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. Immuno-precipitation 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 T98G 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.

OBJECTIVETo study the role of adaptor protein Dok6 in neurite outgrowth in PC12 cells.

METHODSSeries of fusion clones were constructed by fusing different domains of Dok6 into mutant TrkC/Y516F. These constructs were transiently transfected into PC12 cells separately and the expression levels of fusion proteins were detected by Western blot. Neurite outgrowth in these PC12 cells was tested after stimulation of NT-3.

RESULTSEach fusion clone was stably expressed in PC12 cells. The fusion clones that fused both TrkC/Y516F-Dok6 (PTB+C) and TrkC/Y516F-Dok6C rescued the loss of neurite outgrowth in PC12 cells resulting from the mutation in tyrosine 516, while fusion clones that fused with single TrkC/Y516F-Dok6PTB did not show such effect.

CONCLUSIONDok6 can promote neurite outgrowth induced by NT-3 stimulation through its C-terminal in TrkC-positive PC12 cells.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Animals ; Neurites ; drug effects ; physiology ; Neurotrophin 3 ; pharmacology ; PC12 Cells ; Rats ; Receptor, trkC ; metabolism ; Transfection

OBJECTIVETo study the interaction between ShcD and TrkC and to reveal the molecular mechanism of the downstream signal transduction of TrkC.

METHODSYeast two-hybrid assay was used. The intracellular domains of TrkC and TrkC mutants were cloned into pAS2-1, and ShcD and its four domains (CH2, PTB, CH1, and SH2 domains) were cloned into pACT2 vector respectively. The constructs were separately cotransformed into yeast. beta-galactosidase activity was measured to detect their interactions. TrkC was cloned into pmRFP (carrying red fluorescent protein), and ShcD was cloned into pEGFP (carrying green fluorescent protein). pmRFP-TrkC and pEGFP-ShcD were co-transfected into 293T cells, and then the cells were fixed and subjected to confocal analysis to study their subcellular localization.

RESULTSShcD interacted with TrkC but not with kinase dead mutant TrkCM1(K572A). Both PTB and SH2 domains were capable of binding to TrkC, and PTB domain bound NPQY motif of TrkC. ShcD colocalized with TrkC throughout the cytoplasm and in the plasma membrane in 293T cells.

CONCLUSIONShcD binds to TrkC in a kinase-activity-dependent manner through its PTB and SH2 domains.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Binding Sites ; Cells, Cultured ; Genetic Vectors ; Humans ; Plasmids ; genetics ; Protein Binding ; Receptor, trkC ; genetics ; metabolism ; Shc Signaling Adaptor Proteins ; genetics ; metabolism ; Transfection ; Transformation, Bacterial ; Two-Hybrid System Techniques ; src Homology Domains ; genetics

OBJECTIVETo explore the influences of the restoration of neural adhesion molecule NECL1 on the morphology, migration, and invasion of NECL1-deficient glioma cell lines.

METHODSScratch and Transwell assays were used to observe the cell migration and invasion, the activities of extracellular metalloproteinases were measured, and the cell morphology was observed. Astrocytes marker glial fibrillary acidic protein was detected by Western blot after the restoration of NECL1 in glioma U251 cell line.

RESULTSIn NECL1-deficient U251 glioma cell lines, migration and invasion were inhibited. The U251 cells was differentiated potentially to astrocytes, and glial fibrillary acidic protein was up-regulated after the restoration of the NECL1 expression.

CONCLUSIONAs a potential tumor repressor, the neural adhesion molecule NECL1 can inhibit the migration and invasion of glioma cell and induces its differentiation.

Brain Neoplasms ; metabolism ; pathology ; Cell Differentiation ; Cell Line, Tumor ; Cell Movement ; Glioma ; metabolism ; pathology ; Humans ; Neoplasm Invasiveness ; Neural Cell Adhesion Molecules ; metabolism

OBJECTIVETo screen the asymmetric dimethyl arginines (ADMA)-containing proteins which could combine with protein arginine methyltransferase 1 (PRMT1).

METHODSWestern blot was adopted to identify the expression of PRMT1 and the proteins with ADMA in glioma cell lines and normal brain tissues, and then to detect the changes of ADMA level after knock-down of PRMT1 with RNAi transfection in U87MG cells. Co-Immunoprecipitation (Co-IP), western blot, and sliver staining were employed to screen the candidate binding proteins of PRMT1. Then liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify the binding proteins of PRMT1.

RESULTSThe expression of PRMT1 and some levels of ADMA were higher in glioma cell lines than in normal brain tissues. After knocking down PRMT1, some ADMA levels were found declined. After screening the binding proteins of PRMT1 with Co-IP and LC-MS/MS, 26 candidate binding proteins were identified. Among them, 6 candidate proteins had higher ions scores (> 38) and bioinformation analysis predicted that SEC23-IP, ANKHD1-EIF4EBP3 protein, and 1-phosphatidylinositol-3-phosphate 5-kinase isoform 2 had possible methylated aginine sites.

CONCLUSIONSThe high expression of PRMT1 in glioma may induce the change of ADMA levels. Altogether 26 candidate proteins were identified, which contain ADMA and specifically bind with PRMT1.

Arginine ; analogs & derivatives ; analysis ; Cell Line, Tumor ; Chromatography, Liquid ; Glioma ; chemistry ; Humans ; Immunoprecipitation ; Protein-Arginine N-Methyltransferases ; analysis ; physiology ; Repressor Proteins ; analysis ; physiology ; Substrate Specificity ; Tandem Mass Spectrometry

OBJECTIVETo express and purify the recombinant human pigment epithelium-derived factor (PEDF) which inhibits the proliferation of the endothelium cells from blood vessel in E.coli.

METHODSPEDF gene was inserted into the prokaryotic expression vector pGEX-4T-2. The recombinant protein PEDF was expressed in E.coli BL-21, and purified by the GST Sepharose 4B affinity column. The recombinant human PEDF protein was identified by Western blot and mass spectrum. The biological activity of the recombinant human PEDF protein was measured by using MTT.

RESULTSThe 46 kDa recombinant human PEDF protein was obtained. It significantly inhibited the proliferation of the human umbilical vein cell line HUVEC.

CONCLUSIONThe recombinant human PEDF with anti-angiogenesis activity protein may be successfully purify through prokaryotic expression.

Angiogenesis Inhibitors ; pharmacology ; Apoptosis ; drug effects ; Base Sequence ; Cell Division ; drug effects ; Cells, Cultured ; Cloning, Molecular ; Endothelium, Vascular ; cytology ; Eye Proteins ; pharmacology ; Humans ; Molecular Sequence Data ; Nerve Growth Factors ; pharmacology ; Prokaryotic Cells ; metabolism ; Recombinant Proteins ; pharmacology ; Serpins ; pharmacology ; Umbilical Veins ; cytology

OBJECTIVETo screen and identify the possible existence of natural antisense transcript (NAT) within the mouse neocortex.

METHODSSixty-three cerebral cortex layer-specific genes were screened by bioinformatics prediction in mice, among which 31 mice with potential NATs were screened. NAT was identified using reverse transcription polymerase chain reaction (RT-PCR) and then cloned in pGEM-T Vector System for sequencing.

RESULTSAmong 31 genes predicted using bioinformatics, 8 were proved to be NAT positive by RT-PCR.

CONCLUSIONSNATs exist in the mouse neocortex tissue during the development of cerebral cortex. NATs may influence mouse cortical development by regulating the related coding genes.

Animals ; Cell Line ; Cerebral Cortex ; Mice ; Molecular Sequence Data ; RNA, Antisense ; genetics ; RNA, Messenger ; genetics

OBJECTIVETo study the regulation role of tumor suppressor NECL1 on the proliferation of glioma cell line.

METHODSWe detected the expression level of NECL1 in human normal brain tissue and glioma cell lines using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot. T98G cell line in which NECL1 was silent and whose transfection efficiency was relatively high as target cell was chosen, and the effect of NECL1 on the proliferation of T98G cell line in vitro was detected by using cell growth curve, flow cytometry, and Hoechst staining.

RESULTSNECL1 was abrogated or markedly reduced in 6 glioma cell lines. When NECL1 was overexpressed in T98G cell line, the cell growth rate obviously decreased and the number of apoptotic cells remarkably increased when compared with the control group.

CONCLUSIONNECL1 may inhibit the proliferation of T98G cells by inducing its apoptosis.

Apoptosis ; genetics ; physiology ; Blotting, Western ; Cell Line, Tumor ; Cell Proliferation ; Glioma ; metabolism ; pathology ; Humans ; Immunoglobulins ; genetics ; metabolism ; In Vitro Techniques ; Membrane Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo study the role of cell adhesion molecules Necl1 in synaptogenesis in primary cultured neurons.

METHODSSemi-quantitive reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression pattern of Necl1 in the neuronal differentiation cell model in vitro. Western blot was performed to detect the expression pattern of Necl1 in primary cultured rat neurons and in purified synaptosome. Immunofluoresence was used to detect the synapse formation in primary neurons and in 293 cells co-culture and to detect the density of synapses in primary neuron with ectopic expression of Necl1.

RESULTSNecl1 expression increased after retinoic acid (RA) induction in SH-SY5Y and P19 cells. The increase of Necl1 expression was consistent with the days of primary neurons culture in vitro, and Necl1 partly localized in synaptosome. The overexpression of Necl1 in 293 cells induced the synapse formation between cocultured 293 cells and neurons. Ectopic expression of Necl1 in primary neurons increased the density of synapses.

CONCLUSIONNecl1 plays an important role in neuronal synapse formation.

Animals ; Blotting, Western ; Cell Adhesion Molecules, Neuronal ; genetics ; metabolism ; Cell Differentiation ; drug effects ; genetics ; Cell Line ; Cells, Cultured ; Fluorescent Antibody Technique ; Humans ; Neurons ; cytology ; drug effects ; metabolism ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Synapses ; drug effects ; metabolism ; physiology ; Synaptosomes ; drug effects ; metabolism ; Tretinoin ; pharmacology

OBJECTIVETo detect the expression of CBX7 in human glioma and investigate the potential regulatory effect of abnormally expressed microRNAs on CBX7 expression.

METHODSSemi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot were applied to detect the expression pattern of CBX7 in 2 human normal brain tissues, 9 glioma tissues, and 3 glioma cell lines. Miranda algorithm and Ensemble Machine Learning algorithm were combined to predict miRNAs that target human CBX7. The expression of miR-9 in those tissues and cell lines were detected by real-time PCR. After miR-9 overexpression in 293ET and miR-9 knock-down in T98G, luciferase assay and Western blot were used to confirm the effect of miR-9 on CBX7 expression. MTT assay and flow cytometry were applied to detect the effect of miR-9 knock-down on T98G cells.

RESULTSNo obvious difference in the CBX7 mRNA level between normal and tumor tissues was observed, while the protein level of CBX7 was abrogated or markedly reduced in glioma tissues and cell lines. Several miRNAs including miR-9 may target CBX7 by bioinformatics prediction. MiR-9 was up-regulated in glioma tissues and cell lines. In 293ET cell, luciferase activity of CBX7-3'UTR reporter was decreased to 24% after miR-9 overexpression. After miR-9 knock-down in T98G cell, the luciferase activity was increased by 1.8 fold and there was no change of CBX7 mRNA, while the protein level of endogenous CBX7 was significantly increased. The number of survival T98G cells increased and cells in G1 phase decreased after miR-9 knock-down.

CONCLUSIONIn human glioma, CBX7 is down-regulated by the inhibition of miR-9 at posttranscriptional level.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Algorithms ; Blotting, Western ; Brain ; metabolism ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; Child ; Female ; Flow Cytometry ; Glioma ; genetics ; metabolism ; Humans ; In Vitro Techniques ; Male ; MicroRNAs ; genetics ; physiology ; Middle Aged ; Polycomb Repressive Complex 1 ; Repressor Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Young Adult