1.Tumor suppressor genes on frequently deleted chromosome 3p in nasopharyngeal carcinoma.
Juan CHEN ; Li FU ; Li-Yi ZHANG ; Dora L KWONG ; Li YAN ; Xin-Yuan GUAN
Chinese Journal of Cancer 2012;31(5):215-222
Nasopharyngeal carcinoma (NPC) is among the most common malignancies in southern China. Deletion of genomic DNA, which occurs during the complex pathogenesis process for NPC, represents a pivotal mechanism in the inactivation of tumor suppressor genes (TSGs). In many circumstances, loss of TSGs can be detected as diagnostic and prognostic markers in cancer. The short arm of chromosome 3 (3p) is a frequently deleted chromosomal region in NPC, with 3p21.1-21.2 and 3p25.2-26.1 being the most frequently deleted minimal regions. In recent years, our research group and others have focused on the identification and characterization of novel target TSGs at 3p, such as RASSF1A, BLU, RBMS3, and CHL1, in the development and progression of NPC. In this review, we summarize recent findings of TSGs at 3p and discuss some of these genes in detail. A better understanding of TSGs at 3p will significantly improve our understanding of NPC pathogenesis, diagnosis, and treatment.
Cell Adhesion Molecules
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genetics
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Chromosome Deletion
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Chromosomes, Human, Pair 3
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genetics
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Epigenesis, Genetic
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Gene Expression Regulation, Neoplastic
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Genes, Tumor Suppressor
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Heterotrimeric GTP-Binding Proteins
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genetics
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Humans
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Nasopharyngeal Neoplasms
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genetics
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RNA-Binding Proteins
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genetics
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Trans-Activators
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genetics
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Tumor Suppressor Proteins
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genetics
3.The effects of complement C3f segment on expression and secretion of collagen I, III and transforming growth factor-beta1 in human embryonic lung fibroblast.
Wei LIU ; Qing-Bo MA ; Juan-Juan CHEN ; Hai-Xia KONG ; Mao-Ti WEI ; Shi-Xin WANG
Chinese Journal of Industrial Hygiene and Occupational Diseases 2012;30(1):61-63
OBJECTIVETo observe the effects of complement fragment C3f on expression and secretion of collagen I, III and transforming growth factor( TGF)-beta1 in human embryonic lung fibroblast (MRC-5) cells.
METHODSMRC-5 cells were cultured with C3f (the synthetic 17 peptides fragments of complement C3). The extracellular and intracellular expression levels of type I, III collagens and TGF-beta1 in MRC-5 cultures were detected by ELISA and immunohistochemistry, respectively.
RESULTSThe expression levels of type I, III collagen and TGF-beta1 in the supernatant of MRC-5 cultures decreased significantly with the concentrations of C3f as compared with controls (P < 0.05). Also the expression level of TGF-beta1 in MRC-5 cytoplasm reduced significantly as compared with controls (P < 0.05).
CONCLUSIONThe results of present in vitro study showed that the complement fragment C3f could reduce the formation of TGF-beta1 and type I, III collagens in MRC-5 cells, and inhibit the lung tissue fibrosis.
Cell Line ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Complement C3b ; pharmacology ; Fibroblasts ; drug effects ; metabolism ; Humans ; Lung ; cytology ; drug effects ; embryology ; Transforming Growth Factor beta1 ; metabolism
4.Inhibition of mutant-type p53 by a chimeric U6 maxizyme in hepatocellular carcinoma cell lines.
Xin-juan KONG ; Ju-sheng LIN ; Yu-hu SONG ; You-xin JIN
Chinese Journal of Hepatology 2005;13(10):759-762
OBJECTIVETo study the inhibition of maxizyme (Mz) directed against the mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG --> AGT) both in cell-free system and in MHCC97 cell lines.
METHODSMaxizyme and control mutant maxizyme (G5 --> A5) were designed by computer and cloned into the eukaryotic expression vector pBSKneoU6 (pU6Mz, pU6asMz). Mz was driven by T7 RNA polymerase promoter in vitro. In the cell lines, U6 promoter was driven by RNA PolIII. The mutant type p53 gene fragment was cloned into the pGEM-T vector under the T7 promoter control. The 32P-labeled mtp53 transcript was the target RNA. Cold maxizyme transcripts were incubated with 32P-labeled target RNA in vitro. pU6Mz was introduced into MHCC97 cells by Lipofectamine2000 and mtp53 expression was analyzed by RT-PCR and Western blot.
RESULTSIn vitro cleavage showed that pU6Mz was very active with cleavage efficiency of 42% while pU6asMz was not. The wild type p53 was not cleaved. Partial down-regulation of mtp53 mRNA and mtp53 protein were observed in MHCC97 cells transfected with pU6Mz but not those with pU6asMz. The proliferation of MHCC cells was inhibited by MTT analysis.
CONCLUSIONOur findings suggest that the chimeric U6 maxizyme against the mtp53 is a new promising gene therapeutic agent in treating hepatocellular carcinoma.
Carcinoma, Hepatocellular ; genetics ; Cell Line, Tumor ; Genetic Therapy ; methods ; Genetic Vectors ; Humans ; Liver Neoplasms ; genetics ; Nucleic Acid Conformation ; Point Mutation ; Protein Conformation ; RNA, Catalytic ; RNA, Messenger ; chemical synthesis ; metabolism ; Recombinant Fusion Proteins ; Ribonuclease T1 ; pharmacology ; Tumor Suppressor Protein p53 ; genetics
5.The inhibitory effects on hepatitis B virus replication by stable expression of DN mutants of hepatitis B virus X gene pRev X-GFP.
Jia-Wu SONG ; Jiu-Sheng LIN ; Xin-Juan KONG ; Kuo-Huan LIANG
Chinese Journal of Hepatology 2004;12(7):392-394
OBJECTIVEPersistent replication of hepatitis B virus (HBV) is one of the major obstacles in HBV infection treatment. Reduction or clearance of HBV propagation would be one of the aims of HBV therapy. The drugs approved in clinical used such as nucleotide analogs or interferon, were limited effects on HBV replication. The newly developing gene therapy method, dominant negative mutants, were be used as new promising HBV therapy strategy, and a dominant negative mutant of HBVX gene pRev X-GFP which we have reported in our previous study has some effects both on HBV replication and expression in transient expression, but the effects were interfered by persistent secretion of HBV in HepG2 2.2.15 cells without transfection pRev X-GFP in the experiment. To make sure the effects of dominant negative mutant of pRev X-GFP, we established a HBX DN stable express cell clone, and evaluated the effects of HBX dominant negative mutant on HBV replication.
METHODSThe X gene mutant, in which a specific point mutation of 3'-end ATG to AAG and fused with human green fluorescence protein (GFP) were cloned into pRev TRE vector, assigned to pRev HBX-GFP dominant mutant (pRev X-GFP). And the plasmid contains the wild type X gene or GFP gene was cloned into the same vector to construct pRev Xwt, pRev GFP constructs. All the constructs then transfected into HepG2 2.2.15 cells by liposome. After 7 days resistance selection of hygromycin (300 microg/ml), and cell clones which stable expression HBX-GFP, HBXwt, GFP were obtained. After reseeding of 106 cells of each clones in 12 wells with a 12 well cell plate and another 12 wells 2.2.15 cell were serve as blank control. The cells and media were harvested after cultured in DMEM with 10% FBS for 3 days. HBV-related DNA was assayed by dot blot and Southern blot.
RESULTSThe 100% expression of pRev HBX-GFP, GFP and wild type X constructs were obtained. The stable expressed HBX-GFP can significantly reduce HBV DNA level both in cell media and cells by dot blot and Southern blot analysis, but not for pRev Xwt and pRev GFP.
CONCLUSIONThe dominant negative mutant pRev HBX-GFP can significantly inhibit the HBV gene expression. It also suggested that X gene might be one of promising target for HBV gene therapy.
Carcinoma, Hepatocellular ; pathology ; Cloning, Molecular ; DNA Replication ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Hepatitis B virus ; genetics ; physiology ; Humans ; Liver Neoplasms ; pathology ; Point Mutation ; Trans-Activators ; genetics ; Transfection ; Tumor Cells, Cultured ; Virus Replication
6.Clinical study of oligonucleotide microarray on monitoring the lamivudine-resistance mutations in hepatitis B virus.
Jia-wu SONG ; Ju-sheng LIN ; Xin-juan KONG ; Kuo-huan LIANG
Chinese Journal of Hepatology 2003;11(6):361-363
OBJECTIVETo evaluate the mutations of lamivudine-resistance using oligonucleotide microarray in hepatitis B virus (HBV) infected patients.
METHODSA randomized clinical trial was conducted on 20 lamivudine-treated patients for 18 months and 10 patients as controls. The serum HBV DNA was amplified by PCR and the lamivudine-resistance mutations in YMDD region were assayed by 4 sites microarray developed before.
RESULTSThis microarray could clearly differentiate the wide-type from mutated-type HBV with lamivudine-resistance mutations. The rate of mutations in YMDD region increased with the time of lamivudine treatment (chi2=6.69, P<0.01). The most common mutated type was M539V+L515M and next M539I. Continuous administration of lamivudine was no benefit for inhibiting the replication of HBV with YMDD mutation but helpful for wide-type HBV.
CONCLUSIONThe routine serum HBV DNA assay by PCR may introduce prejudice in monitoring HBV inhibitory effect by lamivudine, while the microarray technique can avoid this and is one of the best ways to monitor the lamivudine-resistance mutations in HBV. There is no effect of lamivudine on HBV with YMDD mutation in clinical practice.
Adult ; Antiviral Agents ; pharmacology ; therapeutic use ; Drug Resistance, Viral ; genetics ; Female ; Hepatitis B virus ; drug effects ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Lamivudine ; pharmacology ; therapeutic use ; Male ; Middle Aged ; Oligonucleotide Array Sequence Analysis ; Point Mutation ; genetics ; Polymerase Chain Reaction
7.Arginine supplementation in total parenteral nutrition improves postoperative cellular immune function of patients with hepatocellular carcinoma after operation.
Zhong-xin ZHOU ; Long-juan ZHANG ; Xiao-hui HUANG ; Qing-yu KONG ; Jie ZHOU
Journal of Southern Medical University 2007;27(7):1094-1096
OBJECTIVETo explore the effect of total parenteral nutrition (TPN) supplemented with arginine on cellular immune function of patients with hepatocellular carcinoma (HCC) after radical tumor resection.
METHODSFifty-six HCC patients undergoing radical surgery received fat-free TPN support, routine TPN or TPN with arginine supplementation, and their clinical data were analyzed prospectively. The percentages of T lymphocyte subpopulation and national killer (NK) cells in peripheral blood are determined, and the levels of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) were measured.
RESULTSNo marked changes were noted in peripheral blood CD4+, CD8+ T cells and NK cells, or in IL-2, IL-4 and IFN-gamma levels after fat-free TPN and routine TPN support. TPN supplemented with arginine resulted in significant increase in CD4+ T cells, NK cells and CD4+/CD8+ T cell ratio in the peripheral blood, as well as in IL-2 and IFN-gamma levels. Peripheral blood IL-4 level was decreased significantly.
CONCLUSIONTPN with arginine supplementation can augment the percentages of CD4+ T lymphocytes and NK cells, and increase IL-2 and IFN-gamma levels, suggesting that arginine can enhance cell-mediated immunity in postoperative patients with HCC.
Adult ; Aged ; Arginine ; pharmacology ; Carcinoma, Hepatocellular ; immunology ; metabolism ; surgery ; therapy ; Cytokines ; metabolism ; Dietary Supplements ; Female ; Humans ; Immunity, Cellular ; drug effects ; Liver Neoplasms ; immunology ; metabolism ; surgery ; therapy ; Male ; Middle Aged ; Parenteral Nutrition ; methods ; Postoperative Period ; T-Lymphocyte Subsets ; drug effects ; immunology
8.Correlation analysis between the exposure levels and the serum protein fingerprints in population exposure to silica.
Hai-xia KONG ; Wei LIU ; Qing-bo MA ; Juan-juan CHEN ; Mao-ti WEI ; Shi-xin WANG ; Ya-guang WENG
Chinese Journal of Industrial Hygiene and Occupational Diseases 2012;30(4):258-260
OBJECTIVETo explore the correlation between the exposure levels and serum protein fingerprints in population exposed to silica.
METHODSLiquid chip time-of-flight mass spectrometry technology was used to investigate the serum profiles in control group (30 cases), group exposed to silica (30 cases), silicosis group (I stage, 25 cases) and suspected silicosis group (30 cases), and screen the differential expression proteins. The correlation between the levels of the differential expression proteins and the exposure levels was performed.
RESULTSFive differential expression proteins were found among 4 groups, the expression of 5081 and 5066 proteins was upregulated, and the expression of 3954, 2021 and 1777 proteins was downregulated. There was no the correlation between the exposure levels and the peak with M/Z among those proteins.
CONCLUSIONthe results of present investigation indicated there was no correlation between the exposure levels and protein/peptide peak.
Adult ; Blood Proteins ; analysis ; Case-Control Studies ; Dust ; analysis ; Female ; Humans ; Male ; Mass Spectrometry ; Middle Aged ; Occupational Exposure ; analysis ; Peptide Mapping ; Proteomics ; Silicon Dioxide ; toxicity ; Silicosis ; blood
9.Inhibition of mutant p53 in hepatocellular carcinoma cells by hammerhead ribozyme.
Xin-juan KONG ; Ju-sheng LIN ; Yu-hu SONG ; Xia SHEN ; You-xin JIN
Chinese Journal of Hepatology 2003;11(12):722-724
OBJECTIVESTo investigate the inhibition of mutant type p53 in hepatocellular carcinoma by hammerhead ribozyme in both cell-free system and MHCC97 cells.
METHODSHammerhead ribozyme genes (RZ) and control ribozyme (asRZ) directed against mutant p53 (249 codons, AGG --> AGT) were designed by computer. The in vitro transcription plasmid and eukaryotic expression plasmid were constructed into the vector pBSKU6 and pEGFPC1. Human mutant and wild type p53 gene fragment were cloned into the pGEM-T vector under T7 promoter control. In vitro cleavage reaction was carried out by mixing the RZ and target mRNAs which were labeled with [alpha-32P] dUTP. RZ was introduced into MHCC97 cells by LipofectAMINEAM2000 and mtp53 expression was analyzed by RT-PCR.
RESULTSIn cell-free systems, RZ showed a specific cleavage activity against mtp53 with cleavage efficiency of 42%, while the wild type p53 was not cleaved. The mRNA level of mtp53 in MHCC97 cells after transfection was reduced by RT-PCR analysis.
CONCLUSIONThese findings suggest that the hammerhead ribozyme against the mtp53 is a new promosing gene therapeutic agent against hepatocellular carcinoma.
Cell Line, Tumor ; Genes, p53 ; Genetic Therapy ; Humans ; Liver Neoplasms ; genetics ; therapy ; Mutation ; RNA, Catalytic ; pharmacology ; therapeutic use
10.Hammerhead ribozyme against human telomerase catalytic subunit (hTERT) induced apoptosis of liver cancer cells.
Dong-po SONG ; Ju-sheng LIN ; Gui-lian FU ; Xue-mei SUN ; Xin-juan KONG ; Pei-yuan LI ; Xin MA
Chinese Journal of Hepatology 2004;12(10):616-619
OBJECTIVETo construct vector pEGFP-C1-hTERT-ribozyme (pGTRz-U6) and its mutant (pGTmRz-U6) against hTERT containing U6 promoter, then transfect them into human liver cancer cell line SMMC7721 to observe the action of the human telomerase catalytic subunit (hTERT) hammerhead ribozyme on proliferation and apoptosis of human liver cancer cell SMMC7721.
METHODSEukaryotic expressing vector pGTRz-U6 and mutant pGTmRz-U6 were constructed and transfected into SMMC7721 using Lipofectamine2000 Reagent, with pEGFP-C1 as the control group. After strict screening by G418, positive clones were cultured; the amount of expression of ribozyme and hTERT was detected by RT-PCR; cell proliferation by MTT; telomerase activity by TRAP and silver staining assay; cell apoptosis by FCM.
RESULTSWe found that the two ribozymes were expressed persistently in SMMC7721; different expression levels (P < 0.01) of hTERT among SMMC7721-Rz, SMMC7721-mRz and SMMC7721-pEGFP-C1 was exhibited by the analysis of variance with SPSS software. The difference between SMMC7721-Rz and the others is significant in t-test (P < 0.01), while there was no difference between SMMC7721-mRz and SMMC7721-pEGFP-C1 (P > 0.05). With the advance of cell division, telomerase activities of the cells treated by SMMC7721-Rz and SMMC7721-mRz decreased gradually, and the percentage of apoptosis of the cells transfected with Rz and mRz increased gradually. The apoptosis percentage of 7PDS SMMC7721-Rz was 29.86%, while those of SMMC7721-mRz and SMMC7721-pEGFP-C1 were 9.87% and 3.36%, respectively.
CONCLUSIONThe apoptosis level of SMMC7721 induced by hTERT ribozyme increases as cells divide, and this ribozyme maybe a potential approach for liver cancer gene therapy.
Apoptosis ; physiology ; Cell Line, Tumor ; DNA-Binding Proteins ; genetics ; pharmacology ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Mutation ; RNA, Catalytic ; genetics ; pharmacology ; Telomerase ; genetics ; pharmacology ; Transfection