Objectives:Deubiquitinase OTUB2 plays a critical role in the progression of various tumors.However,its specific role in triple-negative breast cancer(TNBC)remains unclear.This study aims to elucidate the biological fu...Objectives:Deubiquitinase OTUB2 plays a critical role in the progression of various tumors.However,its specific role in triple-negative breast cancer(TNBC)remains unclear.This study aims to elucidate the biological function of OTUB2 in TNBC and uncover the underlying mechanisms.Methods:First,we found that the expression of OTUB2 was upregulated in TNBC by bioinformatics analysis,we then validated its expression in TNBC tissues and cells using immunohistochemistry(IHC)and qPCR and plotted the survival curves by Kaplan-Meier method.Gene set enrichment analysis(GSEA)suggested that OTUB2 may be involved in tumor proliferation and metastasis.Further functional assays,including Cell Counting Kit-8(CCK-8),colony formation,Transwell,and wound healing assays,were performed to assess the effects of OTUB2 overexpression and knockdown on TNBC cell proliferation and migration.Additionally,UbiBrowser 2.0 was used to identify OTUB2 substrate proteins and western blotting was conducted to clarify the molecular mechanisms involved.Results:Our results demonstrated that OTUB2 expression was elevated in TNBC and associated with poor prognosis.Overexpression of OTUB2 enhanced the proliferation and migration of TNBC cells,while its knockdown inhibited these processes.Moreover,OTUB2 stabilized tumor necrosis factor receptor-associated factor 6(TRAF6)by deubiquitinating it,leading to activation of the protein kinase B(AKT)pathway.Conclusions:OTUB2 exerts its promoting effects on the progression of TNBC by activating the TRAF6/AKT pathway.展开更多
BACKGROUND Glomerular endothelial cell(GENC)injury is a characteristic of early-stage diabetic nephropathy(DN),and the investigation of potential therapeutic targets for preventing GENC injury is of clinical importanc...BACKGROUND Glomerular endothelial cell(GENC)injury is a characteristic of early-stage diabetic nephropathy(DN),and the investigation of potential therapeutic targets for preventing GENC injury is of clinical importance.AIM To investigate the role ofβ-arrestin-2 in GENCs under DN conditions.METHODS Eight-week-old C57BL/6J mice were intraperitoneally injected with streptozotocin to induce DN.GENCs were transfected with plasmids containing siRNA-β-arrestin-2,shRNA-activating transcription factor 6(ATF6),pCDNA-β-arrestin-2,or pCDNA-ATF6.Additionally,adeno-associated virus(AAV)containing shRNA-β-arrestin-2 was administered via a tail vein injection in DN mice.RESULTS The upregulation ofβ-arrestin-2 was observed in patients with DN as well as in GENCs from DN mice.Knockdown ofβ-arrestin-2 reduced apoptosis in high glucose-treated GENCs,which was reversed by the overexpression of ATF6.Moreover,overexpression ofβ-arrestin-2 Led to the activation of endoplasmic reticulum(ER)stress and the apoptosis of GENCs which could be mitigated by silencing of ATF6.Furthermore,knockdown ofβ-arrestin-2 by the administration of AAV-shRNA-β-arrestin-2 alleviated renal injury in DN mice.CONCLUSION Knockdown ofβ-arrestin-2 prevents GENC apoptosis by inhibiting ATF6-mediated ER stress in vivo and in vitro.Consequently,β-arrestin-2 may represent a promising therapeutic target for the clinical management of patients with DN.展开更多
AIM To clarify the mechanisms involved in the critical endoplasmic reticulum(ER) stress initiating unfolded protein response pathway modified by melatonin.METHODS Hepatoma cells, Hep G2, were cultured in vitro. Flow c...AIM To clarify the mechanisms involved in the critical endoplasmic reticulum(ER) stress initiating unfolded protein response pathway modified by melatonin.METHODS Hepatoma cells, Hep G2, were cultured in vitro. Flow cytometry and TUNEL assay were used to measure Hep G2 cell apoptosis. Western blotting and quantitative reverse transcription-polymerase chain reaction methods were used to determine the protein and messenger RNA levels of ER stress and apoptosis related genes' expression, respectively. Tissue microarray construction from patients was verified by immunohistochemical analysis.RESULTS In the present study, we first identified that melatoninselectively blocked activating transcription factor 6(ATF-6) and then inhibited cyclooxygenase-2 (COX-2) expression, leading to enhanced liver cancer cell apoptosis under ER stress condition. Dramatically increased CCAAT-enhancer-binding protein homologous protein level, suppressed COX-2 and decreased Bcl-2/Bax ratio by melatonin or ATF-6 si RNA contributed the enhanced Hep G2 cell apoptosis under tunicamycin (an ER stress inducer) stimulation. In clinical hepatocellular carcinoma patients, the close relationship between ATF-6 and COX-2 was further confirmed.CONCLUSION These findings indicate that melatonin as a novel selective ATF-6 inhibitor can sensitize human hepatoma cells to ER stress inducing apoptosis.展开更多
Background and aim： The Krueppel-like transcription factor KLF6 is a novel tumor-suppressor gene. It was inactivated in human prostate cancer and other tumors tissue, as the result of frequent mutation and loss of he...Background and aim： The Krueppel-like transcription factor KLF6 is a novel tumor-suppressor gene. It was inactivated in human prostate cancer and other tumors tissue, as the result of frequent mutation and loss of heterozygosity （LOH）. However, there is no data reporting the levels of KLF6 both mRNA and protein in hepatocellular carcinomas （HCCs）. We therefore detected mutations and expression of KLF6 in HCC tissues and further observed the effect of it on cell growth in HCC cell lines. Methods： We analyzed the exon-2 ofKLF6 gene by direct DNA sequencing, and detected the expression of KLF6 by RT-PCR and Western blot in 23 HCC tissues and corresponding nontumorous tissues. Loss of growth suppressive effect of the HCC-derived KLF6 mutant was characterized by in vitro growth curves plotted, flow cytometry and Western blotting. Results： KLF6 mutations were found in 2 of 23 HCC tissues and one of mutations was missense. Expression ofKLF6 mRNA or protein was down-regulated in 8 （34.7%） or 9 （39.1%） of 23 HCC tissues. Wild-type KLF6 （wtKLF6） inhibited cellular proliferation and prolonged G1 -S transition by inducing the expression of p21WAF 1 following stable transfection into cultured HepG2 cells, but tumor-derived KLF6 mutant （mKLF6） had no effects. Conclusion： Our findings suggest that KLF6 may be involved in pathogenesis of HCC.展开更多
Objective To investigate the role of TNF receptor-associated factor 2 (TRAF-2) and TRAF6 in CD40-induced nuclear factor-κB (NF-κB) signaling pathway and whether CD40 signaling requires TRAF2. Methods Human B cell li...Objective To investigate the role of TNF receptor-associated factor 2 (TRAF-2) and TRAF6 in CD40-induced nuclear factor-κB (NF-κB) signaling pathway and whether CD40 signaling requires TRAF2. Methods Human B cell lines were transfected with plasmids expressing wild type TRAF2 or dominant negative TRAF2,TRAF2-shRNA,or TRAF6-shRNA. The activation of NF-κB was detected by Western blot,kinase assay,transfactor enzyme-linked immunosorbent assay (ELISA),and fluorescence resonance energy transfer (FRET). Analysis of the role of TRAF-2 and TRAF-6 in CD40-mediated NF-κB activity was examined following stimulation with recombinant CD154. Results TRAF2 induced activity of IκB-kinases (IKKα,IKKi/ε),phosphorylation of IκBα,as well as nuclear translocation and phosphorylation of p65/RelA. In contrast,TRAF6 strongly induced NF-κB activation and nuclear translocation of p65 as well as p50 and c-Rel. Engagement of CD154-induced nuclear translocation of p65 was inhibited by a TRAF6-shRNA,but conversely was enhanced by a TRAF2-shRNA. Examination of direct interactions between CD40 and TRAFs by FRET documented that both TRAF2 and TRAF6 directly interacted with CD40. However,the two TRAFs competed for CD40 binding. Conclusions These results indicate that TRAF2 can signal in human B cells,but it is not essential for CD40-mediated NF-κB activation. Moreover,TRAF2 can compete with TRAF6 for CD40 binding,and thereby limit the capacity of CD40 engagement to induce NF-κB activation.展开更多
This study examined the mechanism of the inhibitory effect of parthenolide（PTL） on the activity of NF-κB in multiple myeloma（MM）. Human multiple myeloma cell line RPMI 8226 cells were treated with or without diff...This study examined the mechanism of the inhibitory effect of parthenolide（PTL） on the activity of NF-κB in multiple myeloma（MM）. Human multiple myeloma cell line RPMI 8226 cells were treated with or without different concentrations of PTL for various time periods, and then MTT assay was used to detect cell proliferation. Cell cycle and apoptosis were flow cytometrically detected. The level of protein ubiquitination was determined by using immunoprecipitation. Western blotting was employed to measure the level of total protein ubiquitination, the expression of IκB-α in cell plasma and the content of p65 in nucleus. The content of p65 in nucleus before and after PTL treatment was also examined with immunofluorescence. Exposure of RPMI 8226 cells to PTL attenuated the level of ubiquitinated Nemo, increased the expression of IκB-α and reduced the level of p65 in nucleus, finally leading to the decrease of the activity of NF-κB. PTL inhibited cell proliferation, induced apoptosis and blocked cell cycle. Furthermore, the levels of ubiquitinated tumor necrosis factor receptor-associated factor 6（TRAF6） and total proteins were decreased after PTL treatment. By using Autodock software package, we predicted that PTL could bind to TRAF6 directly and tightly. Taken together, our findings suggest that PTL inhibits the activation of NF-κB signaling pathway via directly binding with TRAF6, thereby suppressing MM cell proliferation and inducing apoptosis.展开更多
BACKGROUND： The tumor necrosis factor recepter associated factor （TRAF） 6 is an important intracellular adapter protein that plays a pivotal role in activating multiple inflammatory and immune related processes ind...BACKGROUND： The tumor necrosis factor recepter associated factor （TRAF） 6 is an important intracellular adapter protein that plays a pivotal role in activating multiple inflammatory and immune related processes induced by cytokines. TRAF6 represents a strong candidate susceptibility factor for sepsis. We investigated whether polymorphisms at the TRAF6 gene are associated with the susceptibility to and severity of sepsis.METHODS： A hospital-based case-control study was conducted with 255 patients with sepsis and 260 controls who were recruited from Zhengzhou, China. Haplotype tagging single nucleotide polymorphisms （htSNPs） were selected from the HapMap database and genotyped using the SNPstream genotyping platform. The associations with the susceptibility and disease severity of sepsis were estimated by logistic regression, and adjusted for age, sex, smoking, drinking, chronic diseases status, APACHEII score and critical illness status.RESULTS： A total of 13 TRAF6 SNPs were tagged by 7 htSNPs. Five htSNPs （rs5030490, rs5030411, rs5030416, rs5030445 and rs3740961） were genotyped in the case control study. Genotype frequencies of the htSNPs were conformed to the Hardy-Weinberg equilibrium in both patients and controls. No significant association was found between the 5 htSNPs and the susceptibility to and severity of sepsis. Compared with the main haplotype -11120A/-10688T/-9423A/805G/12967G, no certain haplotype was associated with the signi？ cantly susceptibility to or severity of sepsis.CONCLUSION： TRAF6 gene polymorphisms might not play a major role in mediating the susceptibility to and severity of sepsis in the Chinese population. A larger population-based case-control study is warranted.展开更多
An enriched environment is used as a behavio ral intervention therapy that applies sensory,motor,and social stimulation,and has been used in basic and clinical research of va rious neurological diseases.In this study,...An enriched environment is used as a behavio ral intervention therapy that applies sensory,motor,and social stimulation,and has been used in basic and clinical research of va rious neurological diseases.In this study,we established mouse models of photothrombotic stroke and,24 hours later,raised them in a standard,enriched,or isolated environment for 4 weeks.Compared with the mice raised in a standard environment,the cognitive function of mice raised in an enriched environment was better and the pathological damage in the hippocampal CA1 region was remarkably alleviated.Furthermore,protein expression levels of tumor necrosis factor receptor-associated factor 6,nuclear factorκB p65,interleukin-6,and tumor necrosis factorα,and the mRNA expression level of tumor necrosis factor receptor-associated factor 6 were greatly lower,while the expression level of miR-146a-5p was higher.Compared with the mice raised in a standard environment,changes in these indices in mice raised in an isolated environment were opposite to mice raised in an enriched environment.These findings suggest that different living environments affect the hippocampal inflammatory response and cognitive function in a mouse model of stro ke.An enriched environment can improve cognitive function following stroke through up-regulation of miR-146a-5p expression and a reduction in the inflammatory response.展开更多
Kruppel-like factor 6 (KLF6) was reported as tumor suppressor in multiple cancers. However, loss of chromosomal locus spanning KLF6 is relatively infrequent in previous published studies. To explore the role of KLF6 i...Kruppel-like factor 6 (KLF6) was reported as tumor suppressor in multiple cancers. However, loss of chromosomal locus spanning KLF6 is relatively infrequent in previous published studies. To explore the role of KLF6 in hepatocellular carcinoma (HCC), we examined the gene for expression change, loss of heterozygosity (LOH) and mutation in 26 HCC samples. The expression levels of KLF6 were significantly down-regulated in HCCs, as detected by qRT-PCR. LOH occurred in 11 (52%) of 21 tumors, and all the samples with LOH showed KLF6 down-regulation. The mutational frequency was 24%, and sequence changes located in activation domain of KLF6. Furthermore, MTT assay showed a significant antiproliferative effect of the wt KLF6 transfected in HepG2 hepatoblastoma cells. Fluorescence-activated cell sorting analysis revealed that KLF6 could induce apoptosis. These findings indicate that deregulation of KLF6, together with genetic abnormalities of allelic imbalance and mutations, may play a role in HCC pathogenesis.展开更多
BACKGROUND Extensive evidence has illustrated the promotive role of integrin binding sialoprotein(IBSP)in the progression of multiple cancers.However,little is known about the functions of IBSP in gastric cancer(GC)pr...BACKGROUND Extensive evidence has illustrated the promotive role of integrin binding sialoprotein(IBSP)in the progression of multiple cancers.However,little is known about the functions of IBSP in gastric cancer(GC)progression.AIM To investigate the mechanism underlying the regulatory effects of IBSP in GC progression,and the relationship between IBSP and cleavage and polyadenylation factor 6(CPSF6)in this process.METHODS The mRNA and protein expression of relevant genes were assessed through realtime quantitative polymerase chain reaction and Western blot,respectively.Cell viability was evaluated by Cell Counting Kit-8 assay.Cell invasion and migration were evaluated by Transwell assay.Pyroptosis was measured by flow cytometry.The binding between CPSF6 and IBSP was confirmed by luciferase reporter and RNA immunoprecipitation(RIP)assays.RESULTS IBSP exhibited higher expression in GC tissues and cell lines than in normal tissues and cell lines.IBSP knockdown suppressed cell proliferation,migration,and invasion but facilitated pyroptosis.In the exploration of the regulatory mechanism of IBSP,potential RNA binding proteins for IBSP were screened with catRAPID omics v2.0.The RNA-binding protein CPSF6 was selected due to its higher expression in stomach adenocarcinoma.Luciferase reporter and RIP assays revealed that CPSF6 binds to the 3’-untranslated region of IBSP and regulates its expression.Knockdown of CPSF6 inhibited cell proliferation,migration,and invasion but boosted pyroptosis.Through rescue assays,it was uncovered that the retarded GC progression mediated by CPSF6 knockdown was reversed by IBSP overexpression.CONCLUSION Our study highlighted the vital role of the CPSF6/IBSP axis in GC,suggesting that IBSP might be an effective biotarget for GC treatment.展开更多
AIM: To investigate the expression of insulin-like growth factor binding protein-6(IGFBP-6) in a proliferative vitreoretinopathy(PVR) model and its effects on proliferation and migration in retinal pigment epithelial(...AIM: To investigate the expression of insulin-like growth factor binding protein-6(IGFBP-6) in a proliferative vitreoretinopathy(PVR) model and its effects on proliferation and migration in retinal pigment epithelial(RPE) cells. ·METHODS: A PVR Wistar rat model was established by the intravitreal injection of RPE-J cells combined with platelet-rich plasma(PRP). The expression levels of IGFBP-6 were tested by ELISA. ARPE-19 cell proliferation was evaluated by the MTS method,and cell migration was evaluated by wound healing assays. ·RESULTS: The success rate of the PVR model was 89.3%(25/28). IGFBP-6 was expressed at higher levels in the vitreous,serum and retina of rats experiencing advanced PVR(grade 3) than in the control group(vitreous: 152.80 ±15.08ng/mL vs 105.44 ±24.81ng/mL,P 】 0.05; serum: 93.48 ±9.27ng/mL vs 80.59 ±5.20ng/mL,P 【 0.05; retina: 3.02±0.38ng/mg vs 2.05±0.53ng/mg,P 【0.05). In vitro,IGFBP-6(500ng/mL) inhibited the IGF-II(50ng/mL) induced ARPE-19 cell proliferation(OD value at 24h: from 1.38±0.05 to 1.30±0.02; 48h: from 1.44±0.06 to 1.35± 0.05). However,it did not affect basal or VEGF-,TGF-β-and PDGF-induced cell proliferation. IGFBP-6(500ng/ml) reduced the IGF-II(50ng/mL)-induced would healing rate [24h: from(43.91 ±3.85)% to(29.76 ±2.49)%; 48h: from(66.09±1.67)% to(59.88±3.43)%]. ·CONCLUSION: Concentrations of IGFBP-6 increased in the vitreous,serum,and retinas only in advanced PVR in vivo. IGFBP-6 also inhibited IGF-II-induced cell proliferation in a not dose or time dependent manner and migration. IGFBP-6 participates in the development of PVR and might play a protective role in PVR.展开更多
基金supported by the National Natural Science Foundation of China(No.82373380,Xinhua Xie).
文摘Objectives:Deubiquitinase OTUB2 plays a critical role in the progression of various tumors.However,its specific role in triple-negative breast cancer(TNBC)remains unclear.This study aims to elucidate the biological function of OTUB2 in TNBC and uncover the underlying mechanisms.Methods:First,we found that the expression of OTUB2 was upregulated in TNBC by bioinformatics analysis,we then validated its expression in TNBC tissues and cells using immunohistochemistry(IHC)and qPCR and plotted the survival curves by Kaplan-Meier method.Gene set enrichment analysis(GSEA)suggested that OTUB2 may be involved in tumor proliferation and metastasis.Further functional assays,including Cell Counting Kit-8(CCK-8),colony formation,Transwell,and wound healing assays,were performed to assess the effects of OTUB2 overexpression and knockdown on TNBC cell proliferation and migration.Additionally,UbiBrowser 2.0 was used to identify OTUB2 substrate proteins and western blotting was conducted to clarify the molecular mechanisms involved.Results:Our results demonstrated that OTUB2 expression was elevated in TNBC and associated with poor prognosis.Overexpression of OTUB2 enhanced the proliferation and migration of TNBC cells,while its knockdown inhibited these processes.Moreover,OTUB2 stabilized tumor necrosis factor receptor-associated factor 6(TRAF6)by deubiquitinating it,leading to activation of the protein kinase B(AKT)pathway.Conclusions:OTUB2 exerts its promoting effects on the progression of TNBC by activating the TRAF6/AKT pathway.
基金Supported by Key Research and Development Program of Shandong Province,No.2021CXGC011101Special Fund for Taishan Scholars Project,No.tsqn202211324+2 种基金National Natural Science Foundation of China,No.81900669Natural Science Foundation of Shandong Province,China,No.ZR2018PH007the Multidisciplinary Innovation Center for Nephrology of the Second Hospital of Shandong University.
文摘BACKGROUND Glomerular endothelial cell(GENC)injury is a characteristic of early-stage diabetic nephropathy(DN),and the investigation of potential therapeutic targets for preventing GENC injury is of clinical importance.AIM To investigate the role ofβ-arrestin-2 in GENCs under DN conditions.METHODS Eight-week-old C57BL/6J mice were intraperitoneally injected with streptozotocin to induce DN.GENCs were transfected with plasmids containing siRNA-β-arrestin-2,shRNA-activating transcription factor 6(ATF6),pCDNA-β-arrestin-2,or pCDNA-ATF6.Additionally,adeno-associated virus(AAV)containing shRNA-β-arrestin-2 was administered via a tail vein injection in DN mice.RESULTS The upregulation ofβ-arrestin-2 was observed in patients with DN as well as in GENCs from DN mice.Knockdown ofβ-arrestin-2 reduced apoptosis in high glucose-treated GENCs,which was reversed by the overexpression of ATF6.Moreover,overexpression ofβ-arrestin-2 Led to the activation of endoplasmic reticulum(ER)stress and the apoptosis of GENCs which could be mitigated by silencing of ATF6.Furthermore,knockdown ofβ-arrestin-2 by the administration of AAV-shRNA-β-arrestin-2 alleviated renal injury in DN mice.CONCLUSION Knockdown ofβ-arrestin-2 prevents GENC apoptosis by inhibiting ATF6-mediated ER stress in vivo and in vitro.Consequently,β-arrestin-2 may represent a promising therapeutic target for the clinical management of patients with DN.
基金grants from the National Natural Science Foundation of China,No.81572430 and No.81272739
文摘AIM To clarify the mechanisms involved in the critical endoplasmic reticulum(ER) stress initiating unfolded protein response pathway modified by melatonin.METHODS Hepatoma cells, Hep G2, were cultured in vitro. Flow cytometry and TUNEL assay were used to measure Hep G2 cell apoptosis. Western blotting and quantitative reverse transcription-polymerase chain reaction methods were used to determine the protein and messenger RNA levels of ER stress and apoptosis related genes' expression, respectively. Tissue microarray construction from patients was verified by immunohistochemical analysis.RESULTS In the present study, we first identified that melatoninselectively blocked activating transcription factor 6(ATF-6) and then inhibited cyclooxygenase-2 (COX-2) expression, leading to enhanced liver cancer cell apoptosis under ER stress condition. Dramatically increased CCAAT-enhancer-binding protein homologous protein level, suppressed COX-2 and decreased Bcl-2/Bax ratio by melatonin or ATF-6 si RNA contributed the enhanced Hep G2 cell apoptosis under tunicamycin (an ER stress inducer) stimulation. In clinical hepatocellular carcinoma patients, the close relationship between ATF-6 and COX-2 was further confirmed.CONCLUSION These findings indicate that melatonin as a novel selective ATF-6 inhibitor can sensitize human hepatoma cells to ER stress inducing apoptosis.
文摘Background and aim： The Krueppel-like transcription factor KLF6 is a novel tumor-suppressor gene. It was inactivated in human prostate cancer and other tumors tissue, as the result of frequent mutation and loss of heterozygosity （LOH）. However, there is no data reporting the levels of KLF6 both mRNA and protein in hepatocellular carcinomas （HCCs）. We therefore detected mutations and expression of KLF6 in HCC tissues and further observed the effect of it on cell growth in HCC cell lines. Methods： We analyzed the exon-2 ofKLF6 gene by direct DNA sequencing, and detected the expression of KLF6 by RT-PCR and Western blot in 23 HCC tissues and corresponding nontumorous tissues. Loss of growth suppressive effect of the HCC-derived KLF6 mutant was characterized by in vitro growth curves plotted, flow cytometry and Western blotting. Results： KLF6 mutations were found in 2 of 23 HCC tissues and one of mutations was missense. Expression ofKLF6 mRNA or protein was down-regulated in 8 （34.7%） or 9 （39.1%） of 23 HCC tissues. Wild-type KLF6 （wtKLF6） inhibited cellular proliferation and prolonged G1 -S transition by inducing the expression of p21WAF 1 following stable transfection into cultured HepG2 cells, but tumor-derived KLF6 mutant （mKLF6） had no effects. Conclusion： Our findings suggest that KLF6 may be involved in pathogenesis of HCC.
基金Supported by Key Projects of the National Science & Technology Pillar Program in the Eleventh Five-year Plan Period (2008-BAI59B02)
文摘Objective To investigate the role of TNF receptor-associated factor 2 (TRAF-2) and TRAF6 in CD40-induced nuclear factor-κB (NF-κB) signaling pathway and whether CD40 signaling requires TRAF2. Methods Human B cell lines were transfected with plasmids expressing wild type TRAF2 or dominant negative TRAF2,TRAF2-shRNA,or TRAF6-shRNA. The activation of NF-κB was detected by Western blot,kinase assay,transfactor enzyme-linked immunosorbent assay (ELISA),and fluorescence resonance energy transfer (FRET). Analysis of the role of TRAF-2 and TRAF-6 in CD40-mediated NF-κB activity was examined following stimulation with recombinant CD154. Results TRAF2 induced activity of IκB-kinases (IKKα,IKKi/ε),phosphorylation of IκBα,as well as nuclear translocation and phosphorylation of p65/RelA. In contrast,TRAF6 strongly induced NF-κB activation and nuclear translocation of p65 as well as p50 and c-Rel. Engagement of CD154-induced nuclear translocation of p65 was inhibited by a TRAF6-shRNA,but conversely was enhanced by a TRAF2-shRNA. Examination of direct interactions between CD40 and TRAFs by FRET documented that both TRAF2 and TRAF6 directly interacted with CD40. However,the two TRAFs competed for CD40 binding. Conclusions These results indicate that TRAF2 can signal in human B cells,but it is not essential for CD40-mediated NF-κB activation. Moreover,TRAF2 can compete with TRAF6 for CD40 binding,and thereby limit the capacity of CD40 engagement to induce NF-κB activation.
基金supported by the National Natural Science Foundation of China(No.81272624)
文摘This study examined the mechanism of the inhibitory effect of parthenolide（PTL） on the activity of NF-κB in multiple myeloma（MM）. Human multiple myeloma cell line RPMI 8226 cells were treated with or without different concentrations of PTL for various time periods, and then MTT assay was used to detect cell proliferation. Cell cycle and apoptosis were flow cytometrically detected. The level of protein ubiquitination was determined by using immunoprecipitation. Western blotting was employed to measure the level of total protein ubiquitination, the expression of IκB-α in cell plasma and the content of p65 in nucleus. The content of p65 in nucleus before and after PTL treatment was also examined with immunofluorescence. Exposure of RPMI 8226 cells to PTL attenuated the level of ubiquitinated Nemo, increased the expression of IκB-α and reduced the level of p65 in nucleus, finally leading to the decrease of the activity of NF-κB. PTL inhibited cell proliferation, induced apoptosis and blocked cell cycle. Furthermore, the levels of ubiquitinated tumor necrosis factor receptor-associated factor 6（TRAF6） and total proteins were decreased after PTL treatment. By using Autodock software package, we predicted that PTL could bind to TRAF6 directly and tightly. Taken together, our findings suggest that PTL inhibits the activation of NF-κB signaling pathway via directly binding with TRAF6, thereby suppressing MM cell proliferation and inducing apoptosis.
文摘BACKGROUND： The tumor necrosis factor recepter associated factor （TRAF） 6 is an important intracellular adapter protein that plays a pivotal role in activating multiple inflammatory and immune related processes induced by cytokines. TRAF6 represents a strong candidate susceptibility factor for sepsis. We investigated whether polymorphisms at the TRAF6 gene are associated with the susceptibility to and severity of sepsis.METHODS： A hospital-based case-control study was conducted with 255 patients with sepsis and 260 controls who were recruited from Zhengzhou, China. Haplotype tagging single nucleotide polymorphisms （htSNPs） were selected from the HapMap database and genotyped using the SNPstream genotyping platform. The associations with the susceptibility and disease severity of sepsis were estimated by logistic regression, and adjusted for age, sex, smoking, drinking, chronic diseases status, APACHEII score and critical illness status.RESULTS： A total of 13 TRAF6 SNPs were tagged by 7 htSNPs. Five htSNPs （rs5030490, rs5030411, rs5030416, rs5030445 and rs3740961） were genotyped in the case control study. Genotype frequencies of the htSNPs were conformed to the Hardy-Weinberg equilibrium in both patients and controls. No significant association was found between the 5 htSNPs and the susceptibility to and severity of sepsis. Compared with the main haplotype -11120A/-10688T/-9423A/805G/12967G, no certain haplotype was associated with the signi？ cantly susceptibility to or severity of sepsis.CONCLUSION： TRAF6 gene polymorphisms might not play a major role in mediating the susceptibility to and severity of sepsis in the Chinese population. A larger population-based case-control study is warranted.
基金financially the National Natural Science Foundation of China,No.82072533the China Postdoctoral Science Foundation,No.2017M621675+1 种基金Huxin Foundation of Jiangsu Key Laboratory of Zoonosis of China,No.HX2003Yangzhou Science and Technology Development Plan Project of China,No.YZ2020201(all to XW)。
文摘An enriched environment is used as a behavio ral intervention therapy that applies sensory,motor,and social stimulation,and has been used in basic and clinical research of va rious neurological diseases.In this study,we established mouse models of photothrombotic stroke and,24 hours later,raised them in a standard,enriched,or isolated environment for 4 weeks.Compared with the mice raised in a standard environment,the cognitive function of mice raised in an enriched environment was better and the pathological damage in the hippocampal CA1 region was remarkably alleviated.Furthermore,protein expression levels of tumor necrosis factor receptor-associated factor 6,nuclear factorκB p65,interleukin-6,and tumor necrosis factorα,and the mRNA expression level of tumor necrosis factor receptor-associated factor 6 were greatly lower,while the expression level of miR-146a-5p was higher.Compared with the mice raised in a standard environment,changes in these indices in mice raised in an isolated environment were opposite to mice raised in an enriched environment.These findings suggest that different living environments affect the hippocampal inflammatory response and cognitive function in a mouse model of stro ke.An enriched environment can improve cognitive function following stroke through up-regulation of miR-146a-5p expression and a reduction in the inflammatory response.
文摘Kruppel-like factor 6 (KLF6) was reported as tumor suppressor in multiple cancers. However, loss of chromosomal locus spanning KLF6 is relatively infrequent in previous published studies. To explore the role of KLF6 in hepatocellular carcinoma (HCC), we examined the gene for expression change, loss of heterozygosity (LOH) and mutation in 26 HCC samples. The expression levels of KLF6 were significantly down-regulated in HCCs, as detected by qRT-PCR. LOH occurred in 11 (52%) of 21 tumors, and all the samples with LOH showed KLF6 down-regulation. The mutational frequency was 24%, and sequence changes located in activation domain of KLF6. Furthermore, MTT assay showed a significant antiproliferative effect of the wt KLF6 transfected in HepG2 hepatoblastoma cells. Fluorescence-activated cell sorting analysis revealed that KLF6 could induce apoptosis. These findings indicate that deregulation of KLF6, together with genetic abnormalities of allelic imbalance and mutations, may play a role in HCC pathogenesis.
文摘BACKGROUND Extensive evidence has illustrated the promotive role of integrin binding sialoprotein(IBSP)in the progression of multiple cancers.However,little is known about the functions of IBSP in gastric cancer(GC)progression.AIM To investigate the mechanism underlying the regulatory effects of IBSP in GC progression,and the relationship between IBSP and cleavage and polyadenylation factor 6(CPSF6)in this process.METHODS The mRNA and protein expression of relevant genes were assessed through realtime quantitative polymerase chain reaction and Western blot,respectively.Cell viability was evaluated by Cell Counting Kit-8 assay.Cell invasion and migration were evaluated by Transwell assay.Pyroptosis was measured by flow cytometry.The binding between CPSF6 and IBSP was confirmed by luciferase reporter and RNA immunoprecipitation(RIP)assays.RESULTS IBSP exhibited higher expression in GC tissues and cell lines than in normal tissues and cell lines.IBSP knockdown suppressed cell proliferation,migration,and invasion but facilitated pyroptosis.In the exploration of the regulatory mechanism of IBSP,potential RNA binding proteins for IBSP were screened with catRAPID omics v2.0.The RNA-binding protein CPSF6 was selected due to its higher expression in stomach adenocarcinoma.Luciferase reporter and RIP assays revealed that CPSF6 binds to the 3’-untranslated region of IBSP and regulates its expression.Knockdown of CPSF6 inhibited cell proliferation,migration,and invasion but boosted pyroptosis.Through rescue assays,it was uncovered that the retarded GC progression mediated by CPSF6 knockdown was reversed by IBSP overexpression.CONCLUSION Our study highlighted the vital role of the CPSF6/IBSP axis in GC,suggesting that IBSP might be an effective biotarget for GC treatment.
文摘AIM: To investigate the expression of insulin-like growth factor binding protein-6(IGFBP-6) in a proliferative vitreoretinopathy(PVR) model and its effects on proliferation and migration in retinal pigment epithelial(RPE) cells. ·METHODS: A PVR Wistar rat model was established by the intravitreal injection of RPE-J cells combined with platelet-rich plasma(PRP). The expression levels of IGFBP-6 were tested by ELISA. ARPE-19 cell proliferation was evaluated by the MTS method,and cell migration was evaluated by wound healing assays. ·RESULTS: The success rate of the PVR model was 89.3%(25/28). IGFBP-6 was expressed at higher levels in the vitreous,serum and retina of rats experiencing advanced PVR(grade 3) than in the control group(vitreous: 152.80 ±15.08ng/mL vs 105.44 ±24.81ng/mL,P 】 0.05; serum: 93.48 ±9.27ng/mL vs 80.59 ±5.20ng/mL,P 【 0.05; retina: 3.02±0.38ng/mg vs 2.05±0.53ng/mg,P 【0.05). In vitro,IGFBP-6(500ng/mL) inhibited the IGF-II(50ng/mL) induced ARPE-19 cell proliferation(OD value at 24h: from 1.38±0.05 to 1.30±0.02; 48h: from 1.44±0.06 to 1.35± 0.05). However,it did not affect basal or VEGF-,TGF-β-and PDGF-induced cell proliferation. IGFBP-6(500ng/ml) reduced the IGF-II(50ng/mL)-induced would healing rate [24h: from(43.91 ±3.85)% to(29.76 ±2.49)%; 48h: from(66.09±1.67)% to(59.88±3.43)%]. ·CONCLUSION: Concentrations of IGFBP-6 increased in the vitreous,serum,and retinas only in advanced PVR in vivo. IGFBP-6 also inhibited IGF-II-induced cell proliferation in a not dose or time dependent manner and migration. IGFBP-6 participates in the development of PVR and might play a protective role in PVR.
