RNA-binding Protein UNR Promotes Glioma Cell Migration and Regulates the Expression of Ribosomal Protein L9 / 中国医学科学杂志(英文版)

Objective To investigate the role of RNA binding protein─upstream-of-N-Ras (UNR) in the development of glioma and its molecular mechanism.Methods First, bioinformatics analysis of CGGA database was performed to detect UNR expression level and prognosis of patients with glioma. Western blot and real-time PCR were used to detect UNR expression level in glioma cell lines and tissues. Next, UNR siRNAs were transfected in glioma cells, and MTS assay and scratch wound-healing assay were used to detect changes in cell proliferation and migration. Then, the candidate UNR target mRNAs were identified by analyzing the sequencing data of UNR iCLIP-seq, RNA sequencing and ribosome profiling databases of human melanoma. RNA immunoprecipitation and biotin pull-down assays were used to identify the UNR target mRNAs in glioma cells. Finally, western blot was used to detect the effect of UNR knockdown on ribosomal protein L9 (RPL9) and RPL9 protein expression level in glioma cell lines. RPL9 siRNA was transfected in A172 and T98G and the expression of vimentin in the cells was detected with western blot.Results Bioinformatics analysis showed that UNR mRNA expression level was significantly higher in high-grade glioma [Grade 2 (n=126), Grade 3 (n=51), Grade 4 (n=128), P<0.001]. UNR high expression levels were associated with poor prognosis (P=0.0177). UNR had high expression level in glioma cell lines and patient samples compared with normal cell lines and normal brain samples (P<0.01). Knockdown of UNR inhibited glioma cells migration (P<0.05), but did not inhibit glioma cells growth in three glioma cell lines. UNR binded the 3' untranslated region (UTR) of PTEN and RPL9 mRNAs. RPL9 protein was significantly highly expressed in most glioma cell lines (n=9) and knockdown of UNR resulted in a downregulation of RPL9 protein expression. Epithelial-mesenchymal transition (EMT)-related marker─vimentin was positively regulated by RPL9.Conclusions UNR could bind to the 3'UTR of PTEN and RPL9 in glioma cell lines, therefore promoting glioma cell migration and regulating the expression of RPL9. Here, we establish a link between UNR and RPL9 protein, which will provide new ideas for the further study of glioma.

Identification of differentially expressed microRNAs by microarray: a possible role for microRNAs gene in medulloblastomas / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>MicroRNAs (miRNAs) are small noncoding regulatory RNAs whose aberrant expression may be observed in many malignancies. However, few data are yet available on human primary medulloblastomas. This work aimed to identify that whether miRNAs would be aberrantly expressed in tumor tissues compared with non-tumorous cerebellum tissues from same patients, and to explore a possible role during carcinogenesis.</p><p><b>METHODS</b>A high throughput microRNA microarray was performed in human primary medulloblastoma specimens to investigate differentially expressed miRNAs, and some miRNAs were validated using real-time quantitative RT-PCR method. In addition, the predicted target genes for the most significantly down- or up-regulated miRNAs were analyzed by using a newly modified ensemble algorithm.</p><p><b>RESULTS</b>Nine miRNA species were differentially expressed in medulloblastoma specimens versus normal non-tumorous cerebellum tissues. Of these, 4 were over expressed and 5 were under expressed. The changes ranged from 0.02-fold to 6.61-fold. These findings were confirmed using real-time quantitative RT-PCR for most significant deregulated miRNAs (miR-17, miR-100, miR-106b, and miR-218) which are novel and have not been previously published. Interestingly, most of the predicted target genes for these miRNAs were involved in medulloblastoma carcinogenesis.</p><p><b>CONCLUSIONS</b>MiRNAs are differentially expressed between human medulloblastoma and non-tumorous cerebellum tissue. MiRNAs may play a role in the tumorigenesis of medulloblastoma and maybe serve as potential targets for novel therapeutic strategies in future.</p>

Neural adhesion molecule NECL1 inhibits migration, invasion, and potentially induces differentiation of glioma cell / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the influences of the restoration of neural adhesion molecule NECL1 on the morphology, migration, and invasion of NECL1-deficient glioma cell lines.</p><p><b>METHODS</b>Scratch 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.</p><p><b>RESULTS</b>In 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.</p><p><b>CONCLUSION</b>As a potential tumor repressor, the neural adhesion molecule NECL1 can inhibit the migration and invasion of glioma cell and induces its differentiation.</p>

MiR-9 regulates the expression of CBX7 in human glioma / 中国医学科学院学报

<p><b>OBJECTIVE</b>To detect the expression of CBX7 in human glioma and investigate the potential regulatory effect of abnormally expressed microRNAs on CBX7 expression.</p><p><b>METHODS</b>Semi-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.</p><p><b>RESULTS</b>No 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.</p><p><b>CONCLUSION</b>In human glioma, CBX7 is down-regulated by the inhibition of miR-9 at posttranscriptional level.</p>

Role of cell adhesion molecules Necl1 in synaptogenesis in primary cultured rat neurons / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the role of cell adhesion molecules Necl1 in synaptogenesis in primary cultured neurons.</p><p><b>METHODS</b>Semi-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.</p><p><b>RESULTS</b>Necl1 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.</p><p><b>CONCLUSION</b>Necl1 plays an important role in neuronal synapse formation.</p>

Effect of NECL1 on the proliferation of T98G glioma cell line / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the regulation role of tumor suppressor NECL1 on the proliferation of glioma cell line.</p><p><b>METHODS</b>We 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.</p><p><b>RESULTS</b>NECL1 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.</p><p><b>CONCLUSION</b>NECL1 may inhibit the proliferation of T98G cells by inducing its apoptosis.</p>

Expression pattern of polycomb gene Nspc1 at the early developmental stage in zebrafish / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the expression pattern of Polycomb gene Nspc1 at the early developmental stage in zebrafish.</p><p><b>METHODS</b>In situ hybridization probe for Nspc1 was designed according to the GenBank information. Collecting zebrafish embryos at different stages including one cell stage, two-cell stage, bud stage, and somites stage, we hybridized them with the prepared probe. Then the hybridization signals at different intervals were observed and photographed at the right time.</p><p><b>RESULTS</b>Nspc1 was expressed globally at the early stage. Its expression specificity began at the somites stage, mainly in the nervous system of the head.</p><p><b>CONCLUSION</b>Nspc1 may play essential roles in the early stage development of zebrafish, especially in the nervous system.</p>

MiRE: a graphical R package for microRNA-related analysis / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To provide a set of useful analysis tools for the researchers to explore the microRNA data.</p><p><b>METHODS</b>The R language was used for generating the Graphical Users Interface and implementing most functions. Some Practical Extraction and Report Language (Perl) scripts were used for parsing source files.</p><p><b>RESULTS</b>We developed a graphical R package named miRE, which was designated for the analysis of microRNA functions, genomic organization, etc. This package provided effective and convenient tools for molecular biologists to deal with routine analyses in microRNA-related research. With its help, the users would be able to build a desktop-centered microRNA research environment quite easily and effectively. miRE is freely available at http://www. biosino.org/-kanghu/WorkPresentation/miRE/miRE.html. A detailed user manual and tutorials with example code and image are also available.</p><p><b>CONCLUSION</b>miRE is a tool providing an open-source, user-friendly, integrated interface for microRNA-related analysis. With its help, researchers can perform microRNA-related analysis more efficiently.</p>

Bioluminescent imaging monitoring of a anti-angiogenesis therapeutic gene vasostatin in tumor cell PC3 / 中国医学科学院学报

<p><b>OBJECTIVE</b>To generate a sensitive tool for noninvasive monitoring of a therapeutic gene vasostatin.</p><p><b>METHODS</b>We fused the bioluminescent reporter gene firefly luciferase to the therapeutic transgene vasostatin and ensured that these two proteins would not interrupt each other and kept their own natural character.</p><p><b>RESULTS</b>We therefore examined clones of PC3 cells stably expressing fusion gene and positive controlfluc with bioluminescence. In vivo imaging of PC3-Fluc subcutaneous tumors showed that the mean tumor bioluminescence increased in animals over several weeks.</p><p><b>CONCLUSION</b>Noninvasive monitoring facilitates the detection of gene expression in vivo and in vitro.</p>

Recombinant human pigment epithelium-derived factor inhibits the proliferation of the endothelial cells from blood vessels / 中国医学科学院学报

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>PEDF 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.</p><p><b>RESULTS</b>The 46 kDa recombinant human PEDF protein was obtained. It significantly inhibited the proliferation of the human umbilical vein cell line HUVEC.</p><p><b>CONCLUSION</b>The recombinant human PEDF with anti-angiogenesis activity protein may be successfully purify through prokaryotic expression.</p>

The expression of collagen IX in the apical disc of idiopathic scoliosis / 中华外科杂志

<p><b>OBJECTIVE</b>To study the distribution of collagen IX gene in the disc and to determine its role in the pathogeny of idiopathic scoliosis (IS).</p><p><b>METHODS</b>The data included apical disc and intermediate disc from 14 cases of adolescent IS, 26 discs from 13 cases of scoliosis of confirmed pathogeny (CPS), which included 10 cases of congenital scoliosis and neurofibromatosis scoliosis. Six discs were obtained from 3 cases of normal young man served as controls. The distribution of collagen IX was studied in the apical disc of IS by immunohistochemistry and in situ hybridization (ISH) with RNA probe. The figure of collagen IX hybridization in the endplate cartilage was input to the figure analysis system. The mRNA content of collagen IX was compared between each group by SPSS software.</p><p><b>RESULTS</b>Collagen IX was mainly distributed in the inner fibrous annulus, nucleus and endplate cartilage. Collagen IX was secreted by the little round chondrocyte-like cells, which was not expressed in the hypertrophic cells. There was significant difference of collagen IX mRNA content between the concave side of apical disc in the IS and the normal disc(P < 0.05), and also between intermediate vertebrae of CS group and normal.</p><p><b>CONCLUSIONS</b>There is no obvious abnormal distribution of collagen IX in the disc of idiopathic scoliosis. Collagen IX may be related to the pathogensis of IS. More investigation such as quantity analysis and protein function determination is needed to confirm its role in the pathogenicity of IS.</p>

cDNAs cloning of SARS-CoV PUMC2 viral genome / 中国医学科学院学报

<p><b>OBJECTIVE</b>To get the cDNA clones which cover the whole genome of SARS-CoV PUMC2 strain.</p><p><b>METHODS</b>Using the SARS-CoV PUMC2 strain genomic RNA as the template, the cDNA fragments were amplified by RT-PCR, the PCR products were further purified and ligated into the pGEM-T vector, and all the clones obtained were sequenced.</p><p><b>RESULTS</b>The cDNA clones which cover the whole genome of SARS-CoV PUMC2 strain were obtained.</p><p><b>CONCLUSIONS</b>These cDNAs can be provided for the function study of SARS-CoV proteins and the construction of full-length infectious cDNA clone of SARS-CoV.</p>

Cloning, expression and purification of SARS coronavirus PUMC2 strain nucleocapsid protein / 中国医学科学院学报

<p><b>OBJECTIVE</b>To clone, express and purify nucleocapsid protein from SARS coronavirus PUMC2 strain.</p><p><b>METHODS</b>According to the published SARS coronavirus genome sequences, the full length cDNA of N protein from SARS coronavirus PUMC2 strain was cloned by RT-PCR and the cDNA was cloned into the pET32a expression vector. The recombinant N protein was expressed in E. coli BL21 (DE3), and purified by Ni(2+)-NTA.</p><p><b>RESULTS</b>Prokaryoticly expressed and purified N protein of SARS coronavirus PUMC2 strain was obtained.</p><p><b>CONCLUSIONS</b>The SARS coronavirus recombinant N protein obtained by genetic engineering methods can be used for further functional study of SARS coronavirus N protein.</p>

Cloning and expression of human single-chain Fv antibody against amyloid beta peptide involved in Alzheimer's disease / 中国医学科学院学报

<p><b>OBJECTIVE</b>To screen out the specific antibody clones against amyloid beta peptide 40, and clone the antibody gene and express it in a bacterial system, so as to provide a solid basis for novel diagnostic and therapeutic methods for Alzheimer's Disease.</p><p><b>METHODS</b>beta amyloid peptide 40 was bound on the solid surface of Nunc plates as antigen to screen the binding clones from a phage-display human single-chain Fv antibody library. After five rounds of bio-panning, the host E. coli TG1 was infected with eluted filamentous phage from the last turn of selection. 55 well-separated colonies were picked randomly from the plates and the specific positive clones were identified by ELISA test. The single-chain Fv antibody gene was sequenced and their amino acids sequence was deduced. The scFv antibody gene was sub-cloned into a protokayotic expression vector pGEX-6P-1 and transformed into bacteria strain BL21 to express the glutathione-S-transferase (GST) fusion single-chain antibody.</p><p><b>RESULTS</b>ELISA test showed that 33 of the 55 clones could bind amyloid beta peptide 40 and 10 of the 33 clones could be inhibited by amyloid beta peptide 40 itself to below 50% of its original binding activities. Five of the 10 clones could also be inhibited by amyloid beta peptide 1-16 to the same level, which meant that the binding epitope of the antibody from the 5 clones was between first to sixteenth amino acids at amino-end of amyloid beta peptide 40. DNA sequencing data demonstrated that the gene of the single-chain antibody specifically against amyloid beta peptide 40 was consisted of 768 bp and the deduced amino acids sequence confirmed its typical antibody structure. The complement determinant regions and framework regions were discriminated empirically. After cloning the antibody gene into a protokayotic system, the GST fusion antibody was expressed as the expected size.</p><p><b>CONCLUSIONS</b>After five rounds of bio-panning and subsequently serial ELISA testing, the specific antibody clones against amyloid beta peptide 40 were screened out successfully. The antibody gene DNA sequence and amino acids sequence were analyzed and confirmed. The fusion antibody was expressed as expected in the bacterial system.</p>

The cloning and prokaryotic expression of human tumor necrosis factor like protein / 中国医学科学院学报

<p><b>OBJECTIVE</b>To identify and clone the gene encoding human TNFLP (tumor necrosis factor like protein) for some functional study on TNFLP.</p><p><b>METHODS</b>The full-length cDNA of TNFLP was isolated from fetal brain cDNA library. Several kinds of software were used to analyze nucleotide sequence and amino acid sequence of TNFLP. TNFLP mRNA distribution was identified by Northern blot. TNFLP-C and TNFLP-N were expressed in E. coli with GST expression system.</p><p><b>RESULTS</b>The cDNA of human TNFLP was 2,112 bp, which encoded protein of 208 amino acid. Hydrophobility analysis found there were two hydrophobility regions of human TNFLP. TNFLP-C (112-207 amino acid) and mouse TNF-alpha were homologous. The identity of their amino acid sequence was 42%. Moreover, both of them had a motif-TYKRL. TNFLP was located in chromosome 16. Human TNFLP was widely expressed in various human tissues. Northern blot showed TNFLP was highly expressed in heart, brain and spleen, only one transcript can be seen. GST-TNFLP-C and GST-TNFLP-N fusion proteins were obtained.</p><p><b>CONCLUSIONS</b>Tissue expression spectrum of TNFLP and prokaryotic expression of TNFLP have been done, which establish the base for the functional analysis of TNFLP.</p>

Cloning, expression and purification of neural specific HuD cDNA / 中国医学科学院学报

<p><b>OBJECTIVE</b>To prokaryoticly express and purify HuD protein and its RNA recognition motifs.</p><p><b>METHODS</b>HuD protein was prokaryoticly expressed and purified by molecular cloning technology. Its biologic activity was testified by Western Blot.</p><p><b>RESULTS</b>Purified HuD protein and its RNA recognized motifs were observed.</p><p><b>CONCLUSIONS</b>The result might aid for basic research and clinical application.</p>

The identification and cloning of human M961 full-length cDNA and its splicing isoform / 中国医学科学院学报

<p><b>OBJECTIVE</b>To identify and clone the gene encoding human M96 gene and study its expression spectrum in several blood cell lines.</p><p><b>METHODS</b>According to the sequence of human EST which was highly homologous to the mouse M96 gene, primers used for library screening were synthesized, then the human adult testis and fetal brain cDNA library were screened. The gene was analyzed by making use of BLAST and CLUSTAL W, and its expression spectrum was studied by multiple-cell lines Northern blot analysis. The expression change of M961 in cell differentiation was observed by use of K562 cell line induced by hemin.</p><p><b>RESULTS</b>Two cDNA clones encoding human M96 gene were isolated, identified and named as M961, and M962. They were found to be isoforms of each other. Northern, blot showed that M961 gene was expressed highly in CEM, Hel, Dami and K562 cell lines. However, during K562 cell line differentiation, process the expression of M961 elevated only slightly.</p><p><b>CONCLUSIONS</b>M961 gene was expressed highly in pluripotent cell lines with erythrocytic and megakaryocytic potentials.</p>