:Role of Calculus bovis

Liver cancer,and in particular hepatocellular carcinoma(HCC)is a disease of rising prevalence and incidence.To date,definitive treatment options include either surgical excision or ablation of the affected area.With increasing research on several pathways that could be involved in the progression of HCC,new elements within these pathways emerge as potential targets for novel therapies.The WNT/β-catenin pathway favors the presence of M2 tumor-associated macrophages which in turn promote tumor growth and metastasis.The inhibition of this pathway is considered a good candidate for such targeted therapeutic interventions.Interestingly,as Huang et al show in their recently published article,Calculus bovis which is used in traditional Chinese medicine can exert an inhibitory effect on theβ-catenin pathway and become a potential candidate for targeted pharmacotherapy against liver cancer.

芪玉三龙方通过Wnt5a/β-catenin信号通路逆转上皮—间充质转化抑制肺癌细胞侵袭和转移

目的从细胞水平探讨芪玉三龙方是否通过Wnt5a/β-catenin信号通路抑制上皮—间充质细胞转化,抑制肺癌转移。方法将重组慢病毒转染小鼠肺癌细胞建立稳定过表达Wnt5a的细胞系,同时构建Wnt5a干扰和空载细胞株。qRT-PCR法检测Wnt5a、β-catenin mRNA的表达水平。CCK8、EdU实验检测肺癌细胞的增殖活性,Transwell实验检测肺癌细胞的迁移和侵袭能力。Western blot法检测Wnt5a、β-catenin、c-Myc、Cyclin D1、Vimentin、N-cadherin、E-cadherin、Snail蛋白表达水平。结果与对照组比较,芪玉三龙方组Wnt5a、β-catenin、c-Myc、Cyclin D1蛋白表达水平下降(P<0.05),细胞增殖活性、侵袭和迁移能力显著降低(P<0.05)。Wnt5a过表达组中Vimentin、N-cadherin、Snail表达水平增加(P<0.05),E-cadherin蛋白表达水平下降(P<0.05),细胞增殖活性、侵袭和迁移能力显著增强(P<0.05);Wnt5a干扰组Vimentin、N-cadherin、Snail表达水平下降(P<0.05),E-cadherin蛋白表达水平升高(P<0.05),细胞增殖活性、侵袭和迁移能力显著下降(P<0.05),明显抑制EMT的发生。芪玉三龙方处理后,Wnt5a过表达组Wnt5a、β-catenin、c-Myc、Cyclin D1、Vimentin、N-cadherin、Snail表达水平下降(P<0.05),E-cadherin蛋白表达水平升高(P<0.05),细胞增殖活性、侵袭和迁移能力得到抑制。结论芪玉三龙方可能通过Wnt5a/β-catenin信号通路逆转上皮—间充质转化,抑制肺癌细胞增殖和转移。

Calculus bovis in hepatocellular carcinoma:Tumor molecular basis,Wnt/β-catenin pathway role,and protective mechanism

In this editorial,we comment on the recent article by Huang et al.The editorial focuses specifically on the molecular mechanisms of hepatocellular carcinoma(HCC),mechanism of Wnt/β-catenin pathway in HCC,and protective mechanism of Calculus bovis(CB)in HCC.Liver cancer is the fourth most common cause of cancer-related deaths globally.The most prevalent kind of primary liver cancer,HCC,is typically brought on by long-term viral infections(hepatitis B and C),non-alcoholic steatohepatitis,excessive alcohol consumption,and other conditions that can cause the liver to become chronically inflamed and cirrhotic.CB is a wellknown traditional remedy in China and Japan and has been used extensively to treat a variety of diseases,such as high fever,convulsions,and stroke.Disturbances in lipid metabolism,cholesterol metabolism,bile acid metabolism,alcohol metabolism,and xenobiotic detoxification lead to fatty liver disease and liver cirrhosis.Succinate,which is a tricarboxylic acid cycle intermediate,is vital to energy production and mitochondrial metabolism.It is also thought to be a signaling molecule in metabolism and in the development and spread of liver malignancies.The Wnt/β-catenin pathway is made up of a group of proteins that are essential for both adult tissue homeostasis and embryonic development.Cancer is frequently caused by the dysregulation of the Wnt/β-catenin signaling pathway.In HCC liver carcinogenesis,Wnt/β-catenin signaling is activated by the expression of downstream target genes.Communication between the liver and the gut exists via the portal vein,biliary tract,and systemic circulation.This"gutliver axis"controls intestinal physiology.One of the main factors contributing to the development,progression,and treatment resistance of HCC is the abnormal activation of the Wnt/β-Catenin signaling pathway.Therefore,understanding this pathway is essential to treating HCC.Eleven ingredients of CB,particularly oleanolic acid,ergosterol,and ursolic acid,have anti-primary liver cancer properties.Additionally,CB is important in the treatment of primary liver cancer through pathways linked to immune system function and apoptosis.CB also inhibits the proliferation of cancer stem cells and tumor cells and controls the tumor microenvironment.In the future,clinicians may be able to recommend one of many potential new drugs from CB ingredients to treat HCC expression,development,and progress.

Silencing of peroxiredoxin 2 suppresses proliferation and Wnt/β-catenin pathway,and induces senescence in hepatocellular carcinoma

Hepatocellular carcinoma(HCC),a common malignancy worldwide,still lacks effective clinical treatment.The study aimed to investigate the oncogenes that affect the progression of HCC and their possible mechanisms.In our study,we initially confirmed a higher level of PRDX2 in the bile of HCC patients compared to those with choledocholithiasis by 2-DE,LC-MS,and ELISA.Subsequently,we demonstrated the high expression of peroxiredoxin 2(PRDX2)in HCC based on the TCGA database and clinical sample analysis.Furthermore,PRDX2 overexpression enhanced the viability of HCC cells.And PRDX2 silencing induced senescence of HCC cells.In vivo,knockdown of PRDX2 significantly reduced the weight of xenograft tumors.PRDX2 also was found to activate the Wnt/β-catenin pathway by inducingβ-catenin nuclear translocation.Consequently,we proved that silencing PRDX2 could inhibit proliferation and Wnt/β-catenin pathway while promoting senescence in HCC cells.

新风胶囊通过结合Wnt5a经Wnt/β-catenin信号通路抑制类风湿性关节炎

目的:本研究将探讨Wnt5a是否可以作为类风湿性关节炎(RA)潜在的诊断和治疗靶点,新风胶囊(XFC)如何通过Wnt5a/β-catenin信号通路改善RA。方法:在体内Adjuvant arthritis(AA)大鼠模型中采用ELISA和RT-qPCR检测炎症因子和病理基因研究XFC对AA大鼠疗效,RT-qPCR检测验证XFC调控通过网络药理学预测的核心基因和关键通路。在体外原代AA成纤维样滑膜细胞(FLS)中采用RT-qPCR、Western blot和免疫荧光等方法研究XFC对Wnt/β-catenin通路的调节机制。结果:XFC显著下调AA大鼠的关节炎评分和足爪肿胀,抑制AA大鼠关节炎症。XFC降低AA大鼠外周血中炎症因子TNF-α和IL-1水平,抑制AA大鼠关节滑膜和AA FLS中病理基因MMP3和fibronectin水平。网络药理学预测出Wnt通路与XFC治疗RA高度相关。细胞水平上,含XFC血清抑制Wnt通路相关基因β-catenin、CCND1和c-Myc的表达。分子对接结果显示XFC关键成分与Wnt5a的结合能力强,在AA FLS中Wnt5a过表达(Wnt5a-ove)干扰了XFC的作用。结论:Wnt5a在AA FLS和RA FLS中表达明显升高,XFC通过与Wn5a结合,抑制Wnt/β-catenin信号通路活化改善RA,为XFC改善RA提供新的治疗机制。

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.

Inhibition of M2 tumor-associated macrophages polarization by modulating the Wnt/β-catenin pathway as a possible liver cancer therapy method

The problem of liver cancer is becoming increasingly important due to the epi-demic of metabolic diseases and persistent high alcohol consumption.This deter-mines great attention to the development and improvement of methods for early diagnosis and treatment of liver cancer.Huang et al presented a study in the World Journal of Gastroenterology,in which they showed that the use of the traditional Chinese medicine Calculus bovis(CB)can suppress tumor growth in mice by inhibiting M2 tumor-associated macrophages(TAM)through modulating the activity of the Wnt/β-catenin pathway.The interaction of CB components with the Wnt/β-catenin pathway,M2 TAM polarization,and tumor dynamics were studied using network pharmacology,transcriptomics,and molecular docking.It is now generally accepted that the polarization of TAM and the differentiation of the functions of M1 and M2 phagocytes are of great importance for the progression of neoplasms.It is assumed that M2 TAM promote proliferation and migration of tumor cells.Attempts to medicinally influence the Wnt/β-catenin pathway in order to modulate phagocyte polarization now belong to one of the most promising areas of immunotherapy of oncological diseases.Undoubtedly,the work of the Chinese authors deserves attention and further development.

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.

IL13RA2 promotes progression of infantile haemangioma by activating glycolysis and the Wnt/β-catenin signaling pathway

Background:Interleukin 13 receptor subunit alpha 2(IL13RA2)plays an essential role in the progression of many cancers.However,the role of IL13RA2 in infantile haemangioma(IH)is still unknown.Materials and Methods:IL13RA2 expression in IH tissues was analyzed using western blot,qRT-PCR,and immunofluorescence.The role of IL13RA2 in haemangioma-derived endothelial cells(HemECs)was determined following knockdown or overexpression of IL13RA2 using CCK-8,colony formation,apoptosis,wound healing,tubule formation,Transwell,and western blot.Results:IL13RA2 expression was upregulated in IH tissues.IL13RA2 overexpression promoted proliferation,migration,and invasion of HemECs and induced glycolysis,which was confirmed with a glycolysis inhibitor.Specifically,IL13RA2 interacted withβ-catenin and activated the Wnt/β-catenin pathway in HemECs,which were involved in the above-mentioned effects of IL13RA2.Conclusions:These findings revealed that targeting IL13RA2 is a potential therapeutic approach for IH.

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.

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.

Complement factor Ⅰ knockdown inhibits colon cancer development by affecting Wnt/β-catenin/c-Myc signaling pathway and glycolysis

BACKGROUND Colon cancer(CC)occurrence and progression are considerably influenced by the tumor microenvironment.However,the exact underlying regulatory mechanisms remain unclear.AIM To investigate immune infiltration-related differentially expressed genes(DEGs)in CC and specifically explored the role and potential molecular mechanisms of complement factor I(CFI).METHODS Immune infiltration-associated DEGs were screened for CC using bioinformatics.Quantitative reverse transcription polymerase chain reaction was used to examine hub DEGs expression in the CC cell lines.Stable CFI-knockdown HT29 and HCT116 cell lines were constructed,and the diverse roles of CFI in vitro were assessed using CCK-8,5-ethynyl-2’-deoxyuridine,wound healing,and transwell assays.Hematoxylin and eosin staining and immunohistochemistry staining were employed to evaluate the influence of CFI on the tumorigenesis of CC xenograft models constructed using BALB/c male nude mice.Key proteins associated with glycolysis and the Wnt pathway were measured using western blotting.RESULTS Six key immune infiltration-related DEGs were screened,among which the expression of CFI,complement factor B,lymphoid enhancer binding factor 1,and SRY-related high-mobility-group box 4 was upregulated,whereas that of fatty acid-binding protein 1,and bone morphogenic protein-2 was downregulated.Furthermore,CFI could be used as a diagnostic biomarker for CC.Functionally,CFI silencing inhibited CC cell proliferation,migration,invasion,and tumor growth.Mechanistically,CFI knockdown downregulated the expression of key glycolysis-related proteins(glucose transporter type 1,hexokinase 2,lactate dehydrogenase A,and pyruvate kinase M2)and the Wnt pathway-related proteins(β-catenin and c-Myc).Further investigation indicated that CFI knockdown inhibited glycolysis in CC by blocking the Wnt/β-catenin/c-Myc pathway.CONCLUSION The findings of the present study demonstrate that CFI plays a crucial role in CC development by influencing glycolysis and the Wnt/β-catenin/c-Myc pathway,indicating that it could serve as a promising target for therapeutic intervention in CC.

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-15a-5p Regulates Oxaliplatin Resistance in Colorectal Cancer through the Wnt/β-catenin Pathway

[Objectives]To explore the effects and mechanism of miR-15a-5p on oxaliplatin resistance in colorectal cancer HCT116/L cells.[Methods]The expression of miR-15a-5p in colorectal cancer sensitive cells HCT116 and resistant cells HCT116/L was detected by RT-qPCR method;the effect of oxaliplatin on the proliferation of HCT116 and HCT116/L cells was detected by MTT,and its IC_(50) and drug resistance fold of HCT116/L cells were calculated;the expressions of Wnt3a,β-Catenin and P-gp in HCT116 and HCT116/L cells were detected by Western Blot method.HCT-116/L cells were divided into 5 groups:blank control group HCT116/L,control group 1 transfected with miR-15a-5p mimics NC,experimental group 1 transfected with miR-15a-5p mimics,control group 2 transfected with miR-15a-5p inhibitor NC,and experimental group 2 transfected with miR-15a-5p inhibitor.The expression of miR-15a-5p in each group was detected by RT-qPCR method and the transfection efficiency was detected.The effect of different concentrations of oxaliplatin on the proliferation of cells in each group after transfection was detected by MTT and the half inhibitory concentration(50%inhibiting concentration,IC_(50))was calculated.The expressions of Wnt3a,β-catenin and P-gp in each group after transfection were detected by Western Blot method,and the expressions of Wnt3a,β-catenin and MDR1 mRNA in each group after transfection were detected by RT-qPCR method.[Results](i)RT-qPCR results showed that the expression of miR-15a-5p in HCT116/L cells was(0.16±0.05)significantly lower than that in HCT116 cells(P<0.05).(ii)The IC50 of oxaliplatin for HCT-116 cells and HCT116/L cells detected by MTT method were(13.51±2.62)and(103.08±12.29)μg/mL,respectively.The drug resistance index of HCT116/L was 7.63.(iii)Western Blot results showed that the expressions of Wnt3a,β-catenin and P-gp in HCT116/L cells were significantly higher than those in HCT-116 cells(P<0.05).(iv)After successful transfection,the IC50 of miR-15a-5p mimics group to oxaliplatin decreased to(40.78±2.47)μg/mL by MTT method,and its sensitivity to the drug was significantly improved compared with the control group.Western Blot results showed that the relative expressions of P-gp,Wnt3a andβ-catenin were significantly down-regulated(all P<0.05);RT-qPCR results showed that the relative expressions of MDR1,Wnt3a andβ-catenin mRNA were significantly down-regulated(all P<0.05).(v)After successful transfection,the expression of miR-15a-5p in the miR-15a-5p inhibitor transfection group was(0.38±0.04);MTT results showed that its IC50 for oxaliplatin was up-regulated to(132.77±7.97)μg/mL,and its sensitivity to chemotherapy drugs was significantly lower than that of the control group;Western Blot results showed that the relative expressions of P-gp,Wnt3a andβ-catenin were significantly up-regulated(all P<0.05);RT-qPCR results showed that the relative expressions of MDR1,Wnt3a andβ-catenin mRNA were significantly up-regulated(all P<0.05).[Conclusions]Up-regulation of miR-15a-5p expression can reverse the resistance of HCT116/L cell line to oxaliplatin.Up-regulation or down-regulation of miR-15a-5p will affect the expression of P-gp and Wnt/β-catenin signaling pathway-related proteins,suggesting that the mechanism of miR-15a-5p reversal of drug resistance may be related to the inhibition of Wnt/β-catenin pathway,thereby down-regulating the expression of P-gp.

肝细胞肝癌中Wnt5a、β-catenin和E-cadherin蛋白的表达及临床意义

目的初步探讨Wnt5 a、β-catenin和E-cadherin的表达与肝细胞肝癌(HCC)发生、发展和转移的关系。方法采用免疫组化ABC及EnVision方法,检测30例HCC(包括10例尸检样本)癌旁、癌组织中Wnt5a、β-catenin和E-cadherin的表达,结合Ki-67进行统计学分析。结果对比于癌旁组织,83%(25/30)的HCC癌组织内Wnt5a蛋白低或缺失表达(P<0.001),其异常表达还相关于高的肿瘤分期(P=0.010)、高的Ki-67指数(P=0.013),以及β-catenin(P=0.025)和E-cadherin(P=0.003)的膜下降表达。癌组织中β-catenin膜下降表达相关于E-cadherin的膜下降表达(P=0.047)。80%(24/30)HCC癌组织中E-cadherin膜下降表达,相关于高的肿瘤分期(P=0.048)、高的K i-67指数(P=0.042)和尸检组肿瘤转移的发生(P=0.033)。结论Wnt5a缺失表达是HCC进展中的频发事件,并相关于β-catenin和E-cadherin的异常表达。Wnt5a在HCC中发挥肿瘤抑制基因样的作用,该蛋白极可能是一个有用的HCC预后不良指标。