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To explore the effect of all-trans-retinoic acid (ATRA) on the growth inhibition and cellular differentiation of C6 glioma cells.Human glioma C6 cells were treated with 5 mg/L ATRA,and the inhibition of cell growth was assessed by methyl thiazolyl tetrazolium assay. The differentiation of C6 cells was determined by flow cytometry, microscopy,transmission electron microscope, and immunohistochemical technique.Treatment of ATRA could result in the growth inhibition of C6 cells, and the cell density significantly decreased(P0.01). The cell cycle distribution was changed, G0/G1 phase was prolonged, and cells at S phase decreased(P0.01). The C6 glioma cells displayed normal fibroblast-like morphology under the microscope before the induction, and the ATRA-treated C6 cells became slightly long, turned into round in the middle, and had protrusions at both ends. The ATRA-treated C6 cells did not display obvious apoptosis by flow cytometry(P0.05).Whereas, early apoptosis was observed under the transmission electron microscope, the vacuoles increased, the mitochondria and endoplasmic reticulum were abundant in the cytoplasm, and the cellular structures tended to be normal.The expression of glial fibrillaryacidic protein in C6 cells increased in the treatment group.ATRA can inhibit the proliferation, and induce the differentiation of C6 glioma cells.

There is obvious allele disequilibrium in nasopharyngeal carcinoma at chromosome 3p, 9p, 6q, 11q, 13q and 14q. Nasopharyngeal carcinoma (NPC) susceptibility/suppressor gene candidates were obtained by molecular biology methods,such as cDNA representational difference ana-lysis. The functional research of NPC susceptibility/ suppressor gene candidates indicated: (1) The increased expression of Cx contributed to obstacles of gap junctional intercellular communication (GJIC), and resulted an aberration of GJIC; (2) BRD7, a transcript factor, was associated with cell cycle regulation; (3) NAG7,an estrogen receptor repressor, inhibited the invasive potential of human NPC cells by regulating ERalpha expression and the H-ras/p-c-Raf and JNK/AP-1/MMP1 signaling pathways; (4) NGX6, a metastasis-associated protein, can negative-regulate EGF/Ras/MAPK signaling transduction pathway, and interact with ezrin protein to inhibit invasion and metastasis of NPC cells; (5) SPLUNC1, a secreted protein, can inhibit the bacterium clone formation, and is an innate immune molecule. These data will lay an important foundation for the NPC mechanism.

To construct the genomic library of Raji cells and screen it by EBV DNA probe.High molecular weight genomic DNA of Raji cells was digested by restriction enzyme BamHI. DNA fragments ranging from 9 to 23 kb were recovered by agarose gel electrophoresis, which were ligated with Lambda DASH II vector BamHI arms pre-treated with calf intestine alkaline phosphatase (CIAP). Ligated DNA was packed in vitro using Gigapack III gold packaging extract. The library was plated on XL1-blue MRA (P2) host strain.Titering and screening of the Raji genomic library were performed.The primary titer of the Raji genomic library was 1.8 x 10(5) pfu/mL, while that of the amplified library was 2.8 x 10(8) pfu/mL. Plaques (1 x 10(5)) were screened with (32)P-labeled EBV DNA probe(EBV genome 5-3271), 4 positive clones were obtained, and 1 of the 4 positive clones was picked out randomly for the second round of plaque screening. All the phage plaques were positive. DNA of the positive clone was extracted and was digested with BamHI. The length of the inserted fragment was 8.5 kb. Sequencing and BLAST analysis revealed that the inserted fragments consisted of the BamHI-W fragment at one end and clone RP11-665A22 on chromosome 15 at the other end.The successfully established genomic library of Raji cells will provide a basis for cloning the sequences of the EBV junction sites and interpreting the mechanism of oncogenesis of EBV integration.

To explore the effect of LRRC4 on the mobility and invasion of glioblastomas U251 cells through the SDF-1alpha/CXCR4 axis.RT-PCR, transfilter cell invasion assay, adhesion assay, scraping test, scrape loading, and dye transfer assay were used to determine the effect of LRRC4 on U251 cells.SDF-1 alpha could increase the invasion in U251 which expressed CXCR4. The reintroduction of LRRC4 in U251 cells could inhibit the expression of CXCR4. LRRC4 also inhibited the adhesion ability of U251 to ECV304 as well as the mobility and invasion ability in vitro, which was mediated by the SDF-1alpha/CXCR4 axis. Furthermore, LRRC4 could greatly enhance the gap junctional intercellular communication of U251 cells.The reintroduction of LRRC4 in U251 cells can inhibit the expression of CXCR4 and the SDF-1alpha/CXCR4 axis-mediated cell invasion in vitro.

To prepare anti-LRRC4 polyclonal antibody and analyze the correlation between the expression of LRRC4 and pathological grades of gliomas in rabbits.Appropriate protein sequence with good hydrophilicity and antigenicity was chosen by analyzing with DS Gene 1.1 software. The corresponding nucleic acid sequence amplified by PCR was used to construct a recombinant pGEX-4T-2/276 bp. E.coli JM109 transformed with the recombinant was induced by IPTG to express GST-fusion protein, and the fusion protein expressed as insoluble inclusion bodies. Then the purified inclusion body was used to immunize rabbits. Once the titer of antiserum reached 1:10(8) by indirect ELISA, the serum was collected and purified. The expression-profile of LRRC4 in embryonic tissues and gliomas with various pathological grades were obtained by western blot and immunohistochemistry with the anti-LRRC4 polyclonal antibody.The highly specific anti-LRRC4 polyclonal antibody whose titer reached 1:10(8) was prepared. The relatively specific expression of LRRC4 was detected in the normal brain, but reduced expression or loss of expression in gliomas was also noticed by immunohistochemistry, and there was a correlation between the expression level of lrrc4 and the pathological grade of gliomas.The anti-LRRC4 polyclonal antibody with high titer and specificity has been obtained. A correlation between the expression level of LRRC4 and the pathological grade of gliomas is detected, which lays the foundation for advanced research of LRRC4.

To examine the expression absence of LRRC4 gene in glioblastoma cell lines.RT-PCR and Northern blot were used to detect the expression of LRRC4 gene in 6 glioblastomas cells lines. Polymerase chain reaction and DNA sequencing were used to screen the LRRC4 gene mutation, while bioinformation assay was used to search for the reason of LRRC4 gene absence in U251 cell lines.The expression of LRRC4 was absent in 6 malignant glioma cell lines (U251, U87, BT325, SF126, SF767 and M17), which were examined by Northern-blot and RT-PCR assay. All sequencing of PCR products from gDNA of SF126, SF767, and M17 cell lines contained the point mutation at the same position ( LRRC4 geneT977A) (3/5), which was a synonymous mutation. However, PCR products from gDNA of U251 and U87 cell lines (2/5) were not obtained. The expression absence of LRRC4 was ascribed to the loss of homozygosity of 7q32-ter in U251 cell lines.The expression of LRRC4 gene is absent in glioblastoma cell lines, and it offers the important experiment proof for LRRC4 to act as a new candidate of brain tumor suppressor gene from glioma. The loss of homozygosity of 7q32-ter contributed to the expression absence of LRRC4 in U251 cell lines.

Omics docking study for polygenic inheritance tumors has become an important strategy in oncology research. This review focuses on the conceptions and technologies of omics, and puts forward the central contents and omics docking for polygenic inheritance tumor to reveal the role of molecular changes at different stages of polygenic inheritance tumor at multidisciplinary and multilayer level. It is a new strategy to explore the mechanism of tumor carcinogenesis, and to regulate the network, key molecules, and drug target by combined biology effects.

Metabolomics is a new science and technology, which it refers to a holistic analytical approach to all the low molecular weight metabolites in an organism or a cell. In this paper, the definition and objective of metabolomics are provided, and the current application of metabolomic research in malignant tumors (diagnosis and therapy) are summarized.

To explore the effect of LRRC4, a glioma suppressive gene, on blocking U251 cells in G0/G1 by MAPK signaling pathway.LRRC4 was transfected into U251 cells, and at 24 hour of post-transfection, cells were split at a 1:3 dilution, challenged with 500 microg /mL G418 and formed a stable transfected clone pool. RT-PCR, Northern blot and Western blot were used to identify the stable transfectants. ERK, JNK and P38 expression changes were analyzed by Western blot. FACS analysis, Luciferase reporter gene assay and Western blot were used to detect the cell cycle and cyclin D1.LRRC4 down-regulated the expression of phosphorylated ERK2 and up-regulated the expression of total protein JNK2 (a key molecule of MAPK signaling pathway) and phosphorylated c-Jun. LRRC4 decreased the expression of mutation P53, cyclin D1 activation and its expression. U251 cells were blocked in G0/G1 by LRRC4.LRRC4 can decrease JNK2, up-regulate the phosphoralated c-Jun, down-regulate mutant P53 and cyclin D1, and therefore block U251 cells in G0/G1.

To express the recombinant SPLUNC1 protein in HNE1 cells and to study its function of bactericidal and binding to lipopolysaccharide (LPS).Full length of SPLUNC1 gene was cloned into pCMV-tag4A vector and stably transfected into HNE1 cell lines, the supernatant of cell cultures was collected. After being treated with the supernatant, the Pseudomonas aeruginosa was seeded to LB soft agar plate, and the bacteria clones were counted and analyzed. For in vitro LPS binding assay, LPS was coated to 96-well plates. We incubated in the plate with SPLUNC1 protein, and detected the binded SPLUNC1 protein by ELISA. Incubating the FITC-LPS with the SPLUNC1 stably transfected or control cells, the intracellular intensity of fluorescence was observed under the fluorescence microscope.SPLUNC1 inhibited the bacteria clone formation obviously. Although the binding efficiency of LPS and SPLUNC1 in vitro was very low, more FITC-LPS entered into the SPLUNC1 stably transfected cells.SPLUNC1 can inhibit the growth of Pseudomonas aeruginosa and bind LPS, and play an important defensive role in innate immunity of the upper airway.

To analyze the expression and location of coding protein of UBAP1 gene and to understand the relationship between the expression pattern of the protein and cell carcinogenesis.Bioinformatics was used to analyze the protein character to provide an available clue of subsequent research. The codon frame cDNA was amplified by PCR, and subcloned into enhance green fluorescence protein (EGFP) of pEGFP-C2. The recombinant plasmid was transfected into HNE1 cells. The expression of coding protein was observed by fluorescence microscopy.The expressed GFP-fusion protein generated striking green fluorescence in the cytoplasm in HNE1 cells. EGFP/UBAP1 was expressed and existed mainly in the nuclear, especially accumulated on the nuclear envelope.The expression difference in HNE1 might be related to the carcinogenesis of NPC.

To filter biomarkers of nasopharyngeal carcinoma (NPC) by constructing the homogenesis tissue gene expression profiling with the whole human genome GeneChip.The epithelium cells of the homogenesis NPC and the pure nasopharyngeal normal tissues microdissected from nasopharyngeal biopsy which was preserved in the RNAlater were used to isolate RNA and then to harvest the aRNA through in vitro transcription, and aRNA prober was labled to hybridize to HG-U133. plus 2.0, so the expression profiling of each homogenesis tissue could be constructed.Some candidate biomarker genes related to the tumorigenesis of NPC had been filtered by comparing the expression profiling of NPC samples with the expression profiling of normal nasopharyngeal epithelia samples. Any genes regarding the metastasis of NPC might have been selected by comparing the expression profiling of no-metastasis samples with those of the metastasis samples.Using the whole genome GeneChip to construct the expression profiling for the microdissected homogenesis tissue is effective to filter the candidate biomarker genes.