OBJECTIVE: Try to identify single nucleotide polymorphism (SNPs), copy number variation (CNVs) and mRNA expression from the genetic or tumor markers that are associated with lymph node metastases in colorectal cancer. METHODS:Targeted next-generation sequencing(Illumina Hiseq) was applied to capture SNPs and CNVs in tumor-related candidate genes of tumor tissues and paired normal tissues adjacent to the tumors from 25 colorectal cancer specimens;real time-PCR was used to detect specific mRNA expression of tumor-related candidate genes (VEGFC、CCNA2、IL2、ABCG2、EGF and NFKB1) on chromosome 4 of 39 colorectal cancer patient specimens. RESULTS: The SNPs in SLC28A3, BRCA1, RRM2, PMS2, CDA, EPHX1, RALY, CD33, BCL10, ETV1, MST1R, KMT2B, BCL2, LSM3, TTF1, MAP3K1 genes were significantly correlated with lymphatic metastasis risk (P<0.05). There were no significant differences of the CNVs in the DDR1, CYP21A2, SULT1A1, XRCC2, POU5F1, FLT1 genes between the positive lymph node metastasis group and the negative lymph node metastasis group(P>0.05). Compared with non lymph node metastasis group, mRNA expressions of EGF and NFKB1 were both down-regulated in the lymph node metastasis group, and the difference was statistically significant(P<0.05). CONCLUSION:SNPs in SLC28A3, BRCA1, RRM2, PMS2, CDA, EPHX1, RALY, CD33, BCL10, ETV1, MST1R, KMT2B, BCL2, LSM3, TTF1, MAP3K1 genes and down-regulated expression of EGF and NFKB1 might be the potential genetic markers in lymph node metastasis of colorectal cancers. No CNV was found to be associated with lymph node metastasis in colorectal cancer in our study.

OBJECTIVE: The aim of our study was to evaluate the potential associations between the single nucleotide polymorphisms (SNPs) of Toll-like receptor (TLR2) rs3804099 and rs3804100 genes and the risk of gastric carcinoma(GC), especially to Epstein-Barr virus-associated gastirc carcinoma (EBVaGC). METHODS:TLR2 rs3804099 and rs3804100 gene polymorphism were assessed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 185 cases of EBV-negative GC, 41 cases of EBVaGC. 100 cases of peripheral blood samples from healthy individuals were also examined. The data was analysed by SPSS. RESULTS:As for the TLR2 gene (rs3804099), there was significant difference between the GC group and the control group in both genotype and allelic frequencies (χ²=5.617, P=0.018;χ²=6.467, P=0.011). The C allele and C allele carriers frequencies of gastric carcinoma group were significantly higher than those of controls (χ²=6.467, P=0.011;χ²=4.444, P=0.035). Compared with the wild type TT, the CC genotype could increase the risk of gastric cancer (OR=3.554, 95%CI=1.179-10.715). As for the TLR2 gene (rs38040100), there were no significant differences between the GC group and the control group in genotype, C allelic frequency and C allele carrier frequency (P>0.05). In all the indicators, no polymorphism was found to be related to EBVaGC in the studied population (P>0.05). CONCLUSION:There was an association between TLR2 rs3804099 gene polymorphism and gastric carcinoma, and the C allele may be a risk factor for gastric carcinoma. Carrying the C allele may increase the risk of gastric carcinoma. No association was observed between TLR2 (rs3804099 and rs38040100) gene polymorphism and EBVaGC.

OBJECTIVE: To investigate the role of ubiquitin protein ligase RING2 in cell cycle and expression of P53 in human bronchial epithelial cells exposed to benzo[a]pyrene. METHODS:The untreated 16HBE cellsgroups were used as negative control groups, the DMSO groups were used assolvent control groups, the MOCK groups were used as sequence control groups. 16HBE cells and 16HBE(siRNA-RING2) cells were exposed to BaP at different concentrations (1, 2, 4, 8, 16, 32 μmol/L) for 24 h, and were exposed to BaP at 16 μmol/L for different time points (1, 2, 4, 8, 12, 24 h). Flow cytometry was used to evaluate the cell cycle. The levels of P53 protein were detected by Western-blot. RESULTS:After BaP treatment, compared with the negative control groups, at each concentration and each time point the proportion of S phase of 16 HBE cells were significantly increased (P<0.01). However at each concentration and each time point 16HBE (siRNA-RING2) cells groups the proportion of S phase cells were significantly decreased (P<0.01). Covariance analysis shows grouping factors (with or without RNAi) and all concentrations had impact on the proportion of S phase, P values were both less than 0.01. The estimated mean of the proportion of S phase in 16HBE (siRNA-RING2) cell group (17.09%) was significantly lower than that of 16HBE cell group (31.55%). Grouping factors (with or without RNAi) and the exposure time all have impacted on theproportion of S phase, P values were both less than 0.01. The estimated mean of the proportion of S phase in 16HBE (siRNA-RING2) cell group (13.07%) was significantly lower than that of 16HBE cell group (28.04%) (P<0.05). After BaP treatment, compared with the negative control group, at each concentration and each time point P53 protein levels at 16 HBE cell groups were significantly increased (P<0.05). However, at each concentration and each time point 16HBE (siRNA-RING2) cell groups (except 16 μmol/L BaP treated 8 h) the levels of P53 protein were significantly decreased (P<0.05). Covariance analysis shows grouping factors (with or without RNAi) and all concentration showed impact on the level of P53 protein, P values were 0.026 and 0.028, respectively. The estimate mean of the levels of P53 protein in 16HBE (siRNA-RING2) cell group (0.989) was significantly lower than that of 16HBE cell group (1.375) (P<0.05). Grouping factors (with or without RNAi) and the exposure time both impacted on the level of P53 protein, P values were 0.007 and 0.035, respectively. The estimate mean of the levels of P53 protein in 16HBE (siRNA-RING2) cell group (0.857) was significantly lower than that of 16HBE cell group (1.541) (P<0.05). CONCLUSION:The histone ubiquitination modifications in which RING2 was involved may regulate DNA repair by affecting the expression of P53 and cell cycle S phase changes.

OBJECTIVE: To analyze TMEM196 gene methylation and expression in lung cancer. METHODS: Methylation-specific PCR was used to detect the DNA methylation status of TMEM196 gene in normal and tumor tissues and cell lines. RT-PCR was used to analyze the expression of TMEM196 gene in tumor cells and also in 5-aza-dC-treated tumor cells. RESULTS:The hypermethylation of TMEM196 was detected in lung cancer tissues (57%, 28/49), but not in normal tissues (0, 0/20). Methylation of TMEM196 gene was significantly correlated with tumor differentiation (P=0.012) and the clinical stages of lung cancer patients (P=0.007), but not correlated with the patient's age, sex, smoking, and pathological classification (P>0.05). TMEM196 showed hypermethylation and its expression decreased in lung cancer cell lines H1975 and H1650. After treatment with demethylation drug, expression of TMEM196 gene increased significantly in these cell lines. CONCLUSION:The expression of TMEM196 gene was inhibited through hypermethylation in lung cancer. This process may play an important role in lung cancer.

OBJECTIVE: To study the effects of vitamin E succinate(VES) on proliferation, apoptosis and expressions of associated protein of human esophageal carcinoma cell line Eca-109 and TE-1．METHODS:Eca-109 and TE-1 human esophageal carcinoma cells were cultured in vitro. Cells were treated with different concentrations of VES for 24 h. MTT was used to measure the proliferation of cells. Morphological changes of cells were examined by inverted microscopy. Apoptosis was assessed by DAPI staining. The expression of poly ADP-ribose polymerase(PARP) of two cell lines among different concentration groups was examined by Western blotting．RESULTS:The inhibitory rate increased gradually with increasing concentration of vitamin E succinate (P<0.05). The number of dead cells decreased with increasing concentration of VES. Apoptosis appeared in different degrees in cells with increasing concentration of VES. The cleavage of PARP protein in two cell lines also increased gradually. CONCLUSION:Vitamin E succinate could inhibit proliferation and induce apoptosis of Eca-109 and TE-1 cell lines．

OBJECTIVE: To explore the correlation between TERT promoter mutations and clinicopathological features in patients with gliomas. METHODS:TERT promoter mutations were screened by direct DNA sequencing in a population-based collection of 78 glioma tissues. The correlation of TERT promoter mutations and the prognosis was analyzed. RESULTS:We identified TERT promoter mutations in 32.1% gliomas, including 28.0% in low grade tumors and 34.0% in high grade tumors. The mutations were much more common in oligoastrocytomas (57.1%) and glioblastomas (44.4%), while much less prevalent in astrocytomas (28.6%) and oligodendrogliomas(23.1%). Median overall survival of the patients harboring mutations in TERT promoter was longer than that without the mutations (P=0.001) in both low (P=0.019) and high grade gliomas (P=0.018). Multivariate analysis revealed TERT promoter mutations and no postoperative adjuvant therapy as significant prognostic factors for shorter survival (P=0.002, HR=3.486, 95%CI: 1.591-7.637;P=0.004, HR=0.331, 95%CI:0.156-0.699). CONCLUSION:TERT promoter mutations frequently occurred in gliomas, which was a prognostic factor of poor outcome for the patients with the same pathological grade gliomas.

OBJECTIVE: To investigate the association of human leukocyte antigen(HLA) DRB1 gene polymorphisms with genetic susceptibility to breast cancer in Uyghur women. METHODS:Genomic DNA was isolated from peripheral blood of 196 patients with breast cancer and 230 healthy Uyghur women, HLA-DRB1 typing was performed by means of polymerase chain reaction-sequence-specific priming (PCR-SSP) and capillary electrophoresis sequencing (CE) method. RESULTS：The frequency of HLA-DRB1*01 genotype in breast cancer was significantly higher than in the control group (χ²=10.180, OR=4.550, P<0.05), while the frequency of HLA-DRB1*16 genotype was significantly lower than in the control group (χ²=4.792, OR=0.492, P<0.05). The frequencies of other HLA-DRB1 alleles did not show statistical significance between the two groups. CONCLUSION:Breast carcinogenesis in Uyghur women was closely associated with the allele polymorphisms of HLA-DRB1, which provides objective evidence for elucidating the molecular mechanism and clinical diagnosis for breast cancer.

OBJECTIVE: To investigate the effects of lutein and epigallocatechin gallate (EGCG) supplementation on the antioxidant activity and DNA damage. METHODS:A total of 40 healthy young adults were randomly divided into four groups (n=10 in each group), including lutein group (20 mg/d), EGCG group (270 mg/d), lutein (20 mg/d)+EGCG (270 mg/d) group and normal control group. Three experimental groups of volunteers were treated with lutein and or EGCG for 20 d while the control group wasn't given any supplement. 5 mL fasting blood was collected at the beginning and the end of the trial. Plasma lutein and EGCG concentrations were measured by HPLC detector systems. Plasma total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) and malondial dehyde (MDA) levels were measured by kits. Peripheral blood lymphocyte DNA damage was analyzed by single-cell gel electrophoresis (SCGE). RESULTS:After supplementation for 20 days, plasma lutein concentration of the subjects in the lutein group and lutein+EGCG group increased by 257% and 175%(P<0.01). Plasma MDA and T-SOD concentrations of the subjects in the lutein group were significantly lower than those of the control group, while plasma GSH-Px concentration was significantly increased (P<0.05). No significant difference was found in the antioxidant activity of the subjects in the EGCG group. The combined supplementation of lutein and EGCG had no interaction on the antioxidant activity. DNA damage analysis showed that low- and medium- concentration H₂O₂-induced DNA damage were markedly lower in either lutein or EGCG group than those in the control group (P<0.05). However high- concentration H₂O₂-induced and spontaneous DNA damage in both groups did not show any significant difference with those in the control group. The combined supplementation of lutein and EGCG had no interaction on the DNA damage (P>0.05). CONCLUSION:Lutein or EGCG supplementation alone could effectively reduce DNA damage. Lutein supplementation enhanced antioxidant activity, but EGCG did not. Lutein and EGCG did not show any interaction on antioxidant activity and DNA damage.

OBJECTIVE: To investigate the effects of mycrocystin-LR(MC-LR) on the neurotoxic mechanisms of the central nervous system, the contents of nitric oxide (NO), nitric oxide synthase (NOS), tumor necrosis factor-α (TNF-α), and interleukin- 6 (IL-6) in BV-2 microglial cells were detected in our study. METHODS:With the methyl thiazolyl tetrazolium (MTT) assay, the cytotoxicity of the inflammatory model in BV-2 cells was performed after exposure to MC-LR. The levels of NO and NOS were measured by the nitric acid reductase assay and nitric oxide synthase assay, respectively. The contents of TNF-α and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS:After exposure to MC-LR for 24 h, the viability in BV-2 cells was decreased with the increase of MC-LR concentration within the range of 0-28 μg/L (r=-0.609, P<0.05), and the median effective concentration (EC50) was 22.49 μg/L. Besides, the levels of IL-6 and NOS in BV-2 cells were increased at the concentration of 5-10 μg/L MC-LR, and the levels of TNF-α and NO were increased at the concentration of 10 μg/L MC-LR. Compared with control group, those differences above mentioned were statistically significant (P<0.05). CONCLUSION:MC-LR induced inflammatory response in BV-2 microglial cells by increases in production of NO, NOS, IL-6 and TNF-α.

OBJECTIVE: To study the genotoxicity of PAMAM dendrimer (ethylenediamine core) generation 5.0 solution. METHODS:In the plate incorporation test of Salmonella typhimurium, the average colony number of spontaneous revertants of TA97, TA98, TA100, TA102, TA1535 were set at 5 concentrations:3 985, 797, 139.4, 31.88 and 6.376 μg per plate. In the cytokinesis-block micronucleus cytome(CBMN Cyt) assay, the final concentration 0.625, 1.25, 2.5, 5, 10 μg/mL of PAMAM dendrimer (ethylenediamine core) generation 5.0 was added to L5178Y cells, then assessed the micronucleus cytome effect, dose-effect and time-effect relationships. RESULTS: The colony number of revertants for the materials tested did not obviously increase, compared with that of spontaneous revertants. The result of Ames test for the subject was negative. In the CBMN Cyt assay, the frequency of nuclear buds increased at concentration of 1.25 μg/mL. The formation of total, type Ⅰ and typeⅡ micronuclei as well as nuclear-cytoplasmic bridges were oberserved at and above concentration of 2.5 μg/mL. At of 5 μg/mL, the time-effect results indicated that the number of total, type Ⅰ amd type Ⅱ micronuclei and nuclear buds increased at 9 h, while the nucleoplasmic bridges started to increase at 18h. The indexes of total, type Ⅰ and type Ⅱ micronuclei, nucleoplasmic bridges and nuclear buds reached their peak at 27 h. CONCLUSION:The PAMAM dendrimer (ethylenediamine core) generation 5.0 solution showed no potential mutagenicity. While could cause four kinds of micronucleus cytome effects with clear dose-effect and time-effect in L5178Y cells. From the dose and time point of view, nuclear bud was the most sensitive index.

OBJECTIVE: To study the impacts of leukocyte elevation on reactive oxygen species (ROS) and sperm DNA damage. METHODS:A total of 52 elevated leukocyte specimen and 50 normal leukocytes specimen were collected. ROS production was measured using luminol as the probe by the Chemiluminescence method. Sperm nuclear DNA damage was assessed by sperm chromatin structure assay (SCSA) and single-cell gel electrophoresis (SCGE). RESULTS:ROS levels (using the log-transformation) was significantly higher in the elevated leukocyte group (P<0.01). Data of SCSA indicated there were significant increases of DNA fragmentation index and high DNA stainable in the elevated leukocytes group (P=0.020, P=0.012). SCGE analysis, in terms of tail moments, was significantly higher in the elevated leukocyte group (P=0.007). CONCLUSION:Leukocyte elevation could result in the increase of reactive oxygen species, which may induce sperm nuclear DNA damage.

OBJECTIVE: To describe the characteristics of toxicokinetics and toxicodynamics of chlorpyifos (CPF) in juvenile rats after repeated oral gavage of chlorpyrifos by using PBTK/TD model. METHODS:21 days-old female SD rats were given daily gavage doses of CPF at doses of 0, 1.0, 2.5, 5.0, 10.0 and 15.0 mg/kg/d for 10 days. Serum and cerebral cortex were collected on days 3, 6, 10 and 11, to determine the concentration of CPF and 3, 5, 6- trichloropyridinol (TCP) in serum and the activity of acetylcholinesterase (AChE) in cerebral cortex. Urine samples were collected on days 3, 6 and 11, to determine the cumulative amount of TCP in urine. PBTK/TD model was used to predict the time-concentration curve of the indexes. RESULTS:The concentrations of serum CPF and TCP first increased and then decreased with time every 24 h during the period of administration, significantly different with the control group (P<0.05). The activities of AChE in serum and cerebral cortex also showed cyclical changes over time. The activities of AChE in cortex and serum both decreased as the CPF dose increased (P<0.05). The first day after exposure stopped, the activity of AChE in serum had a remarkable recovery (P<0.05). CONCLUSION:In repeated exposure, toxicokinetic and toxicodynamic indexes showed periodic variation. Continuous exposure to chlorpyrifos resulted in AChE in cortex and serum to be maintained at a certain level of inhibition, which only recovered when until significantly exposure stopped.

OBJECTIVE: To establish a simple, fast and high throughput screening method for green fluorescent protein (GFP) assay. METHODS:We applied TECAN multi function measuring instrument infinite M200pro to examine the fluorescence intensity of GFP sample in different dilution buffers, and established the standard curve between fluorescence intensity and GFP relative content. Compared the results obtained from multi function measuring instrument, fluorescence-activated cell sorting (FACS) and Western-blotting. RESULTS:50 mmol/L Tris-HCl (pH=10) was more suitable for examining GFP samples with low content than other dilution buffers. The regression coefficient R2 between fluorescent value and GFP content was above 0.99 by using multi function measuring instrument. The results obtained from multi function measuring instrument correlated significantly with those obtained from FACS and Western-blotting. CONCLUSION:The established method of GFP assay using multi function measuring instrument was convenient, fast and could be used to screen large samples in a short time.

OBJECTIVE: To explore the application of partial least squares-quantities structure activity relationship (PLS-QSAR) model to predict the bacterial toxicity of nanometer metal oxide, in order to provide methodological support for toxicology research. METHODS:We built 12 models through 3 ways to determine principal components and 4 ways of variables selection from published data of nanometer metal oxide (EC50) and chose the best model based on internal and external validations. RESULTS:We chose the best model obtained using forward selection of variables combining variable importance in projection(VIP) and cross validation, and CVtest principle components determination. The model had the better fitness(R²=0.974), stability (Q_CV=0.951) and prediction (Q_EXT=0.775). CONCLUSION:PLS-QSAR can be applied to predict the bacterial toxicity of nanometer metal oxide as an alternative method and it may be worthwhile to explore variable selections and determine number of principle component.