The advantages of saliva-based biomarkers detection in cancer were accurate,simple and noninvasive.Currently,biomarkers validated for saliva detection include protein,mRNA,miRNA and DNA,using PAGE,microarray and sequencing,respectively.Analysis of literatures shows;that saliva biopsy plays an important role in cancer diagnosis,clinical treatment and prognosis.Due to the design of experiments reported werediversification,a large number of validations are necessary to standardize saliva collecting,processing,testing methods and quality assurance.

microRNAs(miRNAs)are a class of 18～26 nt small non-coding single strand RNA molecules,which negatively regulate the expression of a variety of genes at post-transcriptional level by binding to complementary sites on target mRNA.Mutation,deletion or high expression of miRNA was found to correlate with various human cancers.miRNAs involved in tumor cell proliferation,differentiation and apoptosis processes as function as oncogenes or tumor supressors It is predictable that microRNA might be applied in the diagnosis and treatment of malignant tumors.

Objective To develop an analyzing technique of single nucleotide polymorphism(SNP) based on non-probe real-time quantitative PCR, and to explore the relationship between SNP in human adiponectin (APM1) gene and type 2 diabetes (T2DM).Methods Design primers with specific 3′-terminal nucleotides and perform Real-time PCR using SYBR Green dye. Genotypes were determined by comparing the amplification efficiency of each pair of primer and verified by sequencing. SNPs 45 and 276 in APM1 of 20 T2DM, 24 obesity and 28 healthy persons were analyzed. Results This technique could discriminate genotype as efficient as sequencing. The genotype of APM1 at +45 site was significantly correlated with T2DM (P

Objective To explore the function of BCRP in ovary cancer and establisment of BCRP expressing ovary cancer cell line 3AO/BCRP.Methods We construct the BCRP expressing cell line 3AO/BCRP.Extract the total RNA of MCF-7 cells,clone the whole length of BCRP gene by RT-PCR and ligate the gene to pcDNA3.1(-),transfect the positive clones to 3AO cells and select positive cell clones with G418,identify the survival cells by MTT,efflux assay,western blot.Results The multidrug resistance index of 3AO/BCRP to mitoxantrone increased to 11.58 times;the efflux of 3AO/BCRP to mitoxantrone enhanced;the relative level of BCRP mRNA in 3AO/BCRP increased and western blot indicate that the 3AO/BCRP cells can express more protein than 3AO cells.Conclusion 3AO/BCRP is a reliable BCRP expressing cell model,the establishment of this cell line has laid basis for further study of the mechanism of BCRP.

Circulating DNA is defined as a kind of extracellular DNA that exists in plasma,cerebrospinal fluid and synovial fluid.The concentration of circulating DNA of cancer patients is significantly higher than that in healthy people.The genetic and epigenetic alterations of circulating cell-free nucleic acids are relevant to cancer development and progression,for example,gene mutation,DNA methylation and microsatellite instability and so on.The quantitative and qualitative detection of circulating DNA shows promising potential value in cancer screening,diagnosis,disease monitoring treatment and prognosis.

AIM: To investigate the anti-cancer effect of cytotoxic T lymphocytes (CTL) activated by sensitized dendritic cells (DCs). METHODS: Immature DCs were induced in vitro from peripheral blood monocytic cells (PBMC) and sensitized by adding tumor cells antigen extract. DCs were identified by their morphology and surface markers. MTT assay was used to evaluate the killing activity of CTL activated by sensitized DCs. The effects of specific CTL cells on inhibiting transplanted tumor HT-29 growth and on preventing HT-29 tumor generation were evaluated by injecting CTL into nude mice. RESULTS: After cultured for seven days, a large number of activated DCs were obtained with typical morphology, extensive stimulatory proliferation capacity and high CD80 (63.5%), CD83 (67.6%) and CD3/HLA-DR (83.2%) expressions. The killing activity of CTL at 20∶1 ratio of effective cells to target cells was more than 75% to tumor cells, 35%-45% to homologous cell line and weaker to other germ cell line (P

microRNAs (miRNAs) are a recently discovered family of small non-coding RNAs, which exist in eukaryotes and regulate gene expression post-transcriptionally by binding to complementary sites on target mRNAs. MiRNAs play important roles in regulation of cell proliferation, development, apoptosis and cell differentiation. It’s predicted that about 1% human known genes encode microRNAs and microRNAs may regulate 10～30%of the genes in the human genome. The paper focus on the knowledge about how to recognize miRNAs and identify their target mRNAs.

Objective To construct the RNA interference(RNAi)recombinant adenovirus vector targeting at human hypoxia-inducible transcription factor 1?(HIF-1?)and to evaluate its effect on human lung adenocarcinoma cell line SPCA-1.Methods The recombinant adenovirus Ad was constructed.HIF-1? inserted with HIF-1? RNAi fragment via AdEasy system.The virus was purifed by CsCl gradient centrifuge.The functional titer of recombinant adenovirus was measured by transfection test in HEK 293 cells.SPCA-1 cells were transducted with 2 multiplicity of infection(MOI)Ad.HIF1? in vitro,the expression rate of green fluorescence protein(GFP)was recorded by flow cytometry,HIF-1? mRNA and protein level was measured by Real-Time RT-PCR and flow cytometry.ResultsThe recombinant shuttle plasmid PAdTrack.HIF-1? and adenovirus plasmid Ad.HIF-1? were all correct shown by enzyme digestion confirmation.The plasmid pAd.HIF-1? was transducted into HEK293 cells,15%GFP expressionwere seen after 3 days.The final titers of recombinant adenovirus were 5.0?1010 TU/mL.SPCA-1 cells was transducted by Ad.HIF-1? in vitro for 48 h,GFP expression rate was 92%,HIF-1? mRNA and protein level decreased 89% and 87%,respectively.Conclusion RNAi adenovirus vector of human HIF-1? gene has been successfully constructed,which could facilitate the research onHIF-1? gene related gene therapy for lung cancer.

AIM: To investigate the inhibitory effect of arsenic trioxide on human glioma cell line U251 growth, change of gene expression and intracellular calcium content. METHODS: MTT method was used to observe the growth inhibition effect. Cell cycle, positive rate of proliferation cell nuclear antigen (PCNA), apoptosis associated protein Fas and Bcl-2, and intracellular calcium ion (IECa~ 2+ ) levels were measured by flow cytometry in U251 cells treated with different doses of As_2O_3. Apoptosis was detected with annexin V-FITC+PI dual parameter. RESULTS: As_2O_3 inhibited the growth of U251 cells dramatically. There were obvious dosage-effect and time-effect correlations (P0.05). The cell cycle was arrested in G_2M phase. Apoptosis occurred in U251 cells treated with As_2O_3 by annexin V-FITC+PI dual parameter detection. CONCLUSION: As_2O_3 inhibits the growth of U251 cells in vitro dramatically and induces apoptosis. The mechanism is probably associated with the improvement of Fas expression and IECa~ 2+ levels, decrease in PCNA protein expression and cell cycle arrest.

Astrocyte elevated gene-1 (AEG-1) was cloned as an human immunodeficiency virus -1-inducible and tumor necrosis factor-α-inducible transcript in primary human fetal astrocytes by a rapid subtraction hybridization approach. AEG-1 down-regulates the expression of the glutamate transporter EAAT2, thus, it is implicated in glutamate-induced excitotoxic damage to neurons as evident in HIV-associated neurodegeneration. Meanwhile, AEG-1 expression is elevated in subsets of breast cancer, prostatic cancer, glioblastoma multiforme and melanoma cells, having a dual specificity phosphatase activity. Overexpression of AEG-1 increases and siRNA inhibition of AEG-1 decreases migration and invasion of human glioma cells, respectively. Recent observations indicate that AEG-1 exerts its effects by activating the nuclear factor kappa B (NF-κB) pathway and AEG-1 is a downstream target of Ha-ras and plays an important role in Ha-ras-mediated tumorigenesis. These findings are intensifying interest in AEG-1 as a crucial regulator of tumor progression and metastasis and as a potential mediator of neurodegeneration.