Objective To screen the proteins associated with four-and-a-half LIM domains 3(FHL3) 3' untranslated region(3'UTR) in glioma cells. Methods Western blot was adopted to detect the regulatory effect of poly(C)-b...Objective To screen the proteins associated with four-and-a-half LIM domains 3(FHL3) 3' untranslated region(3'UTR) in glioma cells. Methods Western blot was adopted to detect the regulatory effect of poly(C)-binding protein 2(PCBP2) on FHL3. Biotin pull-down and sliver staining were employed to screen and verify the candidate binding proteins of FHL3 3'UTR. Then liquid chromatography-tandem mass spectrometry(LC-MS/MS) and molecule annotation system were used to identify and analyze the candidate binding proteins. Immunoprecipitation was conducted to study the interaction between PCBP2 and polypyrimidine tract-binding protein 1(PTBP1), a binding protein identified by LC-MS/MS. Results PCBP2 could bind to FHL3 mRNA 3'UTR-A and inhibited the expression of FHL3 in T98 G glioms cells. 22 candidate binding proteins were identified. Among them, there were 11 RNA binding proteins, including PCBP2. PTBP1 associated with FHL3 mRNA 3'UTR and interacted with PCBP2 protein. Conclusion PCBP2 and PTBP1 can both associate with FHL3 mRNA 3'UTR through forming a protein complex.展开更多
[Objective] To construct prokaryotic expression vectors encoding gene Erb3binding protein （EBP1）, which plays important roles in regulating plant organ size from Nervilia fordii （Hance） Schltr. [Methods] PCR produ...[Objective] To construct prokaryotic expression vectors encoding gene Erb3binding protein （EBP1）, which plays important roles in regulating plant organ size from Nervilia fordii （Hance） Schltr. [Methods] PCR products of NfEBP1 with particular restriction sites and expression vectors, pET-28 and pET-16b were digested. Ligation, transformation and selection were performed to construct the recombinant plasmids pET-28-NfEBP1 and pET-16-NfEBP1. The recombinant plasmids were transformed into E. coli BL21 using heat -shock transformation. [Results] Recombinant plasmids pET-28-NfEBP1-1188 and pET-16-NfEBP1-1188 were constructed and transformed into expressional host cells, E. coli BL21, and validated by colony PCR, sequencing and double digestion. [Conclusion] Prokaryotic expression vectors of EBP1 gene from N. fordii were successfully constructed, which laid the foundation for characterization of the gene function.展开更多
本研究旨在探讨Erb B3结合蛋白1(Erb B3-binding protein 1,Ebp1)在食管癌细胞生长中的作用及其机制。用携带Ebp1基因的慢病毒载体感染食管癌Eca109和KYSE150细胞,用real-time PCR检测食管癌组织中Ebp1 m RNA的表达情况,用MTT和结晶紫...本研究旨在探讨Erb B3结合蛋白1(Erb B3-binding protein 1,Ebp1)在食管癌细胞生长中的作用及其机制。用携带Ebp1基因的慢病毒载体感染食管癌Eca109和KYSE150细胞,用real-time PCR检测食管癌组织中Ebp1 m RNA的表达情况,用MTT和结晶紫分别检测食管癌细胞生长和存活能力,用软琼脂细胞生长实验检测细胞克隆形成能力,用流式细胞术检测细胞凋亡率,用Western blot检测和凋亡有关的蛋白表达变化,用裸鼠皮下成瘤实验检测食管癌细胞的成瘤能力。结果显示,与配对的正常组织相比,食管癌组织中Ebp1 m RNA水平显著下降。过表达Ebp1不仅抑制食管癌细胞Eca109和KYSE150的体外生长和存活能力,而且能诱导这两种食管癌细胞发生凋亡,上调Rb和P53的蛋白表达,下调Cyclin D1的表达。Ebp1过表达还能抑制Eca109细胞的裸鼠皮下成瘤能力。以上结果提示,Ebp1通过诱导细胞凋亡抑制食管癌细胞体外生长能力和体内成瘤能力。展开更多
AIM To elucidate the underlying mechanism that microRNA-22(miR-22) promotes the apoptosis of rat pancreatic acinar cells(AR42 J) and the elements that regulate the expression of miR-22.METHODS One hundred nanomoles pe...AIM To elucidate the underlying mechanism that microRNA-22(miR-22) promotes the apoptosis of rat pancreatic acinar cells(AR42 J) and the elements that regulate the expression of miR-22.METHODS One hundred nanomoles per liter of caerulein(Cae)was administrated to induce the apoptosis of AR42 J cells and the apoptosis rate was detected by flow cytometry analysis. An amylase assay kit was used to measure the amylase expression level in the supernatant. Quantitative real-time PCR(qRT-PCR)was adopted to measure miR-22 expression. We used online tools to predict the potential transcription promoter of miR-22 and the binding sites, which was further identified by using luciferase reporter analysis,chromatin immunoprecipitation(ChIP) and ChIPqP CR assays. Then, a mimic of miR-22, Nr3 c1 plasmid encoding the glucocorticoid receptor(GR), and siNr3 c1 were used to transfect AR42 J cells, respectively.The mRNA expression of miR-22, Nr3 c1, and Erb-b2 receptor tyrosine kinase 3(ErbB3) was confirmed by qRT-PCR and the apoptosis rate of AR42 J cells was detected by flow cytometry analysis. Western blot was used to detect the expression of ErbB3, GR, PI3 k, PI3 kp85α, Akt, p-Akt, Bad, Bax, Bcl-xl, Bcl-2, and cleaved caspase3.RESULTS After inducing apoptosis of AR42 J cells in vitro, the expression of miR-22 was significantly increased by2.20 ± 0.26 and 4.19 ± 0.54 times, respectively, at3 h and 6 h in comparison with the control group.As revealed by qRT-PCR assay, the expression of miR-22 was 78.25 ± 6.61 times higher in the miR-22 mimic group relative to the miRNA control group,accompanied with an obviously increased acinar cell apoptosis rate(32.53 ± 1.15 vs 18.07 ± 0.89, P =0.0006). The upregulation of miR-22 could suppress its target gene, ErbB3, and the phosphorylation of PI3 k and Akt. Furthermore, we predicted the potential transcription promoter of miR-22 and the binding sites using online tools. Luciferase reporter analysis and sitedirected mutagenesis indicated that the binding site(GACAGCCATGTACA) of the GR, which is encoded by the Nr3 c1 gene. Downregulation of the expression of GR could upregulate the expression of miR-22, which further promoted the apoptosis of AR42 J cells.CONCLUSION GR transcriptionally represses the expression of miR-22,which further promotes the apoptosis of pancreatic acinar cells by downregulating the downstream signaling pathway.展开更多
基金Supported by Peking Union Medical College Youth Fundthe Fundamental Research Funds for the Central Universities(3332013052)
文摘Objective To screen the proteins associated with four-and-a-half LIM domains 3(FHL3) 3' untranslated region(3'UTR) in glioma cells. Methods Western blot was adopted to detect the regulatory effect of poly(C)-binding protein 2(PCBP2) on FHL3. Biotin pull-down and sliver staining were employed to screen and verify the candidate binding proteins of FHL3 3'UTR. Then liquid chromatography-tandem mass spectrometry(LC-MS/MS) and molecule annotation system were used to identify and analyze the candidate binding proteins. Immunoprecipitation was conducted to study the interaction between PCBP2 and polypyrimidine tract-binding protein 1(PTBP1), a binding protein identified by LC-MS/MS. Results PCBP2 could bind to FHL3 mRNA 3'UTR-A and inhibited the expression of FHL3 in T98 G glioms cells. 22 candidate binding proteins were identified. Among them, there were 11 RNA binding proteins, including PCBP2. PTBP1 associated with FHL3 mRNA 3'UTR and interacted with PCBP2 protein. Conclusion PCBP2 and PTBP1 can both associate with FHL3 mRNA 3'UTR through forming a protein complex.
基金Supported by Research Fund of the Doctoral Program of Higher Education (200805720004)Scientific Research Foundation for Returned Scholars, Ministry of Education of China ([2009]1001)~~
文摘[Objective] To construct prokaryotic expression vectors encoding gene Erb3binding protein （EBP1）, which plays important roles in regulating plant organ size from Nervilia fordii （Hance） Schltr. [Methods] PCR products of NfEBP1 with particular restriction sites and expression vectors, pET-28 and pET-16b were digested. Ligation, transformation and selection were performed to construct the recombinant plasmids pET-28-NfEBP1 and pET-16-NfEBP1. The recombinant plasmids were transformed into E. coli BL21 using heat -shock transformation. [Results] Recombinant plasmids pET-28-NfEBP1-1188 and pET-16-NfEBP1-1188 were constructed and transformed into expressional host cells, E. coli BL21, and validated by colony PCR, sequencing and double digestion. [Conclusion] Prokaryotic expression vectors of EBP1 gene from N. fordii were successfully constructed, which laid the foundation for characterization of the gene function.
基金supported by the Funds for Creative Research Groups of the National Natural Science Foundation of China(No.81321062)
文摘本研究旨在探讨Erb B3结合蛋白1(Erb B3-binding protein 1,Ebp1)在食管癌细胞生长中的作用及其机制。用携带Ebp1基因的慢病毒载体感染食管癌Eca109和KYSE150细胞,用real-time PCR检测食管癌组织中Ebp1 m RNA的表达情况,用MTT和结晶紫分别检测食管癌细胞生长和存活能力,用软琼脂细胞生长实验检测细胞克隆形成能力,用流式细胞术检测细胞凋亡率,用Western blot检测和凋亡有关的蛋白表达变化,用裸鼠皮下成瘤实验检测食管癌细胞的成瘤能力。结果显示,与配对的正常组织相比,食管癌组织中Ebp1 m RNA水平显著下降。过表达Ebp1不仅抑制食管癌细胞Eca109和KYSE150的体外生长和存活能力,而且能诱导这两种食管癌细胞发生凋亡,上调Rb和P53的蛋白表达,下调Cyclin D1的表达。Ebp1过表达还能抑制Eca109细胞的裸鼠皮下成瘤能力。以上结果提示,Ebp1通过诱导细胞凋亡抑制食管癌细胞体外生长能力和体内成瘤能力。
基金National Natural Science Foundation of China,No.31671440
文摘AIM To elucidate the underlying mechanism that microRNA-22(miR-22) promotes the apoptosis of rat pancreatic acinar cells(AR42 J) and the elements that regulate the expression of miR-22.METHODS One hundred nanomoles per liter of caerulein(Cae)was administrated to induce the apoptosis of AR42 J cells and the apoptosis rate was detected by flow cytometry analysis. An amylase assay kit was used to measure the amylase expression level in the supernatant. Quantitative real-time PCR(qRT-PCR)was adopted to measure miR-22 expression. We used online tools to predict the potential transcription promoter of miR-22 and the binding sites, which was further identified by using luciferase reporter analysis,chromatin immunoprecipitation(ChIP) and ChIPqP CR assays. Then, a mimic of miR-22, Nr3 c1 plasmid encoding the glucocorticoid receptor(GR), and siNr3 c1 were used to transfect AR42 J cells, respectively.The mRNA expression of miR-22, Nr3 c1, and Erb-b2 receptor tyrosine kinase 3(ErbB3) was confirmed by qRT-PCR and the apoptosis rate of AR42 J cells was detected by flow cytometry analysis. Western blot was used to detect the expression of ErbB3, GR, PI3 k, PI3 kp85α, Akt, p-Akt, Bad, Bax, Bcl-xl, Bcl-2, and cleaved caspase3.RESULTS After inducing apoptosis of AR42 J cells in vitro, the expression of miR-22 was significantly increased by2.20 ± 0.26 and 4.19 ± 0.54 times, respectively, at3 h and 6 h in comparison with the control group.As revealed by qRT-PCR assay, the expression of miR-22 was 78.25 ± 6.61 times higher in the miR-22 mimic group relative to the miRNA control group,accompanied with an obviously increased acinar cell apoptosis rate(32.53 ± 1.15 vs 18.07 ± 0.89, P =0.0006). The upregulation of miR-22 could suppress its target gene, ErbB3, and the phosphorylation of PI3 k and Akt. Furthermore, we predicted the potential transcription promoter of miR-22 and the binding sites using online tools. Luciferase reporter analysis and sitedirected mutagenesis indicated that the binding site(GACAGCCATGTACA) of the GR, which is encoded by the Nr3 c1 gene. Downregulation of the expression of GR could upregulate the expression of miR-22, which further promoted the apoptosis of AR42 J cells.CONCLUSION GR transcriptionally represses the expression of miR-22,which further promotes the apoptosis of pancreatic acinar cells by downregulating the downstream signaling pathway.
