AIM: To investigate the subcellular localization and the function of mouse transducin β-like 3(Tbl3).METHODS: The coding sequence of mouse Tbl3 was cloned from the c DNAs of a promyelocyte cell line by reverse transc...AIM: To investigate the subcellular localization and the function of mouse transducin β-like 3(Tbl3).METHODS: The coding sequence of mouse Tbl3 was cloned from the c DNAs of a promyelocyte cell line by reverse transcription-polymerase chain reaction. Fusion constructs of Tbl3 and enhanced green fluorescent protein(EGFP) were transfected into fibroblasts and examined by fluorescence microscopy to reveal the subcellular localization of tbl3. To search for nucleolar targeting sequences, scanning deletions of Tbl3-EGFP were constructed and transfected into fibroblasts. To explore the possible function of Tbl3, small hairpin RNAs(sh RNAs) were used to knock down endogenous Tbl3 in mouse promyelocytes and fibroblasts. The effects of Tbl3 knockdown on ribosomal RNA(r RNAs) synthesis or processing were studied by labeling cells with 5,6-3H-uridine followed by a chase with fresh medium for various periods. Total RNAs were purified from treated cells and subjected to gel electrophoresis and Northern analysis. Ribosome profiling by sucrose gradient centrifugation was used to compare the amounts of 40 S and 60 S ribosome subunits as well as the 80 S monosome. The impact of Tbl3 knockdown on cell growth and proliferation was examined by growth curves and colony assays.RESULTS: The largest open reading frame of mouse Tbl3 encodes a protein of 801 amino acids(AA) with an apparent molecular weight of 89-90 kilodalton. It contains thirteen WD40 repeats(an ancient protein-protein interaction motif) and a carboxyl terminus that is highly homologous to the corresponding region of the yeast nucleolar protein, utp13. Virtually nothing is known about the biological function of Tbl3. All cell lines surveyed expressed Tbl3 and the level of expression correlated roughly with cell proliferation and/or biosynthetic activity. Using Tbl3-EGFP fusion constructs we obtained the first direct evidence that Tbl3 is targeted to the nucleoli in mammalian cells. However, no previously described nucleolar targeting sequences were found in Tbl3, suggesting that the WD40 motif and/or other topological features are responsible for nucleolar targeting. Partial knockdown(by 50%-70%) of mouse Tbl3 by shR NA had no discernable effects on the processing of the 47 S pre-ribosomal RNA(pre-r RNA) or the steady-state levels of the mature 28 S, 18 S and 5.8S r RNAs but consistently increased the expression level of the 47 S pre-rR NA by two to four folds. The results of the current study corroborated the previous finding that there was no detectable rR NA processing defects in zebra fish embryos with homozygous deletions of zebra fish Tbl3. As ribosome production consumes the bulk of cellular energy and biosynthetic precursors, dysregulation of pre-rR NA synthesis can have negative effects on cell growth, proliferation and differentiation. Indeed, partial knockdown of Tbl3 in promyelocytes severely impaired their proliferation. The inhibitory effect of Tbl3 knockdown was also observed in fibroblasts, resulting in an 80% reduction in colony formation. Taken together, these results indicate that Tbl3 is a newly recognized nucleolar protein with regulatory roles at very early stages of ribosome biogenesis, perhaps at the level of rR NA gene transcription. CONCLUSION: Tbl3 is a newly recognized nucleolar protein with important regulatory roles in ribosome biogenesis.展开更多
Cellular NDA and AgNOR Protein contents were evaluated byautomatic image analysis in tissue sections stained hy combined Feulgen-AgNOR staining method in 9 normal colonic mucosae, 45 colorectal adenomas and 27 adenoca...Cellular NDA and AgNOR Protein contents were evaluated byautomatic image analysis in tissue sections stained hy combined Feulgen-AgNOR staining method in 9 normal colonic mucosae, 45 colorectal adenomas and 27 adenocarcinomas. The results indicated that during the course that the normal colonical mucosa developed to colorectal adenocarcinoma via adenoma the DNA and AgNOR protein contents increased gradually and there were very significant correlationships between the DNA and the AgNOR protein contents of adenoma group and adenocarcinoma group. However, there were considerahle overlaping in the DNA or AgNOR Protein content and considerahle overlaping cases between adenoma and normal colonic mucosa groups and between adenoma and adenocarcinoma groups. But the overlaping scope in NDA and AgNOR Protein content and the number of overlaping cases were reduced significantly by assessing the correlationship between the DNA and AgNOR protein content. Therefore, it is much more reliable to distinguish colorectal adenomas from adnocarcinomas by using the correlationship between the cellular DNA and the AgNOR Protein contents in the same specimens.展开更多
目的 研究核仁纺锤体相关蛋白1 (Nucleolar and spindle associated protein 1,NUSAP1)在胰腺癌组织中的表达及临床预后分析。方法 利用GEPIA数据库检索NUSAP1基因在胰腺癌组织和正常组织中的表达水平,TCGA数据库及组织芯片研究及验证NU...目的 研究核仁纺锤体相关蛋白1 (Nucleolar and spindle associated protein 1,NUSAP1)在胰腺癌组织中的表达及临床预后分析。方法 利用GEPIA数据库检索NUSAP1基因在胰腺癌组织和正常组织中的表达水平,TCGA数据库及组织芯片研究及验证NUSAP1在胰腺癌的临床预后分析。结果 GEPIA数据库分析结果显示胰腺癌组织中NUSAP1的表达高于正常对照组(P<0.05)。TCGA数据库结果显示,高表达NUSAP1的胰腺癌患者生存时间较低表达NUSAP1的患者更短(HR=2.02,P<0.05)。在病理分级G3-G4组、残余瘤R1和R2组、胰腺癌治疗进展组中NUSAP1的表达均分别高于病理分级G1-G2组、残余瘤R0组、治疗缓解组(P<0.05)。在死亡组中,NUSAP1的表达高于存活组(P<0.05)。组织芯片免疫组化结果显示,与癌旁组织相比,NUSAP1在胰腺癌中呈高表达(χ^(2)=44.10,P<0.001),NUSAP1高表达的患者生存时间更短,预后越差。单因素回归分析结果显示NUSAP1的表达量、性别、病理分级、肿瘤大小、T分期、N分期、TNM分期是影响胰腺癌患者总生存期的危险因素(P<0.05)。多因素回归分析结果显示NUSAP1的表达量、TNM分期是影响总生存期的独立危险因素(P<0.05)。结论 NUSAP1在胰腺癌组织中高表达,与胰腺癌患者不良预后相关。展开更多
目的:探讨核仁纺锤体相关蛋白1(nucleolar and spindle-associated protein 1,NUSAP1)在食管癌(esophageal carci⁃noma,ESCA)中的表达情况、临床意义及功能机制,为ESCA的早期诊断和治疗提供新思路。方法:从GEPIA2和TIMER数据库下载ESCA...目的:探讨核仁纺锤体相关蛋白1(nucleolar and spindle-associated protein 1,NUSAP1)在食管癌(esophageal carci⁃noma,ESCA)中的表达情况、临床意义及功能机制,为ESCA的早期诊断和治疗提供新思路。方法:从GEPIA2和TIMER数据库下载ESCA样本与癌旁样本,进行NUSAP1的表达分析,STRING和Cytoscape软件进行NUSAP1相关基因的PPI网络构建,并进行GO、KEGG及GSEA富集分析。TIMER数据库被用来分析NUSAP1与免疫细胞的相关性。最后通过RT-PCR、Western blot和免疫组化验证NUSAP1在ESCA组织和细胞中的表达,使用慢病毒包被的小干扰RNA转染ESCA细胞,进行NUSAP1的表达敲减。通过CKK-8和Ki67免疫荧光评估NUSAP1对ESCA细胞增殖的影响。结果:NUSAP1在ESCA组织和细胞中高表达。NUSAP1的表达与CDK1、B细胞和巨噬细胞浸润正相关,而与CD4+T细胞、CD8+T细胞、中性粒细胞和树突状细胞浸润负相关。GSEA分析发现NUSAP1的高表达组主要富集于Wnt/β-catenin信号通路、过氧化物酶通路、PI3K/Akt/mTOR信号通路。在体外,敲减NUSAP1后抑制了ESCA细胞增殖。结论:NUSAP1在ESCA中高表达,并与ESCA细胞增殖和免疫细胞浸润密切相关。因此,NUSAP1可能有助于ESCA的诊断和治疗。展开更多
基金Supported by In part by a grant from the St.Perres Fund,No.11-02011
文摘AIM: To investigate the subcellular localization and the function of mouse transducin β-like 3(Tbl3).METHODS: The coding sequence of mouse Tbl3 was cloned from the c DNAs of a promyelocyte cell line by reverse transcription-polymerase chain reaction. Fusion constructs of Tbl3 and enhanced green fluorescent protein(EGFP) were transfected into fibroblasts and examined by fluorescence microscopy to reveal the subcellular localization of tbl3. To search for nucleolar targeting sequences, scanning deletions of Tbl3-EGFP were constructed and transfected into fibroblasts. To explore the possible function of Tbl3, small hairpin RNAs(sh RNAs) were used to knock down endogenous Tbl3 in mouse promyelocytes and fibroblasts. The effects of Tbl3 knockdown on ribosomal RNA(r RNAs) synthesis or processing were studied by labeling cells with 5,6-3H-uridine followed by a chase with fresh medium for various periods. Total RNAs were purified from treated cells and subjected to gel electrophoresis and Northern analysis. Ribosome profiling by sucrose gradient centrifugation was used to compare the amounts of 40 S and 60 S ribosome subunits as well as the 80 S monosome. The impact of Tbl3 knockdown on cell growth and proliferation was examined by growth curves and colony assays.RESULTS: The largest open reading frame of mouse Tbl3 encodes a protein of 801 amino acids(AA) with an apparent molecular weight of 89-90 kilodalton. It contains thirteen WD40 repeats(an ancient protein-protein interaction motif) and a carboxyl terminus that is highly homologous to the corresponding region of the yeast nucleolar protein, utp13. Virtually nothing is known about the biological function of Tbl3. All cell lines surveyed expressed Tbl3 and the level of expression correlated roughly with cell proliferation and/or biosynthetic activity. Using Tbl3-EGFP fusion constructs we obtained the first direct evidence that Tbl3 is targeted to the nucleoli in mammalian cells. However, no previously described nucleolar targeting sequences were found in Tbl3, suggesting that the WD40 motif and/or other topological features are responsible for nucleolar targeting. Partial knockdown(by 50%-70%) of mouse Tbl3 by shR NA had no discernable effects on the processing of the 47 S pre-ribosomal RNA(pre-r RNA) or the steady-state levels of the mature 28 S, 18 S and 5.8S r RNAs but consistently increased the expression level of the 47 S pre-rR NA by two to four folds. The results of the current study corroborated the previous finding that there was no detectable rR NA processing defects in zebra fish embryos with homozygous deletions of zebra fish Tbl3. As ribosome production consumes the bulk of cellular energy and biosynthetic precursors, dysregulation of pre-rR NA synthesis can have negative effects on cell growth, proliferation and differentiation. Indeed, partial knockdown of Tbl3 in promyelocytes severely impaired their proliferation. The inhibitory effect of Tbl3 knockdown was also observed in fibroblasts, resulting in an 80% reduction in colony formation. Taken together, these results indicate that Tbl3 is a newly recognized nucleolar protein with regulatory roles at very early stages of ribosome biogenesis, perhaps at the level of rR NA gene transcription. CONCLUSION: Tbl3 is a newly recognized nucleolar protein with important regulatory roles in ribosome biogenesis.
文摘Cellular NDA and AgNOR Protein contents were evaluated byautomatic image analysis in tissue sections stained hy combined Feulgen-AgNOR staining method in 9 normal colonic mucosae, 45 colorectal adenomas and 27 adenocarcinomas. The results indicated that during the course that the normal colonical mucosa developed to colorectal adenocarcinoma via adenoma the DNA and AgNOR protein contents increased gradually and there were very significant correlationships between the DNA and the AgNOR protein contents of adenoma group and adenocarcinoma group. However, there were considerahle overlaping in the DNA or AgNOR Protein content and considerahle overlaping cases between adenoma and normal colonic mucosa groups and between adenoma and adenocarcinoma groups. But the overlaping scope in NDA and AgNOR Protein content and the number of overlaping cases were reduced significantly by assessing the correlationship between the DNA and AgNOR protein content. Therefore, it is much more reliable to distinguish colorectal adenomas from adnocarcinomas by using the correlationship between the cellular DNA and the AgNOR Protein contents in the same specimens.
文摘目的:探讨核仁纺锤体相关蛋白1(nucleolar and spindle-associated protein 1,NUSAP1)在食管癌(esophageal carci⁃noma,ESCA)中的表达情况、临床意义及功能机制,为ESCA的早期诊断和治疗提供新思路。方法:从GEPIA2和TIMER数据库下载ESCA样本与癌旁样本,进行NUSAP1的表达分析,STRING和Cytoscape软件进行NUSAP1相关基因的PPI网络构建,并进行GO、KEGG及GSEA富集分析。TIMER数据库被用来分析NUSAP1与免疫细胞的相关性。最后通过RT-PCR、Western blot和免疫组化验证NUSAP1在ESCA组织和细胞中的表达,使用慢病毒包被的小干扰RNA转染ESCA细胞,进行NUSAP1的表达敲减。通过CKK-8和Ki67免疫荧光评估NUSAP1对ESCA细胞增殖的影响。结果:NUSAP1在ESCA组织和细胞中高表达。NUSAP1的表达与CDK1、B细胞和巨噬细胞浸润正相关,而与CD4+T细胞、CD8+T细胞、中性粒细胞和树突状细胞浸润负相关。GSEA分析发现NUSAP1的高表达组主要富集于Wnt/β-catenin信号通路、过氧化物酶通路、PI3K/Akt/mTOR信号通路。在体外,敲减NUSAP1后抑制了ESCA细胞增殖。结论:NUSAP1在ESCA中高表达,并与ESCA细胞增殖和免疫细胞浸润密切相关。因此,NUSAP1可能有助于ESCA的诊断和治疗。
