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
Repair of DNA double-strand break(DSB)is critical for the maintenance of genome integrity.A class of DSB-induced small RNAs(di RNAs)has been shown to play an important role in DSB repair.In humans,di RNAs are associat...Repair of DNA double-strand break(DSB)is critical for the maintenance of genome integrity.A class of DSB-induced small RNAs(di RNAs)has been shown to play an important role in DSB repair.In humans,di RNAs are associated with Ago2 and guide the recruitment of Rad51 to DSB sites to facilitate repair by homologous recombination(HR).Ago2 activity has been reported to be regulated by phosphorylation under normal and hypoxic conditions.However,the role of Ago2 phosphorylation in DNA damage repair is unexplored.Here,we show that S672,S828,T830,and S831 of human Ago2 are phosphorylated in response to ionizing radiation(IR).S672 A mutation of Ago2 leads to significant reduction in Rad51 foci formation and HR efficiency.We further show that defective association of Ago2 S672 A variant with DSB sites,instead of defects in di RNA and Rad51 binding,may account for decreased Rad51 foci formation and HR efficiency.Our study reveals a novel regulatory mechanism for the function of Ago2 in DNA repair.展开更多
目的探讨m^(7)G相关基因能否作为肝细胞性肝癌预后的生物标志物。方法采用癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库筛选肝细胞性肝癌组织和癌旁组织中有表达差异的m^(7)G相关基因组,m^(7)G相关基因和临床数据中的生存时间...目的探讨m^(7)G相关基因能否作为肝细胞性肝癌预后的生物标志物。方法采用癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库筛选肝细胞性肝癌组织和癌旁组织中有表达差异的m^(7)G相关基因组,m^(7)G相关基因和临床数据中的生存时间及生存状态按照样本ID号匹配合并后筛选预后基因组,将两组的交集基因纳入lasso回归,对筛选出来的基因进行风险评分,基于风险评分的中位数值将所有肝细胞性肝癌患者分为高、低两个风险组,并对高分险组和低风险组进行单因素和多因素的Cox回归分析,评价风险评分在预后中的差异。结果经lasso回归筛选出4个模型基因(AGO2、NCBP1、NCBP2、WDR4),高风险组的生存率显著低于低风险组(P=0.027),ROC曲线显示风险模型对患者1,2,3年生存预测的曲线下面积(AUC)分别为0.683,0.604,0.602。肿瘤分期、T分期、M分期和风险评分是肝细胞性肝癌预后的相关因素(P<0.05),其中风险评分是影响肝细胞性肝癌患者生存率的独立预后因素(P=0.044,HR=1.637)。结论m^(7)G相关基因可作为肝细胞性肝癌预后的生物标志物。AGO2、NCBP1、NCBP2和WDR4构建的风险模型对肝细胞性肝癌具有重要的预后价值。展开更多
[目的]探讨乏氧条件下长链非编码RNA LINC00488通过miR-376a-3p/AGO2调控甲状腺乳头状癌细胞凋亡的潜在机制。[方法]乏氧处理后,高通量测序和过表达筛选影响乏氧条件下甲状腺乳头状癌细胞B-CPAP凋亡水平的长链非编码RNA。过表达或敲低LI...[目的]探讨乏氧条件下长链非编码RNA LINC00488通过miR-376a-3p/AGO2调控甲状腺乳头状癌细胞凋亡的潜在机制。[方法]乏氧处理后,高通量测序和过表达筛选影响乏氧条件下甲状腺乳头状癌细胞B-CPAP凋亡水平的长链非编码RNA。过表达或敲低LINC00488、miR-376a-3p、AGO2后检测B-CPAP的凋亡水平。[结果]乏氧条件下,LINC00488的表达水平显著上升。敲低LINC00488后乏氧条件下B-CPAP细胞的凋亡水平显著上升[(24.59±3.55 vs 65.69±8.99)%,P<0.05]。过表达miR-376a-3p后乏氧条件下B-CPAP细胞的凋亡水平显著上升[(25.56±3.86 vs 69.04±9.52)%,P<0.05]。此外,LINC00488和miR-376a-3p存在碱基间的相互作用。敲低AGO2后乏氧条件下B-CPAP细胞的凋亡水平显著上升[(26.91±3.77 vs 60.72±8.45)%,P<0.05]。同时,miR-376a-3p靶向结合AGO2 mRNA的3′端非翻译区。过表达miR-376a-3p后乏氧条件下B-CPAP细胞中AGO2的mRNA和蛋白水平均下降(0.84±0.18 vs 0.11±0.01,P<0.05);敲低miR-376a-3p后乏氧条件下B-CPAP细胞中AGO2的mRNA和蛋白水平均上升(0.84±0.18 vs 1.43±0.23,P<0.05)。[结论]乏氧条件下,长链非编码RNA LINC00488结合miR-376a-3p后抑制了miR-376a-3p调节下游基因AGO2表达的能力,从而抑制了甲状腺乳头状癌细胞的凋亡。展开更多
The ultraviolet (UV) photoresponses of Wurtzite GaN, ZnO, and 6H-SiC-based Optical Field Effect Transistor (OPFET) detectors are estimated with an in-depth analysis of the same considering the generalized model and th...The ultraviolet (UV) photoresponses of Wurtzite GaN, ZnO, and 6H-SiC-based Optical Field Effect Transistor (OPFET) detectors are estimated with an in-depth analysis of the same considering the generalized model and the front-illuminated model for high resolution imaging and UV communication applications. The gate materials considered for the proposed study are gold (Au) and Indium-Tin-Oxide (ITO) for GaN, Au for SiC, and Au and silver dioxide (AgO2) for ZnO. The results indicate significant improvement in the Linear Dynamic Range (LDR) over the previously investigated GaN OPFET (buried-gate, front-illuminated and generalized) models with Au gate. The generalized model has superior dynamic range than the front-illuminated model. In terms of responsivity, all the models including buried-gate OPFET exhibit high and comparable photoresponses. Buried-gate devices on the whole, exhibit faster response than the surface gate models except in the AgO2-ZnO generalized OPFET model wherein the switching time is the lowest. The generalized model enables faster switching than the front-illuminated model. The switching times in all the cases are of the order of nanoseconds to picoseconds. The SiC generalized OPFET model shows the highest 3-dB bandwidths of 11.88 GHz, 36.2 GHz, and 364 GHz, and modest unity-gain cut-off frequencies of 4.62 GHz, 8.71 GHz, and 5.71 GHz at the optical power densities of 0.575 μW/cm2, 0.575 mW/cm2, and 0.575 W/cm2 respectively. These are in overall, the highest detection-cum-amplifi-cation bandwidths among all the investigated devices. The same device exhibits the highest LDR of 73.3 dB. The device performance is superior to most of the other existing detectors along with comparable LDR, thus, emerging as a high performance photodetector for imaging and communication applications. All the detectors show considerably high detectivities owing to the high responsivity values. The results have been analyzed by the photovoltaic and the photoconductive effects, and the series resistance effects and will aid in conducting further research. The results are in line with the experiments and the commercially available software simulations. The devices will greatly contribute towards single photon counting, high resolution imaging, and UV communication applications.展开更多
目的探讨Ago2和微小RNA(miRNA,miR)-145-5p对肝癌细胞增殖和转移的影响。方法选取2018年6月至2022年12月商丘市第一人民医院收治的98例肝癌患者肿瘤组织和癌旁组织作为研究对象,采用荧光定量聚合酶链反应(PCR)和蛋白质免疫印迹分析miR-1...目的探讨Ago2和微小RNA(miRNA,miR)-145-5p对肝癌细胞增殖和转移的影响。方法选取2018年6月至2022年12月商丘市第一人民医院收治的98例肝癌患者肿瘤组织和癌旁组织作为研究对象,采用荧光定量聚合酶链反应(PCR)和蛋白质免疫印迹分析miR-145-5p和Ago2表达水平。采用慢病毒在人肝癌细胞系HepG2建立miR-145-5p过表达细胞系,分别为miRNA对照组和miR-145-5p组,采用蛋白质印迹法(Western blot)分析Ago2表达水平;采用慢病毒构建Ago2敲降细胞系,为对照组和Ago2组,采用荧光定量PCR分析miR-145-5p表达水平。采用细胞计数试剂盒(CCK-8)分析不同细胞的增殖能力;采用划痕实验和Transwell实验分析不同细胞的迁移和侵袭能力;分析Ago2和miR-145-5p表达的关系。组间比较采用t检验。结果肝癌组织中Ago2蛋白表达水平(1.99±0.18)高于癌旁组织(1.20±0.12),差异有统计学意义(t=25.260,P<0.05)。肝癌组织中miR-145-5p表达水平(0.64±0.13)低于癌旁组织(1.04±0.15),差异有统计学意义(t=14.060,P<0.05)。miRNA对照组细胞48 h吸光度(A)值(2.01±0.12)高于miR-145-5p组(1.45±0.13),差异有统计学意义(t=7.616,P<0.05)。对照组细胞48 h A值(1.90±0.15)低于Ago2组(2.44±0.13),差异有统计学意义(t=6.603,P<0.05)。miRNA对照组划痕愈合率[(72.74±3.06)%]高于miR-145-5p组[(51.50±5.85)%],差异有统计学意义(t=7.879,P<0.05)。对照组细胞划痕愈合率[(76.31±3.95)%]低于Ago2组[(88.69±2.63)%],差异有统计学意义(t=6.387,P<0.05)。miRNA对照组侵袭数量[(97.17±9.02)个]高于miR-145-5p组[(77.50±6.98)个],差异有统计学意义(t=4.224,P<0.05)。对照组细胞侵袭数量[(93.17±8.18)个]低于Ago2组[(132.83±5.34)个],差异有统计学意义(t=9.941,P<0.05)。Ago2是miR-145-5的靶基因。miRNA对照组Ago2蛋白表达水平(1.46±0.14)高于miR-145-5p组(0.74±0.12),差异有统计学意义(t=9.625,P<0.05)。结论miR-145-5p调节Ago2蛋白表达水平,进而调节肝癌细胞的增殖和转移等恶性细胞生物学行为。展开更多
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.
基金supported by the National Natural Science Foundation of China(31401202)。
文摘Repair of DNA double-strand break(DSB)is critical for the maintenance of genome integrity.A class of DSB-induced small RNAs(di RNAs)has been shown to play an important role in DSB repair.In humans,di RNAs are associated with Ago2 and guide the recruitment of Rad51 to DSB sites to facilitate repair by homologous recombination(HR).Ago2 activity has been reported to be regulated by phosphorylation under normal and hypoxic conditions.However,the role of Ago2 phosphorylation in DNA damage repair is unexplored.Here,we show that S672,S828,T830,and S831 of human Ago2 are phosphorylated in response to ionizing radiation(IR).S672 A mutation of Ago2 leads to significant reduction in Rad51 foci formation and HR efficiency.We further show that defective association of Ago2 S672 A variant with DSB sites,instead of defects in di RNA and Rad51 binding,may account for decreased Rad51 foci formation and HR efficiency.Our study reveals a novel regulatory mechanism for the function of Ago2 in DNA repair.
文摘目的探讨m^(7)G相关基因能否作为肝细胞性肝癌预后的生物标志物。方法采用癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库筛选肝细胞性肝癌组织和癌旁组织中有表达差异的m^(7)G相关基因组,m^(7)G相关基因和临床数据中的生存时间及生存状态按照样本ID号匹配合并后筛选预后基因组,将两组的交集基因纳入lasso回归,对筛选出来的基因进行风险评分,基于风险评分的中位数值将所有肝细胞性肝癌患者分为高、低两个风险组,并对高分险组和低风险组进行单因素和多因素的Cox回归分析,评价风险评分在预后中的差异。结果经lasso回归筛选出4个模型基因(AGO2、NCBP1、NCBP2、WDR4),高风险组的生存率显著低于低风险组(P=0.027),ROC曲线显示风险模型对患者1,2,3年生存预测的曲线下面积(AUC)分别为0.683,0.604,0.602。肿瘤分期、T分期、M分期和风险评分是肝细胞性肝癌预后的相关因素(P<0.05),其中风险评分是影响肝细胞性肝癌患者生存率的独立预后因素(P=0.044,HR=1.637)。结论m^(7)G相关基因可作为肝细胞性肝癌预后的生物标志物。AGO2、NCBP1、NCBP2和WDR4构建的风险模型对肝细胞性肝癌具有重要的预后价值。
文摘[目的]探讨乏氧条件下长链非编码RNA LINC00488通过miR-376a-3p/AGO2调控甲状腺乳头状癌细胞凋亡的潜在机制。[方法]乏氧处理后,高通量测序和过表达筛选影响乏氧条件下甲状腺乳头状癌细胞B-CPAP凋亡水平的长链非编码RNA。过表达或敲低LINC00488、miR-376a-3p、AGO2后检测B-CPAP的凋亡水平。[结果]乏氧条件下,LINC00488的表达水平显著上升。敲低LINC00488后乏氧条件下B-CPAP细胞的凋亡水平显著上升[(24.59±3.55 vs 65.69±8.99)%,P<0.05]。过表达miR-376a-3p后乏氧条件下B-CPAP细胞的凋亡水平显著上升[(25.56±3.86 vs 69.04±9.52)%,P<0.05]。此外,LINC00488和miR-376a-3p存在碱基间的相互作用。敲低AGO2后乏氧条件下B-CPAP细胞的凋亡水平显著上升[(26.91±3.77 vs 60.72±8.45)%,P<0.05]。同时,miR-376a-3p靶向结合AGO2 mRNA的3′端非翻译区。过表达miR-376a-3p后乏氧条件下B-CPAP细胞中AGO2的mRNA和蛋白水平均下降(0.84±0.18 vs 0.11±0.01,P<0.05);敲低miR-376a-3p后乏氧条件下B-CPAP细胞中AGO2的mRNA和蛋白水平均上升(0.84±0.18 vs 1.43±0.23,P<0.05)。[结论]乏氧条件下,长链非编码RNA LINC00488结合miR-376a-3p后抑制了miR-376a-3p调节下游基因AGO2表达的能力,从而抑制了甲状腺乳头状癌细胞的凋亡。
文摘The ultraviolet (UV) photoresponses of Wurtzite GaN, ZnO, and 6H-SiC-based Optical Field Effect Transistor (OPFET) detectors are estimated with an in-depth analysis of the same considering the generalized model and the front-illuminated model for high resolution imaging and UV communication applications. The gate materials considered for the proposed study are gold (Au) and Indium-Tin-Oxide (ITO) for GaN, Au for SiC, and Au and silver dioxide (AgO2) for ZnO. The results indicate significant improvement in the Linear Dynamic Range (LDR) over the previously investigated GaN OPFET (buried-gate, front-illuminated and generalized) models with Au gate. The generalized model has superior dynamic range than the front-illuminated model. In terms of responsivity, all the models including buried-gate OPFET exhibit high and comparable photoresponses. Buried-gate devices on the whole, exhibit faster response than the surface gate models except in the AgO2-ZnO generalized OPFET model wherein the switching time is the lowest. The generalized model enables faster switching than the front-illuminated model. The switching times in all the cases are of the order of nanoseconds to picoseconds. The SiC generalized OPFET model shows the highest 3-dB bandwidths of 11.88 GHz, 36.2 GHz, and 364 GHz, and modest unity-gain cut-off frequencies of 4.62 GHz, 8.71 GHz, and 5.71 GHz at the optical power densities of 0.575 μW/cm2, 0.575 mW/cm2, and 0.575 W/cm2 respectively. These are in overall, the highest detection-cum-amplifi-cation bandwidths among all the investigated devices. The same device exhibits the highest LDR of 73.3 dB. The device performance is superior to most of the other existing detectors along with comparable LDR, thus, emerging as a high performance photodetector for imaging and communication applications. All the detectors show considerably high detectivities owing to the high responsivity values. The results have been analyzed by the photovoltaic and the photoconductive effects, and the series resistance effects and will aid in conducting further research. The results are in line with the experiments and the commercially available software simulations. The devices will greatly contribute towards single photon counting, high resolution imaging, and UV communication applications.
文摘目的探讨Ago2和微小RNA(miRNA,miR)-145-5p对肝癌细胞增殖和转移的影响。方法选取2018年6月至2022年12月商丘市第一人民医院收治的98例肝癌患者肿瘤组织和癌旁组织作为研究对象,采用荧光定量聚合酶链反应(PCR)和蛋白质免疫印迹分析miR-145-5p和Ago2表达水平。采用慢病毒在人肝癌细胞系HepG2建立miR-145-5p过表达细胞系,分别为miRNA对照组和miR-145-5p组,采用蛋白质印迹法(Western blot)分析Ago2表达水平;采用慢病毒构建Ago2敲降细胞系,为对照组和Ago2组,采用荧光定量PCR分析miR-145-5p表达水平。采用细胞计数试剂盒(CCK-8)分析不同细胞的增殖能力;采用划痕实验和Transwell实验分析不同细胞的迁移和侵袭能力;分析Ago2和miR-145-5p表达的关系。组间比较采用t检验。结果肝癌组织中Ago2蛋白表达水平(1.99±0.18)高于癌旁组织(1.20±0.12),差异有统计学意义(t=25.260,P<0.05)。肝癌组织中miR-145-5p表达水平(0.64±0.13)低于癌旁组织(1.04±0.15),差异有统计学意义(t=14.060,P<0.05)。miRNA对照组细胞48 h吸光度(A)值(2.01±0.12)高于miR-145-5p组(1.45±0.13),差异有统计学意义(t=7.616,P<0.05)。对照组细胞48 h A值(1.90±0.15)低于Ago2组(2.44±0.13),差异有统计学意义(t=6.603,P<0.05)。miRNA对照组划痕愈合率[(72.74±3.06)%]高于miR-145-5p组[(51.50±5.85)%],差异有统计学意义(t=7.879,P<0.05)。对照组细胞划痕愈合率[(76.31±3.95)%]低于Ago2组[(88.69±2.63)%],差异有统计学意义(t=6.387,P<0.05)。miRNA对照组侵袭数量[(97.17±9.02)个]高于miR-145-5p组[(77.50±6.98)个],差异有统计学意义(t=4.224,P<0.05)。对照组细胞侵袭数量[(93.17±8.18)个]低于Ago2组[(132.83±5.34)个],差异有统计学意义(t=9.941,P<0.05)。Ago2是miR-145-5的靶基因。miRNA对照组Ago2蛋白表达水平(1.46±0.14)高于miR-145-5p组(0.74±0.12),差异有统计学意义(t=9.625,P<0.05)。结论miR-145-5p调节Ago2蛋白表达水平,进而调节肝癌细胞的增殖和转移等恶性细胞生物学行为。
