目的旨在通过生物信息学分析结合实验验证,探讨PCBP1(Poly(rC)-Binding Protein 1)在胃癌组织中的表达特征及其临床意义,对其与铁死亡主要调控因子STUB1(STIP1 Homology and U-Box Containing Protein 1)的关系研究。并通过免疫组化实...目的旨在通过生物信息学分析结合实验验证,探讨PCBP1(Poly(rC)-Binding Protein 1)在胃癌组织中的表达特征及其临床意义,对其与铁死亡主要调控因子STUB1(STIP1 Homology and U-Box Containing Protein 1)的关系研究。并通过免疫组化实验验证PCBP1与STUB1在胃癌中的表达模式及其与临床病理特征的关系。为新型胃癌靶向治疗策略提供重要的理论支撑和潜在的干预靶点。方法从TIMER 2.0在线分析网站中获取PCBP1在胃癌及癌旁组织的基因表达数据。利用癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库中胃癌数据(STAD)进行KEGG通路富集分析,并揭示其潜在作用机制;在铁死亡调控通路中找出主要调控因子STUB1。随后,采用免疫组化法检测33例胃癌组织及对应癌旁组织中PCBP1和STUB1的表达情况。将所收集的病例依据不同的分化程度、年龄、性别、肿瘤浸润深度、TNM分期及病理学形态进行分组,观察二者的阳性表达率,采用χ^(2)检验分析两者之间及其与临床和病理特征间的相关性,进一步探讨PCBP1和STUB1与胃癌恶性程度的关系。结果免疫组化结果显示PCBP1在癌组织中的阳性表达率为69.7%,明显高于癌旁组织中的阳性表达率48.5%;STUB1在癌组织中的阳性表达率为39.4%,低于癌旁组织中的阳性表达率54.5%,两者差异均有统计学意义(P<0.05)。PCBP1的阳性表达率与肿瘤分化程度、淋巴结转移及Lauren分型有相关性(P<0.05);与患者的年龄、性别、浸润深度、临床分期、神经浸润、脉管侵犯无相关性(P>0.05)。STUB1的阳性表达率与肿瘤分化程度、浸润深度、淋巴结转移及Lauren分型有相关性(P<0.05),但与患者的年龄、性别、临床分期、神经浸润、脉管侵犯无相关性(P>0.05)。PCBP1(癌)与STUB1(癌)的Spearman相关系数为-0.413,P为0.017,表明两者之间存在显著的负相关性。结论PCBP1可通过调控铁死亡通路中主要调控因子STUB1参与胃癌的恶性进展。为胃癌的分子机制探索及潜在治疗靶点提供了新的理论依据。展开更多
Objective Cerebral palsy(CP)is a prevalent neurodevelopmental disorder acquired during the perinatal period,with periventricular white matter injury(PWMI)serving as its primary pathological hallmark.PWMI is characteri...Objective Cerebral palsy(CP)is a prevalent neurodevelopmental disorder acquired during the perinatal period,with periventricular white matter injury(PWMI)serving as its primary pathological hallmark.PWMI is characterized by the loss of oligodendrocytes(OLs)and the disintegration of myelin sheaths,leading to impaired neural connectivity and motor dysfunction.Neural stem cells(NSCs)represent a promising regenerative source for replenishing lost OLs;however,conventional twodimensional(2D)in vitro culture systems lack the three-dimensional(3D)physiological microenvironment.Microfluidic chip technology has emerged as a powerful tool to overcome this limitation by enabling precise spatial and temporal control over 3D microenvironmental conditions,including the establishment of stable concentration gradients of bioactive molecules.Catalpol,an iridoid glycoside derived from traditional medicinal plants,exhibits dual antioxidant and anti-apoptotic properties.Despite its therapeutic potential,the capacity of catalpol to drive NSC differentiation toward OLs under biomimetic 3D conditions,as well as the underlying molecular mechanisms,remains poorly understood.This study aims to develop a microfluidic-based 3D biomimetic platform to systematically investigate the concentration-dependent effects of catalpol on promoting NSCs-to-OLs differentiation and to elucidate the role of the caveolin-1(Cav-1)signaling pathway in this process.Methods We developed a novel multiplexed microfluidic device featuring parallel microchannels with integrated gradient generators capable of establishing and maintaining precise linear concentration gradients(0-3 g/L catalpol)across 3D NSCs cultures.This platform facilitated the continuous perfusion culture of NSC-derived 3D spheroids,mimicking the dynamic in vivo microenvironment.Real-time cell viability was assessed using Calcein-AM/propidium iodide(PI)dual staining,with fluorescence imaging quantifying live/dead cell ratios.Oligodendrocyte differentiation was evaluated through quantitative reverse transcription polymerase chain reaction(qRT-PCR)for MBP and SOX10 gene expression,complemented by immunofluorescence staining to visualize corresponding protein changes.To dissect the molecular mechanism,the Cav-1-specific pharmacological inhibitor methyl‑β‑cyclodextrin(MCD)was employed to perturb the pathway,and its effects on differentiation markers were analyzed.Results Catalpol demonstrated excellent biocompatibility,with cell viability exceeding 96%across the entire tested concentration range(0-3 g/L),confirming its non-cytotoxic nature.At the optimal concentration of 0-3 g/L,catalpol significantly upregulated both MBP and SOX10 expression(P<0.05,P<0.01),indicating robust promotion of oligodendroglial differentiation.Intriguingly,Cav-1 mRNA expression was progressively downregulated during NSC differentiation into OLs.Further inhibition of Cav-1 with MCD further enhanced this effect,leading to a statistically significant increase in OL-specific gene expression(P<0.05,P<0.01),suggesting Cav-1 acts as a negative regulator of OLs differentiation.Conclusion This study established an integrated microfluidic gradient chip-3D NSC spheroid culture system,which combines the advantages of precise chemical gradient control with physiologically relevant 3D cell culture.The findings demonstrate that 3 g/L catalpol effectively suppresses Cav-1 signaling to drive NSC differentiation into functional OLs.This work not only provides novel insights into the Cav-1-dependent mechanisms of myelination but also delivers a scalable technological platform for future research on remyelination therapies,with potential applications in cerebral palsy and other white matter disorders.The platform’s modular design permits adaptation for screening other neurogenic compounds or investigating additional signaling pathways involved in OLs maturation.展开更多
RNA interference(RNAi)is an intrinsic antiviral immune mechanism conserved in diverse eukaryotic organisms.However,the mechanism by which antiviral RNAi in mammals is regulated is poorly understood.In this study,we un...RNA interference(RNAi)is an intrinsic antiviral immune mechanism conserved in diverse eukaryotic organisms.However,the mechanism by which antiviral RNAi in mammals is regulated is poorly understood.In this study,we uncovered that the E3 ubiquitin ligase STIP1 homology and U-box-containing protein 1(STUB1)was a new regulator of the RNAi machinery in mammals.We found that STUB1 interacted with and ubiquitinated AGO2,and targeted it for degradation in a chaperon-dependent manner.STUB1 promoted the formation of Lys48(K48)-linked polyubiquitin chains on AGO2,and facilitated AGO2 degradation through ubiquitin-proteasome system.In addition to AGO2,STUB1 also induced the protein degradation of AGO1,AGO3 and AGO4.Further investigation revealed that STUB1 also regulated Dicer's ubiquitination via K48-linked polyubiquitin and induced the degradation of Dicer as well as its specialized form,termed antiviral Dicer(avi Dicer)that expresses in mammalian stem cells.Moreover,we found that STUB1 deficiency up-regulated Dicer and AGO2,thereby enhancing the RNAi response and efficiently inhibiting viral replication in mammalian cells.Using the newborn mouse model of Enterovirus A71(EV-A71),we confirmed that STUB1 deficiency enhanced the virus-derived si RNAs production and antiviral RNAi,which elicited a potent antiviral effect against EV-A71 infection in vivo.In summary,our findings uncovered that the E3 ubiquitin ligase STUB1 was a general regulator of the RNAi machinery by targeting Dicer,avi Dicer and AGO1–4.Moreover,STUB1 regulated the RNAi response through mediating the abundance of Dicer and AGO2 during viral infection,thereby providing novel insights into the regulation of antiviral RNAi in mammals.展开更多
凡纳滨对虾的主要选育目标分为两个方面:一是培育具有较强抗病、抗逆性的“高抗系”(GK),二是培育具有快速生长特性的“快大系”(KD)。然而,国内缺少针对这两个选育群体的遗传多样性特别是基因组近交水平的调查分析研究。基于液相芯片...凡纳滨对虾的主要选育目标分为两个方面:一是培育具有较强抗病、抗逆性的“高抗系”(GK),二是培育具有快速生长特性的“快大系”(KD)。然而,国内缺少针对这两个选育群体的遗传多样性特别是基因组近交水平的调查分析研究。基于液相芯片“黄海芯1号”(55 K SNP)的基因分型数据,首次分析了GK(1064尾个体)和KD(564尾个体)选育群体的遗传结构和遗传多样性,调查了连续性纯合片段(ROH)的基因组分布特征,并重点评估了两个群体的基因组近交水平。PCA及进化树分析表明GK及KD群体可明确分层,亲缘关系热图表明KD群体内个体间的亲缘关系比GK群体更近。GK群体包括的家系数量更多,导致其遗传多样性高于KD群体;两群体间的F_(st)为0.09,存在中等遗传分化。GK和KD群体每个ROH的平均长度分别为(1.70±0.34)Mb和(1.65±0.38)Mb,每个样本ROH的平均数量分别为1.98±1.30和2.07±1.37。GK和KD群体0.8~1.25 Mb长度的ROH占比分别为11.41%和19.17%,表明KD群体的选育历史比GK群体更长。两个群体>2.25 Mb长度的ROH片段占比分别为10.26和9.74%,表明两个群体短期内未发生近亲交配。七种基因组近交系数评估结果表明,KD群体的近交水平高于GK群体。不依赖基础群体等位基因频率的F_(ROH)和F_(HOM)方法可准确地评价育种群体的真实近交水平,而F_(VR1)、F_(YA1)和F_(LH1)等依赖基础群体等位基因频率的方法可以用来比较群体及个体间的相对近交水平。上述结果为准确地评估育种群体的近交水平和优化育种方案提供了重要参考依据。展开更多
Objective: To study differentially expressed genes by silencing cyclin B1, and to sift out autophagy-related genes. Methods: Double thymidine deoxyribonucleoside blocking was used to synchronize nasopharyngeal carcino...Objective: To study differentially expressed genes by silencing cyclin B1, and to sift out autophagy-related genes. Methods: Double thymidine deoxyribonucleoside blocking was used to synchronize nasopharyngeal carcinoma cell (CNE-2) to S phase, then flow cytometry was applied to test transfection efficiency. The mRNA and protein expression level of cyclin B1 was assessed by q-PCR and western blot, respectively. Differentially expressed genes were screened by high-throughput gene chip. Results: Double thymidine deoxyribonucleoside (2.5 mmol/L) blocking was used to synchronize the cell cycle to S phase. The transfection efficiency of CNE-2 cells was 85.6%. Compared with negative group,cyclin B1-siRNA treated group significantly down-regulated mRNA expression of cyclin B1 (80%) and protein level (75.3%). Totally, 2408 differentially expressed genes were found in CNE-2, including 1245 up-regulated genes and 1163 down-regulated genes. Moreover, PTEN, an autophagy-related gene, was preliminarily sifted out. Conclusions: Cyclin B1-siRNA significantly down-regulated the expression of cyclin B1 and yielded a total of 2408 differentially expressed genes, including PETN (an autophagy-related gene).展开更多
Autophagy is a major degradation system which processes substrates through the steps of auto- phagosome formation, autophagosome-lysosome fusion, and substrate degradation. Aberrant autophagic flux is present in many ...Autophagy is a major degradation system which processes substrates through the steps of auto- phagosome formation, autophagosome-lysosome fusion, and substrate degradation. Aberrant autophagic flux is present in many pathological conditions including neurodegeneration and tumors. CHIP/STUB1, an E3 ligase, plays an important role in neurodegeneration. In this study, we identified the regulation of autophagic flux by CHIP （carboxy-terminus of HscT0-interacting protein）. Knockdown of CHIP induced autophagosome formation through increasing the PTEN protein level and decreasing the AKT/mTOR activity as well as decreasing phosphorylation of ULK1 on Ser757. However, degradation of the autophagic substrate p62 was disturbed by knockdown of CHIP, suggesting an abnormality of autophagic flux. Furthermore, knockdown of CHIP increased the susceptibility of cells to autophagic cell death induced by bafilomycin AI. Thus, our data suggest that CHIP plays roles in the regulation of autophagic flux.展开更多
文摘目的旨在通过生物信息学分析结合实验验证,探讨PCBP1(Poly(rC)-Binding Protein 1)在胃癌组织中的表达特征及其临床意义,对其与铁死亡主要调控因子STUB1(STIP1 Homology and U-Box Containing Protein 1)的关系研究。并通过免疫组化实验验证PCBP1与STUB1在胃癌中的表达模式及其与临床病理特征的关系。为新型胃癌靶向治疗策略提供重要的理论支撑和潜在的干预靶点。方法从TIMER 2.0在线分析网站中获取PCBP1在胃癌及癌旁组织的基因表达数据。利用癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库中胃癌数据(STAD)进行KEGG通路富集分析,并揭示其潜在作用机制;在铁死亡调控通路中找出主要调控因子STUB1。随后,采用免疫组化法检测33例胃癌组织及对应癌旁组织中PCBP1和STUB1的表达情况。将所收集的病例依据不同的分化程度、年龄、性别、肿瘤浸润深度、TNM分期及病理学形态进行分组,观察二者的阳性表达率,采用χ^(2)检验分析两者之间及其与临床和病理特征间的相关性,进一步探讨PCBP1和STUB1与胃癌恶性程度的关系。结果免疫组化结果显示PCBP1在癌组织中的阳性表达率为69.7%,明显高于癌旁组织中的阳性表达率48.5%;STUB1在癌组织中的阳性表达率为39.4%,低于癌旁组织中的阳性表达率54.5%,两者差异均有统计学意义(P<0.05)。PCBP1的阳性表达率与肿瘤分化程度、淋巴结转移及Lauren分型有相关性(P<0.05);与患者的年龄、性别、浸润深度、临床分期、神经浸润、脉管侵犯无相关性(P>0.05)。STUB1的阳性表达率与肿瘤分化程度、浸润深度、淋巴结转移及Lauren分型有相关性(P<0.05),但与患者的年龄、性别、临床分期、神经浸润、脉管侵犯无相关性(P>0.05)。PCBP1(癌)与STUB1(癌)的Spearman相关系数为-0.413,P为0.017,表明两者之间存在显著的负相关性。结论PCBP1可通过调控铁死亡通路中主要调控因子STUB1参与胃癌的恶性进展。为胃癌的分子机制探索及潜在治疗靶点提供了新的理论依据。
基金supported by grants from the Liaoning Province Excellent Talent Program Project(XLYC1902031)Dalian Science and Technology Talent Innovation Plan Grant(2022RG18)Basic Research Project of the Department of Education of Liaoning Province(LJKQZ20222395)。
文摘Objective Cerebral palsy(CP)is a prevalent neurodevelopmental disorder acquired during the perinatal period,with periventricular white matter injury(PWMI)serving as its primary pathological hallmark.PWMI is characterized by the loss of oligodendrocytes(OLs)and the disintegration of myelin sheaths,leading to impaired neural connectivity and motor dysfunction.Neural stem cells(NSCs)represent a promising regenerative source for replenishing lost OLs;however,conventional twodimensional(2D)in vitro culture systems lack the three-dimensional(3D)physiological microenvironment.Microfluidic chip technology has emerged as a powerful tool to overcome this limitation by enabling precise spatial and temporal control over 3D microenvironmental conditions,including the establishment of stable concentration gradients of bioactive molecules.Catalpol,an iridoid glycoside derived from traditional medicinal plants,exhibits dual antioxidant and anti-apoptotic properties.Despite its therapeutic potential,the capacity of catalpol to drive NSC differentiation toward OLs under biomimetic 3D conditions,as well as the underlying molecular mechanisms,remains poorly understood.This study aims to develop a microfluidic-based 3D biomimetic platform to systematically investigate the concentration-dependent effects of catalpol on promoting NSCs-to-OLs differentiation and to elucidate the role of the caveolin-1(Cav-1)signaling pathway in this process.Methods We developed a novel multiplexed microfluidic device featuring parallel microchannels with integrated gradient generators capable of establishing and maintaining precise linear concentration gradients(0-3 g/L catalpol)across 3D NSCs cultures.This platform facilitated the continuous perfusion culture of NSC-derived 3D spheroids,mimicking the dynamic in vivo microenvironment.Real-time cell viability was assessed using Calcein-AM/propidium iodide(PI)dual staining,with fluorescence imaging quantifying live/dead cell ratios.Oligodendrocyte differentiation was evaluated through quantitative reverse transcription polymerase chain reaction(qRT-PCR)for MBP and SOX10 gene expression,complemented by immunofluorescence staining to visualize corresponding protein changes.To dissect the molecular mechanism,the Cav-1-specific pharmacological inhibitor methyl‑β‑cyclodextrin(MCD)was employed to perturb the pathway,and its effects on differentiation markers were analyzed.Results Catalpol demonstrated excellent biocompatibility,with cell viability exceeding 96%across the entire tested concentration range(0-3 g/L),confirming its non-cytotoxic nature.At the optimal concentration of 0-3 g/L,catalpol significantly upregulated both MBP and SOX10 expression(P<0.05,P<0.01),indicating robust promotion of oligodendroglial differentiation.Intriguingly,Cav-1 mRNA expression was progressively downregulated during NSC differentiation into OLs.Further inhibition of Cav-1 with MCD further enhanced this effect,leading to a statistically significant increase in OL-specific gene expression(P<0.05,P<0.01),suggesting Cav-1 acts as a negative regulator of OLs differentiation.Conclusion This study established an integrated microfluidic gradient chip-3D NSC spheroid culture system,which combines the advantages of precise chemical gradient control with physiologically relevant 3D cell culture.The findings demonstrate that 3 g/L catalpol effectively suppresses Cav-1 signaling to drive NSC differentiation into functional OLs.This work not only provides novel insights into the Cav-1-dependent mechanisms of myelination but also delivers a scalable technological platform for future research on remyelination therapies,with potential applications in cerebral palsy and other white matter disorders.The platform’s modular design permits adaptation for screening other neurogenic compounds or investigating additional signaling pathways involved in OLs maturation.
基金the National Natural Science Foundation of China(31970169 to X.Z.and 82172269 and 81873964 to Y.Q.)the International Partnership Program of Chinese Academy of Sciences(153B42KYSB20200004 to X.Z.)+3 种基金the Young Top-notch Talent Cultivation Program of Hubei Province(Y.Q.)the Grant from the CAS Youth Innovation Promotion Association(2020332 to Y.Q.)the Hubei Province Natural Science Funds for Distinguished Young Scholar(2021CFA047 to Y.Q.)the Young Top-notch Talent Cultivation Program of Hubei Province(Y.Q.)。
文摘RNA interference(RNAi)is an intrinsic antiviral immune mechanism conserved in diverse eukaryotic organisms.However,the mechanism by which antiviral RNAi in mammals is regulated is poorly understood.In this study,we uncovered that the E3 ubiquitin ligase STIP1 homology and U-box-containing protein 1(STUB1)was a new regulator of the RNAi machinery in mammals.We found that STUB1 interacted with and ubiquitinated AGO2,and targeted it for degradation in a chaperon-dependent manner.STUB1 promoted the formation of Lys48(K48)-linked polyubiquitin chains on AGO2,and facilitated AGO2 degradation through ubiquitin-proteasome system.In addition to AGO2,STUB1 also induced the protein degradation of AGO1,AGO3 and AGO4.Further investigation revealed that STUB1 also regulated Dicer's ubiquitination via K48-linked polyubiquitin and induced the degradation of Dicer as well as its specialized form,termed antiviral Dicer(avi Dicer)that expresses in mammalian stem cells.Moreover,we found that STUB1 deficiency up-regulated Dicer and AGO2,thereby enhancing the RNAi response and efficiently inhibiting viral replication in mammalian cells.Using the newborn mouse model of Enterovirus A71(EV-A71),we confirmed that STUB1 deficiency enhanced the virus-derived si RNAs production and antiviral RNAi,which elicited a potent antiviral effect against EV-A71 infection in vivo.In summary,our findings uncovered that the E3 ubiquitin ligase STUB1 was a general regulator of the RNAi machinery by targeting Dicer,avi Dicer and AGO1–4.Moreover,STUB1 regulated the RNAi response through mediating the abundance of Dicer and AGO2 during viral infection,thereby providing novel insights into the regulation of antiviral RNAi in mammals.
文摘凡纳滨对虾的主要选育目标分为两个方面:一是培育具有较强抗病、抗逆性的“高抗系”(GK),二是培育具有快速生长特性的“快大系”(KD)。然而,国内缺少针对这两个选育群体的遗传多样性特别是基因组近交水平的调查分析研究。基于液相芯片“黄海芯1号”(55 K SNP)的基因分型数据,首次分析了GK(1064尾个体)和KD(564尾个体)选育群体的遗传结构和遗传多样性,调查了连续性纯合片段(ROH)的基因组分布特征,并重点评估了两个群体的基因组近交水平。PCA及进化树分析表明GK及KD群体可明确分层,亲缘关系热图表明KD群体内个体间的亲缘关系比GK群体更近。GK群体包括的家系数量更多,导致其遗传多样性高于KD群体;两群体间的F_(st)为0.09,存在中等遗传分化。GK和KD群体每个ROH的平均长度分别为(1.70±0.34)Mb和(1.65±0.38)Mb,每个样本ROH的平均数量分别为1.98±1.30和2.07±1.37。GK和KD群体0.8~1.25 Mb长度的ROH占比分别为11.41%和19.17%,表明KD群体的选育历史比GK群体更长。两个群体>2.25 Mb长度的ROH片段占比分别为10.26和9.74%,表明两个群体短期内未发生近亲交配。七种基因组近交系数评估结果表明,KD群体的近交水平高于GK群体。不依赖基础群体等位基因频率的F_(ROH)和F_(HOM)方法可准确地评价育种群体的真实近交水平,而F_(VR1)、F_(YA1)和F_(LH1)等依赖基础群体等位基因频率的方法可以用来比较群体及个体间的相对近交水平。上述结果为准确地评估育种群体的近交水平和优化育种方案提供了重要参考依据。
基金This study was funded by the Natural Science Foundation of Fujian Province(2015J01457).
文摘Objective: To study differentially expressed genes by silencing cyclin B1, and to sift out autophagy-related genes. Methods: Double thymidine deoxyribonucleoside blocking was used to synchronize nasopharyngeal carcinoma cell (CNE-2) to S phase, then flow cytometry was applied to test transfection efficiency. The mRNA and protein expression level of cyclin B1 was assessed by q-PCR and western blot, respectively. Differentially expressed genes were screened by high-throughput gene chip. Results: Double thymidine deoxyribonucleoside (2.5 mmol/L) blocking was used to synchronize the cell cycle to S phase. The transfection efficiency of CNE-2 cells was 85.6%. Compared with negative group,cyclin B1-siRNA treated group significantly down-regulated mRNA expression of cyclin B1 (80%) and protein level (75.3%). Totally, 2408 differentially expressed genes were found in CNE-2, including 1245 up-regulated genes and 1163 down-regulated genes. Moreover, PTEN, an autophagy-related gene, was preliminarily sifted out. Conclusions: Cyclin B1-siRNA significantly down-regulated the expression of cyclin B1 and yielded a total of 2408 differentially expressed genes, including PETN (an autophagy-related gene).
基金supported in part by the National Natural Science Foundation of China (31330030 and 31471012)the National Basic Research Development Program of China (2012CB947602)a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions
文摘Autophagy is a major degradation system which processes substrates through the steps of auto- phagosome formation, autophagosome-lysosome fusion, and substrate degradation. Aberrant autophagic flux is present in many pathological conditions including neurodegeneration and tumors. CHIP/STUB1, an E3 ligase, plays an important role in neurodegeneration. In this study, we identified the regulation of autophagic flux by CHIP （carboxy-terminus of HscT0-interacting protein）. Knockdown of CHIP induced autophagosome formation through increasing the PTEN protein level and decreasing the AKT/mTOR activity as well as decreasing phosphorylation of ULK1 on Ser757. However, degradation of the autophagic substrate p62 was disturbed by knockdown of CHIP, suggesting an abnormality of autophagic flux. Furthermore, knockdown of CHIP increased the susceptibility of cells to autophagic cell death induced by bafilomycin AI. Thus, our data suggest that CHIP plays roles in the regulation of autophagic flux.
