Effects of signal regulatory proteinα1 on proliferation of hepatocellular carcinoma: a preliminary study

BACKGROUND: Signal regulatory protein alpha1 (Sirpα1) is a negative regulatory factor, and inhibits receptor tyro- sine kinase-dependent cell proliferating signal. This study was undertaken to observe the effect of signal regulatory proteinα1 ( Sirpα1) on gankyrin, cyclin D1, CDK4 and Fas expression in Sk-hep1 mouse hepatoma carcinoma cell line. METHODS: BOSC 23 packed cells were respectively trans- fected by means of recombinated retrovirus including pLX- SN, pLXSN-Sirpα1 and pLXSN-Sirpα1Δ4Y2 with lipofec- tin, and various plasmid virus media (viral titer 2.1 × 106 CFU/ml) were collected and infected respectively in 80% confluent Sk-hepl cells. Transfected Sk-hep1 cells were se- lectively screened with G418 (1200 μg/ml), and Sk-hep1 cell lines transfected with various plasmids were obtained. The protein expressions of gankyrin, cyclin D1, CDK4 and Fas in various Sk-hep1 lines were determined by Western blotting. Various Sk-hep1 lines were recovered to culture with 10% fetal bovine serum at 12 hours and 24 hours after starving culture with free serum for 72 hours, and cells were collected to determine the percentage of S phase cells of proliferating cycle by flow cytometry. RESULTS: Sirpα1 transfection remarkably downregulated gankyrin and cyclin D1 expression. Sirpα1Δ4Y2 downregu- lation of gankyrin expression was greater than that of Sirpα1(P <0.05), but no significant effect of Sirpα1 and Sirpα1Δ4Y2 on CDK4 and Fas protein expression was ob- served in transfected Sk-hep1 lines (P >0.05). The per- centage of S phase cells significantly decreased in Sk-hep1 cells transfected with Sirpα1 and Sirpα1Δ4Y2 plasmids (vs pLXSN Sk-hep1, P <0.05). The percentage of S phase cells in various Sk-hep1 cells increased when recovering to culture with 10% fetal bovine serum at 12 hours, but the percentage of S phase cells in Sk-hep1 cells transfected with Sirpα1 was the lowest ( vs pLXSN and Sirpα1Δ4Y2 Sk- hepl, P<0.05). The percentage of S phase cells in trans- fected pLSXN Sk-hep1 cells was the largest (vs Sirpα1 and Sirpα1Δ4Y2 Sk-hepl, P <0. 05). There was no significant difference between the transfected Sirpα1 Sk-hepl cells and Sirpα1Δ4Y2 Sk-hep1 cells (P>0.05). CONCLUSIONS: Sirpα1 decreases gankyrin and cyclin D1 expression, and inhibits proliferation of liver carcinoma cells. It may be one of the forms for an Sirpα1 negative regulation of carcinogenesis and development of hepatocel- lular carcinoma.

Basic regulatory science behind drug substance and drug product specifications of monoclonal antibodies and other protein therapeutics

In this review,we focus on providing basics and examples for each component of the protein therapeutic specifications to interested pharmacists and biopharmaceutical scientists with a goal to strengthen understanding in regulatory science and compliance.Pharmaceutical specifications comprise a list of important quality attributes for testing,references to use for test procedures,and appropriate acceptance criteria for the tests,and they are set up to ensure that when a drug product is administered to a patient,its intended therapeutic benefits and safety can be rendered appropriately.Conformance of drug substance or drug product to the specifications is achieved by testing an article according to the listed tests and analytical methods and obtaining test results that meet the acceptance criteria.Quality attributes are chosen to be tested based on their quality risk,and consideration should be given to the merit of the analytical methods which are associated with the acceptance criteria of the specifications.Acceptance criteria are set forth primarily based on efficacy and safety profiles,with an increasing attention noted for patient-centric specifications.Discussed in this work are related guidelines that support the biopharmaceutical specification setting,how to set the acceptance criteria,and examples of the quality attributes and the analytical methods from 60 articles and 23 pharmacopeial monographs.Outlooks are also explored on process analytical technologies and other orthogonal tools which are on-trend in biopharmaceutical characterization and quality control.

Calmodulins and calmodulin-like proteins-mediated plant organellar calcium signaling networks under abiotic stress

Plant calmodulins(CaMs)and calmodulin-like proteins(CMLs)mediate Ca~(2+)signaling in response to abiotic stresses.Manipulation of this signaling in crops could increase stress tolerance.We review methods for detecting Ca~(2+)signals,regulatory roles of Ca Ms and CMLs,binding targets,and Ca~(2+)networks under abiotic stress in organelles.

Subtraction of liposome signals in cryo-EM structural determination of protein-liposome complexes

Reconstituting membrane proteins in liposomes and determining their structure is a common method for determining membrane protein structures using single-particle cryo-electron microscopy(cryo-EM).However,the strong signal of liposomes under cryo-EM imaging conditions often interferes with the structural determination of the embedded membrane proteins.Here,we propose a liposome signal subtraction method based on single-particle two-dimensional(2D)classification average images,aimed at enhancing the reconstruction resolution of membrane proteins.We analyzed the signal distribution characteristics of liposomes and proteins within the 2D classification average images of protein–liposome complexes in the frequency domain.Based on this analysis,we designed a method to subtract the liposome signals from the original particle images.After the subtraction,the accuracy of single-particle three-dimensional(3D)alignment was improved,enhancing the resolution of the final 3D reconstruction.We demonstrated this method using a PIEZO1-proteoliposome dataset by improving the resolution of the PIEZO1 protein.

Glucokinase regulatory protein rs780094 polymorphism is associated with type 2 diabetes mellitus, dyslipidemia, non-alcoholic fatty liver disease, and nephropathy

In this editorial,we comment on the article by Liu et al published in the recent issue of the World Journal of Diabetes(Relationship between GCKR gene rs780094 polymorphism and type 2 diabetes with albuminuria).Type 2 diabetes mellitus(T2DM)is a chronic disorder characterized by dysregulated glucose homeostasis.The persistent elevated blood glucose level in T2DM significantly increases the risk of developing severe complications,including cardiovascular disease,re-tinopathy,neuropathy,and nephropathy.T2DM arises from a complex interplay between genetic,epigenetic,and environmental factors.Global genomic studies have identified numerous genetic variations associated with an increased risk of T2DM.Specifically,variations within the glucokinase regulatory protein(GCKR)gene have been linked to heightened susceptibility to T2DM and its associated complications.The clinical trial by Liu et al further elucidates the role of the GCKR rs780094 polymorphism in T2DM and nephropathy development.Their findings demonstrate that individuals carrying the CT or TT genotype at the GCKR rs780094 locus are at a higher risk of developing T2DM with albuminuria compared to those with the CC genotype.These findings highlight the importance of genetic testing and risk assessment in T2DM to develop effective preventive strategies and personalized treatment plans.

Effects of invigorating-spleen and anticancer prescription on extracellular signal-regulated kinase/mitogen-activated protein kinase signaling pathway in colon cancer mice model

BACKGROUND Colon cancer(CC)is one of the most common malignant tumors in the gastrointestinal system.Overall,CC had the third highest incidence but the second highest mortality rate globally in 2020.Nowadays,CC is mainly treated with capecitabine chemotherapy regimen,supplemented by radiotherapy,immunotherapy and targeted therapy,but there are still limitations,so Chinese medicine plays an important role.AIM To investigate the effects of invigorating-spleen and anticancer prescription(ISAP)on body weight,tumor inhibition rate and expression levels of proteins in extracellular-signal-regulated kinase(ERK)/mitogen-activated protein kinase(MAPK)signaling pathway in CC mice model.METHODS The CC mice model were established and the mice were randomly divided into 5 groups,including the control group,capecitabine group,the low-dose,mediumdose and high-dose groups of ISAP,with 8 mice in each group,respectively.After 2 weeks of intervention,the body weight and tumor inhibition rate of mice were observed,and the expression of RAS,ERK,phosphorylated ERK(p-ERK),C-MYC and matrix metalloproteinase 2(MMP2)proteins in the tissues of tumors were detected.RESULTS Compared with the control group,the differences of body weight before and after treatment was much smaller in the groups of ISAP,with the smallest difference in the high-dose group of ISAP,while the capecitabine group had the greatest difference,indicating ISAP had a significant inhibiting effect on the growth of transplanted tumor in mice.The expression of RAS protein was decreased in the low-and medium-dose groups of ISAP,and the change of p-ERK was significant in the medium-and high-dose groups of ISAP.MMP2 protein expression was significantly decreased in both the low-dose and medium-dose groups of ISAP.There were no significant changes in ERK in the ISAP group compared to the capecitabine group,while RAS,MMP2,and C-MYC protein expression were reduced in the ISAP group.The expression level of C-MYC protein decreased after treated with ISAP,and the decrease was the most significant in the medium-dose group of ISAP.CONCLUSION ISAP has a potential inhibiting effect on transplanted tumor in mice,and could maintain the general conditions,physical strength and body weight of mice.The expression levels of RAS,p-ERK,MMP2 and c-myc were also decreased to a certain extent.By inhibiting the expression of upstream proteins,the expression levels of downstream proteins in ERK/MAPK signaling pathway were significantly decreased.Therefore,it can be concluded that ISAP may exert an anti-tumor effect by blocking the ERK/MAPK signaling pathway and inhibiting the expression of MMP2 and c-myc proteins.

Regulatory role of CREB/BDNF signaling pathway in acute sleep deprivation-induced anxiety-like behavior mice

Objective:To investigate the regulatory role of cyclic adenosine monophosphate responsive element binding protein(CREB)/brain-derived neurotrophic factor(BDNF)signaling pathway in acute sleep deprivation(SD)-induced anxiety-like behavior mice(SD group)to study the mechanism of anxiety-like behavior better.Methods:The SD chamber was used to deprive the mice of sleep,and the anxiety-like behavior of the mice was verified using an open field test(OFT),elevated plus maze(EPM),forced swim test(FST),and tail suspension test(TST).Finally,proteins were detected by Western blotting.Result:OFT showed that the active distance and the time of stay in the central area were significantly reduced(P<0.05).EPM showed that the time and number of open arms in the SD group were significantly lower than in the control group(P<0.05).The FST showed that the forced swimming immobility time of the SD group was significantly lower than that of the control(P<0.05).Moreover,the TST showed that the immobility time of the tail suspension experiment in the SD group was significantly higher than that in the control group(P<0.05).Conclusion:Acute SD can regulate anxiety-like behavior in mice through the CREB/BDNF signaling pathway.

Calcitriol attenuates liver fibrosis through hepatitis C virus nonstructural protein 3-transactivated protein 1-mediated TGF β1/Smad3 and NF-κB signaling pathways

BACKGROUND Hepatic fibrosis is a serious condition,and the development of hepatic fibrosis can lead to a series of complications.However,the pathogenesis of hepatic fibrosis remains unclear,and effective therapy options are still lacking.Our group identified hepatitis C virus nonstructural protein 3-transactivated protein 1(NS3TP1) by suppressive subtractive hybridization and bioinformatics analysis,but its role in diseases including hepatic fibrosis remains undefined.Therefore,additional studies on the function of NS3TP1 in hepatic fibrosis are urgently needed to provide new targets for treatment.AIM To elucidate the mechanism of NS3TP1 in hepatic fibrosis and the regulatory effects of calcitriol on NS3TP1.METHODS Twenty-four male C57BL/6 mice were randomized and separated into three groups,comprising the normal,fibrosis,and calcitriol treatment groups,and liver fibrosis was modeled by carbon tetrachloride(CCl4).To evaluate the level of hepatic fibrosis in every group,serological and pathological examinations of the liver were conducted.TGF-β1 was administered to boost the in vitro cultivation of LX-2 cells.NS3TP1,α-smooth muscle actin(α-SMA),collagen I,and collagen Ⅲ in every group were examined using a Western blot and real-time quantitative polymerase chain reaction.The activity of the transforming growth factor beta 1(TGFβ1)/Smad3 and NF-κB signaling pathways in each group of cells transfected with pcDNA-NS3TP1 or siRNA-NS3TP1 was detected.The statistical analysis of the data was performed using the Student’s t test.RESULTS NS3TP1 promoted the activation,proliferation,and differentiation of hepatic stellate cells(HSCs)and enhanced hepatic fibrosis via the TGFβ1/Smad3 and NF-κB signaling pathways,as evidenced by the presence of α-SMA,collagen I,collagen Ⅲ,p-smad3,and p-p65 in LX-2 cells,which were upregulated after NS3TP1 overexpression and downregulated after NS3TP1 interference.The proliferation of HSCs was lowered after NS3TP1 interference and elevated after NS3TP1 overexpression,as shown by the luciferase assay.NS3TP1 inhibited the apoptosis of HSCs.Moreover,both Smad3 and p65 could bind to NS3TP1,and p65 increased the promoter activity of NS3TP1,while NS3TP1 increased the promoter activity of TGFβ1 receptor I,as indicated by coimmunoprecipitation and luciferase assay results.Both in vivo and in vitro,treatment with calcitriol dramatically reduced the expression of NS3TP1.Calcitriol therapy-controlled HSCs activation,proliferation,and differentiation and substantially suppressed CCl4-induced hepatic fibrosis in mice.Furthermore,calcitriol modulated the activities of the above signaling pathways via downregulation of NS3TP1.CONCLUSION Our results suggest that calcitriol may be employed as an adjuvant therapy for hepatic fibrosis and that NS3TP1 is a unique,prospective therapeutic target in hepatic fibrosis.

Melatonin improves synapse development by PI3K/Akt signaling in a mouse model of autism spectrum disorder

Autism spectrum disorders are a group of neurodevelopmental disorders involving more than 1100 genes,including Ctnnd2 as a candidate gene.Ctnnd2knockout mice,serving as an animal model of autis m,have been demonstrated to exhibit decreased density of dendritic spines.The role of melatonin,as a neuro hormone capable of effectively alleviating social interaction deficits and regulating the development of dendritic spines,in Ctnnd2 deletion-induced nerve injury remains unclea r.In the present study,we discove red that the deletion of exon 2 of the Ctnnd2 gene was linked to social interaction deficits,spine loss,impaired inhibitory neurons,and suppressed phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt) signal pathway in the prefrontal cortex.Our findings demonstrated that the long-term oral administration of melatonin for 28 days effectively alleviated the aforementioned abnormalities in Ctnnd2 gene-knockout mice.Furthermore,the administration of melatonin in the prefro ntal cortex was found to improve synaptic function and activate the PI3K/Akt signal pathway in this region.The pharmacological blockade of the PI3K/Akt signal pathway with a PI3K/Akt inhibitor,wo rtmannin,and melatonin receptor antagonists,luzindole and 4-phenyl-2-propionamidotetralin,prevented the melatonin-induced enhancement of GABAergic synaptic function.These findings suggest that melatonin treatment can ameliorate GABAe rgic synaptic function by activating the PI3K/Akt signal pathway,which may contribute to the improvement of dendritic spine abnormalities in autism spectrum disorders.

Effects of Ginseng Protein on Gut Microbiota and BDNF/TrkB Signaling Pathway in Alzheimer s Disease Mice

[Objectives] To investigate the effects of ginseng protein on gut microbiota and BDNF/TrkB signaling pathway in Alzheimer s disease (AD) mice. [Methods] D-galactose/AlCl 3 co-induction was used to establish AD model, and mice were randomly divided into normal group 1, normal group 2, model group 1, model group 2, ginseng protein group, and microbiota transplantation group. Morris water maze experiment was used to evaluate learning and memory ability, and Western blot method was used to detect the expression of APP, p-Tau, BDNF, TrkB, p-TrkB proteins in brain tissue, and 16S rDNA was used to detect diversity of fecal microbiota. [Results] Ginseng protein and microbiota transplantation can shorten the escape latency of mice ( P <0.05), increase the number of crossing platforms ( P <0.05), reduce the expression of APP and p-Tau proteins in brain tissue ( P <0.05, P <0.01), increase the expression of BDNF, p-TrkB, p-TrkB/TrkB proteins ( P <0.05, P <0.01), and reduce the abundance of Alloprevotella, Ruminococcaceae _UCG-014, Prevotellaceae _UCG-001, and Ruminococcus _1 ( P <0.05, P <0.01). [Conclusions] The action mechanism of ginseng protein anti AD may be through regulating gut microbiota diversity and activating the BDNF/TrkB signaling pathway.

Osteopontin promotes gastric cancer progression via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

BACKGROUND Gastric cancer(GC)is one of the most common malignant tumors.Osteopontin(OPN)is thought to be closely related to the occurrence,metastasis and prognosis of many types of tumors.AIM To investigate the effects of OPN on the proliferation,invasion and migration of GC cells and its possible mechanism.METHODS The mRNA and protein expression of OPN in the GC cells were analyzed by realtime quantitative-reverse transcription polymerase chain reaction and western blotting,and observe the effect of varying degree expression OPN on the proliferation and other behaviors of GC.Next,the effects of OPN knockdown on GC cells migration and invasion were examined.The short hairpin RNA(shRNA)and negative control shRNA targeting OPN-shRNA were transfected into the cells according to the manufacturer’s instructions.Non transfected cells were classified as control in the identical transfecting process.24 h after RNA transfection cell proliferation activity was detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay,and cell invasiveness and migration were detected by Trans well assay.Meanwhile,the expression of protein kinase B(AKT),matrix metalloproteinase 2(MMP-2)and vascular endothelial growth factor(VEGF)in the human GC cell lines was detected by reverse transcription polymerase chain reaction and western blotting.RESULTS The results of this study revealed that OPN mRNA and protein expression levels were highly expressed in SGC-7901 cells.OPN knockdown by specific shRNA noticeably reduced the capabilities of proliferation,invasion and migration of SGC-7901 cells.Moreover,in the experiments of investigating the underlying mechanism,results showed that OPN knockdown could down-regulated the expression of MMP-2 and VEGF,it also decreased the phosphorylation of AKT.Meanwhile,the protein expression levels of MMP-2,VEGF and phosphorylated AKT was noticeable lower than that in control group in the GC cells after they were added to phosphatidylinositol-3-kinase(PI3K)inhibitor(LY294002).CONCLUSION These results suggested that OPN though PI3K/AKT/mammalian target of rapamycin signal pathway to upregulate MMP-2 and VEGF expression,which contribute SGC-7901 cells to proliferation,invasion and migration.Thus,our results demonstrate that OPN may serve as a novel prognostic biomarkers as well as a potential therapeutic targets for GC.

Barley Protein LFBEP-C1 from Lactiplantibacillus plantarum dy-1 Fermented Barley Extracts by Inhibiting Lipid Accumulation in a Caenorhabditis elegans Model

Objective This study aimed to investigate the lipid-lowering activity of LFBEP-C1 in high glucose-fed Caenorhabditis elegans(C.elegans).Methods In this study,the fermented barley protein LFBEP-C1 was prepared and tested for its potential anti-obesity effects on C.elegans.The worms were fed Escherichia coli OP50(E.coli OP50),glucose,and different concentrations of LFBEP-C1.Body size,lifespan,movement,triglyceride content,and gene expression were analyzed.The results were analyzed using ANOVA and Tukey's multiple comparison test.Results Compared with the model group,the head-swing frequency of C.elegans in the group of LFBEP-C1 at 20μg/mL increased by 33.88%,and the body-bending frequency increased by 27.09%.This indicated that LFBEP-C1 improved the locomotive ability of C.elegans.The average lifespan of C.elegans reached 13.55 days,and the body length and width of the C.elegans decreased after LFBEP-C1 intake.Additionally,LFBEP-C1 reduced the content of lipid accumulation and triglyceride levels.The expression levels of sbp-1,daf-2,and mdt-15 significantly decreased,while those of daf-16,tph-1,mod-1,and ser-4 significantly increased after LFBEP-C1 intake.Changes in these genes explain the signaling pathways that regulate lipid metabolism.Conclusion LFBEP-C1 significantly reduced lipid deposition in C.elegans fed a high-glucose diet and alleviated the adverse effects of a high-glucose diet on the development,lifespan,and exercise behavior of C.elegans.In addition,LFBEP-C1 regulated lipid metabolism mainly by mediating the expression of genes in the sterol regulatory element-binding protein,insulin,and 5-hydroxytryptamine signaling pathways.

PHD17 acts as a target of miR1320 to negatively control cold tolerance via JA-activated signaling in rice

Plant Homeo Domain(PHD)proteins are involved in diverse biological processes during plant growth.However,the regulation of PHD genes on rice cold stress response remains largely unknown.Here,we reported that PHD17 negatively regulated cold tolerance in rice seedlings as a cleavage target of miR1320.PHD17 expression was greatly induced by cold stress,and was down-regulated by miR1320 overexpression and up-regulated by miR1320 knockdown.Through 5'RACE and dual luciferase assays,we found that miR1320 targeted and cleaved the 3'UTR region of PHD17.PHD17 was a nuclearlocalized protein and acted as a transcriptional activator in yeast.PHD17 overexpression reduced cold tolerance of rice seedlings,while knockout of PHD17 increased cold tolerance,partially via the CBF cold signaling.By combining transcriptomic and physiological analyses,we demonstrated that PHD17 modulated ROS homeostasis and flavonoid accumulation under cold stress.K-means clustering analysis revealed that differentially expressed genes in PHD17 transgenic lines were significantly enriched in the jasmonic acid(JA)biosynthesis pathway,and expression of JA biosynthesis and signaling genes was verified to be affected by PHD17.Cold stress tests applied with MeJA or IBU(JA synthesis inhibitor)further suggested the involvement of PHD17 in JA-mediated cold signaling.Taken together,our results suggest that PHD17 acts downstream of miR1320 and negatively regulates cold tolerance of rice seedlings through JA-mediated signaling pathway.

Presence of a long nuclear-localization signal sequence in homeodomain transcription factor Nkx 1.2

Homeodomains,a 60-amino acid sequence encoded by 180 nucleotides,are highly conserved DNA-binding motifs that are present in a variety of transcription factors in species ranging from yeast to humans.The NKX proteins belong to the homeodomain(HD)-containing transcription factor family.They play vital roles in the regulation of morphogenesis.NKX1-2 is one member of the NKX subfamily.At present,information about its nuclear localization signal(NLS)sequence is limited.We studied the NLS sequence of zebrafish Nkx1.2 by introducing sequence changes such as deletion,mutation,and truncation,and identified an NLS motif(QNRRTKWKKQ)that is localized at the C-terminus of the homeodomain.Moreover,the deletion of two amino acid residues(RR)in this NLS motif prevents Nkx1.2 from entering the nucleus,indicating that the two amino acids are essential for Nkx1.2 nuclear localization.However,the NLS motif alone is unable to target cytoplasmic protein glutathione S-transferase(GST)to the nucleus.An intact homeodomain is necessary for mediating the complete nuclear transport of cytoplasmic protein.Unlike most nuclear import proteins with short NLS sequences,a long NLS is present in zebrafish Nkx1.2.We also demonstrated that the sequences of homeodomain of NKX1.2 are well conserved among different species.This study is informative to verify the function of the NKX1.2 protein.

Phosphorylated protein chip combined with artificial intelligence tools for precise drug screening

We have developed a protein array system,named"Phospho-Totum",which reproduces the phosphorylation state of a sample on the array.The protein array contains 1471 proteins from 273 known signaling pathways.According to the activation degrees of tyrosine kinases in the sample,the corresponding groups of substrate proteins on the array are phosphorylated under the same conditions.In addition to measuring the phosphorylation levels of the 1471 substrates,we have developed and performed the artificial intelligence-assisted tools to further characterize the phosphorylation state and estimate pathway activation,tyrosine kinase activation,and a list of kinase inhibitors that produce phosphorylation states similar to that of the sample.The Phospho-Totum system,which seamlessly links and interrogates the measurements and analyses,has the potential to not only elucidate pathophysiological mechanisms in diseases by reproducing the phosphorylation state of samples,but also be useful for drug discovery,particularly for screening targeted kinases for potential drug kinase inhibitors.

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.

IKIP downregulates THBS1/FAK signaling to suppress migration and invasion by glioblastoma cells

Background:Inhibitor of NF-κB kinase-interacting protein(IKIP)is known to promote proliferation of glioblastoma(GBM)cells,but how it affects migration and invasion by those cells is unclear.Methods:We compared levels of IKIP between glioma tissues and normal brain tissue in clinical samples and public databases.We examined the effects of IKIP overexpression and knockdown on the migration and invasion of GBM using transwell and wound healing assays,and we compared the transcriptomes under these different conditions to identify the molecular mechanisms involved.Results:Based on data from our clinical samples and from public databases,IKIP was overexpressed in GBM tumors,and its expression level correlated inversely with survival.IKIP overexpression in GBM cells inhibited migration and invasion in transwell and wound healing assays,whereas IKIP knockdown exerted the opposite effects.IKIP overexpression in GBM cells that were injected into mouse brain promoted tumor growth but inhibited tumor invasion of surrounding tissue.The effects of IKIP were associated with downregulation of THBS1 mRNA and concomitant inhibition of THBS1/FAK signaling.Conclusions:IKIP inhibits THBS1/FAK signaling to suppress migration and invasion of GBM cells.

Neural Wiskott-Aldrich syndrome protein(N-WASP)promotes distant metastasis in pancreatic ductal adenocarcinoma via activation of LOXL2

Pancreatic ductal adenocarcinoma(PDAC)is one of the most aggressive solid malignancies.A specific mechanism of its metastasis has not been established.In this study,we investigated whether Neural Wiskott-Aldrich syndrome protein(N-WASP)plays a role in distant metastasis of PDAC.We found that N-WASP is markedly expressed in clinical patients with PDAC.Clinical analysis showed a notably more distant metastatic pattern in the N-WASP-high group compared to the N-WASP-low group.N-WASP was noted to be a novel mediator of epithelialmesenchymal transition(EMT)via gene expression profile studies.Knockdown of N-WASP in pancreatic cancer cells significantly inhibited cell invasion,migration,and EMT.We also observed positive association of lysyl oxidase-like 2(LOXL2)and focal adhesion kinase(FAK)with the N-WASP-mediated response,wherein EMT and invadopodia function were modulated.Both N-WASP and LOXL2 depletion significantly reduced the incidence of liver and lung metastatic lesions in orthotopic mouse models of pancreatic cancer.These results elucidate a novel role for N-WASP signaling associated with LOXL2 in EMT and invadopodia function,with respect to regulation of intercellular communication in tumor cells for promoting pancreatic cancer metastasis.These findings may aid in the development of therapeutic strategies against pancreatic cancer.

Maintaining cholesterol homeostasis: Sterol regulatory element-binding proteins

The molecular mechanism of how hepatocytes maintain cholesterol homeostasis has become much more transparent with the discovery of sterol regulatory element binding proteins (SREBPs) in recent years. These membrane proteins aremembers of the basic helix-loop-helix-leucine zipper (bHLHZip) family of transcription factors. They activate the expression of at least 30 genes involved in the synthesis of cholesterol and lipids. SREBPs are synthesized as precursor proteins in the endoplasmic reticulum (ER), where they form a complex with another protein, SREBP cleavage activating protein (SCAP). The SCAP molecule contains a sterol sensory domain. In the presence of high cellular sterol concentrations SCAP confines SREBP to the ER. With low cellular concentrations, SCAP escorts SREBP to activation in the Golgi. There, SREBP undergoes two proteolytic cleavage steps to release the mature, biologically active transcription factor, nuclear SREBP (nSREBP). nSREBP translocates to the nucleus and binds to sterol response elements (SRE) in the promoter/enhancer regions of target genes. Additional transcription factors are required to activate transcription of these genes. Three different SREBPs are known, SREBPs-1a, -1c and -2. SREBP-1a and -1c are isoforms produced from a single gene by alternate splicing. SREBP-2 is encoded by a different gene and does not display any isoforms. It appears that SREBPs alone, in the sequence described above, can exert complete control over cholesterol synthesis, whereas many additional factors (hormones, cytokines, etc.) are required for complete control of lipid metabolism. Medicinal manipulation of the SREBP/SCAP system is expected to prove highly beneficial in the management of cholesterol-related disease.