Abstract:Epigenetics is the important component of functional genomics. It can be defined as the study of the interplay between environment and genetics, and the study of the heritable change that is not strictly dependent on DNA sequence. Nasopharyngeal carcinoma (NPC) is the common malignant tumor in south China. As a polygenetic inheritance tumor, NPC has the characteristic of obvious tendency of familial aggregation, possesses genomic instability, and is vulnerable to physical, chemical and biological carcinogenic factors. This specific etiological system of NPC refers that it is one of the best model for studying cancer epigenetics. The main point of this review focuses on the progress of studying on the effects of DNA methylation, histone modification, chromatin remodeling and non-coding RNA regulation on the pathologenesis of NPC, and suggests future directions to thoroughly explore the epigenetic pathologenesis of NPC. Moreover, it will open a new prerequisite foundation for finding the epigenetic molecular marks for screening the high-risk susceptibility population, early diagnosis, treatment and prognostic indentification of NPC.

Abstract:Entry of enveloped viruses into host cells requires fusion of the viral envelope with a cellular membrane. This step is mediated by viral glycoproteins that undergo a dramatic conformational change. Recent advances in structure and function of the fusion proteins of the class Ⅱ viruses, Rhabdoviruses and Herpesviruses were described. Proteomics computational analyses to locate the functional domain of fusion protein were introduced. The fusion proteins of class Ⅱ and class Ⅰ viruses differ radically in their initial structures but refold toward similar final conformation (trimer of hairpin). The Rhabdoviruses and Herpesviruses have a novel fold combining features of fusion proteins from class Ⅰ and class Ⅱ. The fusion proteins of these viruses have a different conformation change and mediate a different fusion process, therefore, the proteins belong to a novel class of fusion proteins. The potent inhibitor of virus entry should be new strategies for developing antiviral drugs.

Abstract:Hutchinson-Gilford progeria syndrome (HGPS) is an early onset severe premature aging disorder due to a point mutation in LMNA gene which encodes nuclear lamin A/C. The mutation activates a cryptic splice site within exon 11 of LMNA, resulting in a 50-amino acid in-frame deletion in prelamin A. However, it is not clear how the mutation in a structural protein under the nuclear envelope could give rise to premature aging phenotypes. Recent studies showed that various abnormalities have been found in nuclear structures and functions of HGPS cells, mainly including progerin accumulation and nuclear morphology abnormalities, altered nuclear mechanical properties, changes of histone methylation patterns and epigenetic control, gene misregulation, p53 signalling activation, and increased genomic instability. Two hypotheses recently emerged in the explanation of the pathogenic mechanisms contributing to HGPS. No effective clinical intervention has been developed so far for HGPS. Several fascinating therapeutic strategies have recently been provided, such as farnesyltransferase inhibitors, antisense oligonucleotides and RNA interference. HGPS has been considered to be a model for studying the mechanisms responsible for normal aging. This study will help to elucidate the physiological functions of lamin A and nuclear envelope, together with their roles in normal aging process and diseases.

Abstract:Mitogen-activated protein kinase (MAPK) signaling pathways are involved in multiple important cellular responses. To activate their downstream protein kinases by phosphorylation is a crucial manner for MAPK family members to fulfill their physiological functions. Downstream of MAPKs, there exist three structurally related MAPK-activated protein kinases (MAPKAPKs or MKs), i.e., MK2, MK3 and MK5. Once upon activated by MAPKs, MKs signal to different cellular targets, to regulate gene expression at the levels of transcription and translation, control cytoskeleton remodelling and cell cycle, and mediate cell migration and embryonic development. Recently, based on the gene knockout studies, the function divisions among different MK subfamily members are gradually clear, leading to tremendous advancements of our knowledge on MKs.

Abstract:Growing evidences show that mesenchymal stem cells (MSCs) home to tumorgenesis and inhibit tumor cells, however, the molecular mechanisms underlying are unclear. The human mesenchymal stem cells (hMSCs) derived from human fetal bone marrow in 4 months were established without immortalization, designated BMMS-03 cells. In order to clarify the molecular mechanism underlying, the effect of conditioned media from hMSCs on human breast cancer MCF-7 cells was examined. The results showed that conditioned media from BMMS-03 cells were able to decrease the colony-forming units and proliferation of MCF-7 cells by colony-forming assay and MTT assay. Western blot analysis revealed that β-catenin and its target gene c-Myc, Bcl-2, PCNA and survivin were downregulated in MCF-7 cells treated with conditioned media from BMMS-03 cells. In addition, the treatment significantly reduced β-catenin nuclear assembly in MCF-7 cells by immunoflorescence staining. The finding demonstrated that the expression level of Dkk-1 in hMSCs was much higher than that in MCF-7 cells. Moreover, treatment with the antibody of rabbit against Dkk-1 abolished the inhibitory effects of conditioned media from BMMS-03 on the tumor cells. Conditioned media from BMMS-03 cells transiently transfected with pcDNA3.1(-)-Dkk-1 produced stronger downregulation of β-catenin and c-Myc expression in MCF-7 cells. The transfection with pcDNA3.1(-)-Dkk-1 in MCF-7 cells resulted in the downregulation of β-catenin and c-Myc as well．The data suggest that hMSCs are able to decrease the malignant phenotype of tumor cells in vitro. Taken together, hMSCs may suppress tumor growth via Wnt/β-catenin pathway, in which Dkk-1 released from the hMSCs is responsible for the depression.

Abstract:Polyamine biosynthesis is controlled primarily by ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC). Antisense ODC and AdoMetDC sequences were cloned into an adenoviral vector (Ad-ODC-AdoMetDCas). To evaluated the effect of recombinant adenovirus Ad-ODC-AdoMetDCas which can simultaneously express both antisense ornithine decarboxylase (ODC) and sadenosylmethionine decarboxylase (AdoMetDC), the human lung cancer cell line A-549, was infected with Ad-ODC-AdoMetDCas as well as with control vector. Viable cell counting, determination of polyamine concentrations, cell apoptosis, and Matrigel invasion assays were performed in order to assess properties of tumor growth and invasiveness. Furthermore, Ad-ODC-AdoMetDCas's anti-tumor effect was also evaluated in vivo in a nude mice xenograft model. It was demonstrated that adenovirus-mediated ODC and AdoMetDC antisense expression could inhibit tumor cell growth, lead to cell apoptosis and reduce tumor cell invasiveness. Polyamine levels were significantly decreased in Ad-ODC-AdoMetDCas-treated cells compared with controls. This adenovirus also induced tumor regression in established tumors in nude mice. It was suggested that as a new anticancer reagent, the recombinant adenovirus Ad-ODC-AdoMetDCas holds promising hope for the therapy of lung cancers.

Abstract:In order to study the effect of mechanical strain on mesenchymal stem cells (MSCs) differentiation into osteogenic, the cyclic substrate deformation instructment was applicated to MSCs line (D1 cell) in osteogenic media for different strain and times. The cells were cyclically stretched for periods of 1, 2, 3, 5 and 10 min and treated with cytochalasin B(CB) and EGTA. Ca²⁺, which were loaded with (15 μl) Fluo-3 AM , were detected by confocal laser scanning microscope(CLSM). The signaling inhibitors, such as SB203580(p38MAPK specific inhibitor), PD98059(MEK-1/2MAPK specific inhibitor), LY294002(PI3Ks specific inhibitor), cytochalasin B(microfilament specific inhibitor), EGTA (Ca²⁺ specific inhibitor), were used to investigate mechanical signaling pathway by (3% 0.5Hz) Cyclic strain. The results indicated that 3% strain stimulus could induce increasement of [Ca²⁺]_i - dependent fluorescence at 2 min. Its fluorescence intensity were 143.68,which were significantly different compared with 1,3,5,10min groups and 1% and 10% strain groups (P < 0.01). The increasement of Ca²⁺ levels were delayed upto 10 min, when the cells were treated with CB. EGTA (5 mmol/L) could inhibited strain induction increasment of Ca²⁺ levels. Strain (3%, 0.5 Hz) could induce Ca²⁺ spark event in D1 cells at 2 min, however the cells were treated with CB Ca²⁺ spark event were delayed until 5min. The expression of OCN and OSX mRNA were completely inhibited by five specific inhabitors in unstrained condition. Inhibition of ERK1/2 and p38 pathway promoted the expression of OSX and OCN mRNA by strain.The expression of OSX and OCN mRNA to inhibit PI₃Ks pathway were partly activated by strain. Application of strain could not activate OSX and OCN mRNA expression when extracellular Ca²⁺ greatly inhibited with EGTA (5 mmol/L) and microfilament were damaged with CB.These results demonstrate that mechanical signals regulate MSCs function, suggested Ca²⁺ signaling, PI₃Ks pathway and microfilament play an important role in transduction mechanical signal in MSCs differentiation.

Abstract:Horizontal gene transfer (HGT), also Lateral gene transfer (LGT), is any process in which an organism transfers genetic material to another species that is not its offspring. With the increase of available genomic data, it has become more convenient to study the way to detect the genes, which are products of horizontal transfers among a given genome. There are few data about known horizontal gene transfers in three bacterium genomes under consideration, so the experiments, which simulated gene transfer by artificially inserting phage genes, were carried out. Combining the feature analysis methods of gene sequences with support vector machine (SVM), a novel method was developed for identifying horizontal gene transfers (HGT) in 3 fully sequenced bacterium genomes (Escherichia coli K12, Borrelia burgdorferi, Bacillus cereus ZK). According to our previous work, codon use frequency (FCU) was selected as the sequence feature, in respect that it is inherently the fusion of both codon usage bias and amino acid composition signals. In addition, another computational method was proposed considering strand asymmetry and predicting horizontal gene transfers of leading strand and lagging strand of genomes under consideration, respectively. To avoid the occasionality of simulating gene transfer through artificially inserting phage genes, 100 times of the transfer-and-recover experiment were repeated and arithmetic average of measurement for each genome being considered were reported to evaluate algorithm's performance. Ten-fold cross-validation was used for both parameter and accuracy estimation. The best results were obtained for C-Support Vector Classification (C-SVC) type by using the radial basis function kernel with γ=100, while for one-class SVM type the best performance was obtained using the polynomial kernel of three degree. The performance of the approach was compared with that of Tsirigos' method ,which is one of the best predictive approachs to date in detecting of horizontal transfer genes. Firstly, for the original method that did not consider the strand asymmetry, the C-SVC type has a high relative improvement(RI) of 31.47% on hit ratio for Escherichia coli K12, while the one-class SVM type has RI of 11.61% for Borrelia burgdorferi. Moreover, as theoretically expected, the method considering the strand asymmetry resulted in higher RI than the original method. In order to examine the approach's performance in detecting factual gene transfer events, the approach was applied in genome of Enterococcus faecalis V583. It is not only succeed in recovering all the seven factual horizontally transferred genes, also found that the whole segment from 7 kb upstream of gene EF2293 to 38 kb downstream of gene EF2299 was probably transferred into E. faecalis V583 genome simultaneously with the above seven genes.

Abstract:With the success of human genome project, a large number of predicted genes were sequenced, requiring functional assays for their characterization in a high-throughput manner. To identify novel human genes associated with cell apoptosis, a high-throughput assay was established. Candidate sequences were amplified and cloned in pcDNA3.1/myc-His(-)B, then were transfected into HeLa cells respectively. The expression vector encoding BAX was used as the positive control, which was wildly-known to effectively induce programmed cell death. JC-1 staining was utilized to assess the mitochondrial membrane potential, which could collapse in the early stage of apoptosis. 600 human novel genes were screened and seven positive genes (CHMP6, CGI-38, hCAP-H2, NUDT16L1, ARMC1, PHF17, and FLJ21103) were found out. A subsequent validation by flow cytometry revealed that three of the seven genes (CHMP6, CGI-38, hCAP-H2) were with functions related to cell apoptosis. In HeLa cells transfected with the above three expression vectors, the proportion of single annexin-V-positive cells was evidently increased (8.01%, 6.88%, 5.01%) compared with PCDB-transfected cells (3.43%). Bioinformatics analysis of the three positive genes reveals that their functions are known little and the relationships between the three genes and cell apoptosis have not previously been reported. These results therefore indicate that a rapid and effective screening system has been studied. Further studies will perform on the 3 genes associated with cell apoptosis.

Abstract:To reveal molecular mechanisms responsible for that over-expression of heat shock protein 27 (HSP27) is associated with poor prognosis in liver cancer, expression of HSP27 was analyzed by RT-PCR and Western blot in Hep3B, MHCC97L and MHCC97H, which had higher metastasis potential than Hep3B, and after HSP27 RNA interference, MHCC97H migratory and invasive capability were evaluated by in vitro migration and invasion assay, apoptosis ratio was detected by flow cytometry(FCM) and TUNEL method, prominent altered signal pathway was analyzed through a Human Q Series Signal Transduction in Cancer Gene Array. The results showed that the up-regulation of HSP27 expression in HCC cell lines was consistent with metastasis potentials. Using RNA interference technique, mRNA and protein level of HSP27 in MHCC97H cell line were specifically reduced by up to 80%. In vitro migration and invasion assay showed the average migratory and invaded cell numbers per field were 21.36 ± 2.92, 19.88 ± 2.23, 11.40 ± 2.05 and 26.35 ± 3.29, 24.43 ± 3.17, 10.92 ± 2.27 respectively in MHCC97H, control RNAi, RNAi groups. Moreover, MHCC97H cell apoptosis ration was 15.12%, 17.56%, 27.64% (MHCC97H, control RNAi, RNAi) respectively by FCM analysis, which was reconfirmed by TUNEL morphological assessment. Furthermore cDNA microarray analysis revealed that NF-κB pathway activation was inhibited after HSP27 RNAi. Immunoblotting analysis showed that the decreasing of nuclear activated NF-κB p65 and phosphorylated IκBα, meanwhile reducing of the association between IKKβ and IKKα in MHCC97H cells after HSP27 RNAi, further co-immunoprecipitation assay showed interaction of IKKβ, IκBα with HSP27 in three HCC cell lines. Altogether, these findings revealed possible roles of HSP27 in being of HCC cell lines metastatic potentials through involving in cellular NF-κB pathway activation.

Abstract:The immunogenicity and protective efficacy of combined DNA priming, Bacillus Calmeette Guerin(BCG) boosting vaccination in mice were examined. Following intravenous challenge with virulent M. tuberculosis H37Rv, the BCG boost approach resulted in significant protection in both lungs (1.3, P < 0.01) and spleens (1.1, P < 0.01) compared with the saline control. In addition, this approach also have much better protection than that vaccination with combined DNA or BCG alone (P < 0.05). The elevated CD4⁺ and CD8⁺ T cells percentage (P < 0.05) in PBMC and higher IFN-γ concentration (1250 ng/L, P < 0.01), IL-2 concentration (230 ng/L, P < 0.05) stimulated by Ag85B protein in spleen cells supernatant in BCG boosted mice 21 days after third vaccination, indicating that the DNA prime-BCG boost strategy significantly enhanced the Th1 type cell response, which is correlated with the protective efficacy against tuberculosis. Furthermore, immuno-histochemistry assay showed that there were more Perforin expression cells, secreted mainly by CD8⁺ T cells, in the lung tissue of the mice primed with DNA prior to BCG. Taken together, the heterogonous boost combination provided superior protection by stronger CD4⁺ (Th1) and CD8⁺T cell mediated immune response, which suggested that it will be a promising regimen for MTB vaccine development.

Abstract:To explore a new way to purify EPCs, hybridoma dish was used to isolate EPCs from rat bone marrow -derived cells,according to the morphology of endothelial progenitor cells colony-forming units (EPCs-CFUs) and special markers of EPCs. The bone marrow derived cells, from rat femoral bone and shinbone, were plated and cultured on hybridoma dish which was maded of polystyrene. Between 4th and 7th days in culture, stem cells colony-forming units were picked out respectively under microscope. Then one part of this cells was identified by immunofluorescence staining of CD133⁺vEGFR-2⁺, the special markers of EPCs. If the both markers of this part was positive, the rest part of the cells was continuously cultured for passaging. This method was named as “Micropore-Method”. The conspicuous stem cells colony-forming units were observed under microscope after four days in cluture, about 7% CFUs were CD133⁺/VEGFR-2⁺ positive EPCs-CFUs. After further culturing 7 days, Purity of cells with special markers of CD133⁺/VEGFR-2⁺/CD34⁺ was over 70% identified by flow cytometry. Passaging cells can form capillary tube like formation and differentiate to endothelial like cells expressing EC special marker vWF. It can be concluded that “Micropore-Method” is a new successful way to isolate EPCs from rat bone marrow.

Abstract:In order to elucidate the mechanisms of p53 overexpression in nasopharyngeal carcinoma (NPC) and detect proteins associated with the function of p53 in high throughout screening, p53 which knockdown human NPC CNE2 cell line (CNE2sip53) were successfully established by using stable RNA interference (RNAi). Two-dimensional gel electrophoresis (2-DE) was used to separate the total proteins of CNE2sip53 and its control cell line CNE2/pSUPER, and PDQuest software was applied to analyze 2-DE images. Twenty-two differential protein spots were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS, some of which are known to be associated with the p53 function (HSP27, hnRNP K, 14-3-3σ etc.) , and others may be novel proteins associated with p53 function (eIF4B, TPT1, hnRNP H3, SFRS1 etc.). Furthermore, the differential expression levels of the partial proteins (HSP27, 14-3-3σ, GRP75) were confirmed by Western blot analysis and compared with CNE2 and CNE2 cells transfected with pcDNA3.1-FLAG, CNE2 cells transfected with pcDNA3.1-FLAG-p53 had obvious down-regulations of HSP27 and 14-3-3σ, and an up-regulation of GRP75. The 22 differentially expressed proteins could be divided into five groups based on their functions: signal transduction, chaperone, transcription and translation, metabolism and cytoskeleton, which were involved in cell cycle, the transcription regulation, cell adherence，cellular metabolism and so on. The data suggest that these differential proteins may be associated with the function of p53 in NPC, and will be valuable for further to study the mechanisms of p53 overexpression and inactivation in NPC.

Abstract:Microcell mediated chromosome transfer (MMCT) is a challenging technique for introducing exogenous chromosomes into interested mammalian cells. Combined with the somatic cell nuclear transfer technique, MMCT has been employed for producing transchromosomic animals of medical and agricultural value. Producing high quality of microcells is a key step in the success of MMCT. Eamamined by fluorescin staining and Giemsa staining, 0.2 mg/L colcemid was considered suitable for inducing high percentage of micronuclei in A9 (neo12) cells, without causing death of a mass of cells. Microcells were produced by centrifugation of micronucleated A9 (neo12) cells in Percoll density gradient containing 20 mg/L Cytochalasin B at 39 000 g. The resulting mixture of microcells, whole cells, karyoplasts and cytoplast fragments was filtered through 8 μm and 5 μm size membrane pores sequentially to obtain pure preparation of microcells. Microcells were then characterized by Giemsa staining and microcell PCR was first applied for examination of the quality of microcell preparation. The result showed that microcells containing our interest chromosomes-human chromosome 12 were equally distributed in the preparation, the preparation was suitable for use in generation of transchromosomic animals.