OBJECTIVE: To investigate the role and mechanism of histone H3 phosphorylation at Ser10[p-H3(Ser10)] on maintainence of malignancy in tumor cells. METHODS: Hepatic tumor cell lines including HepG2,Bel7402,and SMMC-7721 as well as human liver cell line L02 were used to detect the levels of p-H3(Ser10) based on Western blot analysis. In addition,a histone H3 mutant cell line which contained Ser10 mutated at Ala10 (Bel-H3S10A) was constructed. Soft agar assay was conducted to assess the effect of down-regulation of p-H3(Ser10) on the malignant phenotype of tumor cells. The level of histone H3 phosphorylation at Ser10 and its impact on the expression of α4 were detected by western blotting. Bioinformatics was used to identify the adaptor protein 1 (AP-1) binding site of IGBP1 promoter. The relationship between p-H3(Ser10) and the expression of α4 as well as its effect on the transcriptional level of α4 were determined by double luciferase reporter assay and western blot analysis in the constructed Bel7402 and A549 cells overexpressing AP-1and mutated histone H3. RESULTS: p-H3(Ser10) was over-expressed by 2.61 folds and α4 was increased by 2.0-6.3 folds in HCC cell lines(P < 0.05). Moreover,the level of histone H3 phosphorylation at Ser10 in Bel-H3S10A cells was decreased and formed 30% less colonies in soft agar than the control Bel-Vector cells. Cadmium chloride (CdCl₂) induced expression of p-H3(Ser10) and α4 in a dose-dependent manner(P < 0.05). However,the protein expression of α4 decreased by 47% in A549-AP-1-H3S10A cells compared to that in control cells upon CdCl₂ treatment(P < 0.05). CONCLUSION: p-H3(Ser10) played an important role in maintaining malignancy of tumor cells,perhaps through regulating the expression of α4 by activating transcription factor AP-1.

OBJECTIVE: To study the expression and clinical significance of thioredoxin (Trx) and thioredoxin reductase-1 (TrxR1) in tongue squamous cell carcinoma. METHODS: Twenty-eight cases of tongue squamous cell carcinoma and 10 cases of normal epithelial tissues were collected. Expression levels of Trx and TrxR1 in two tissues were detected by the SP immunohistochemical three-step method. The relationship between their expression and clinicopathological features was analyzed. RESULTS: The expression levels of Trx and TrxR1 in tongue squamous cell carcinoma tissues were significantly higher than those in adjacent normal epithelial tissues. Trx IOD values in cancer tissues and adjacent normal tissues were (2.84±0.34)×10⁸ and (3.91±3.00)×10⁷,respectively (P < 0.01). TrxR1 IOD was (1.88±0.29)×10⁸ and (0.69±0.32)×10⁷ (P < 0.05). Trx and TrxR1 protein expression levels were between tumor size and differentiation;the differences in expression were statistically significant (P < 0.05). The expression levels of Trx and TrxR1 were moderately correlated (r=0.504,P < 0.05),and the high and low expression groups of Trx and TrxR1 were expressed. The difference in survival was statistically significant (P < 0.05). CONCLUSION: Thioredoxin and its reductase-1 can possibly be targets of drug therapy for tongue squamous cell carcinoma and important clinical indicators for early diagnosis and tumor screening.

OBJECTIVE: DNA glycosylase 1 (OGG1) is a critical factor involved in oxidative DNA damage repair,the aim of this study was to investigate the role of OGG1 in Helicobacter pylori (HP)-induced DNA damage in gastric epithelial cells (GES-1). METHODS: Cultured cells were divided into four treatment groups:control,HP infection,OGG1 siRNA,HP infection plus OGG1 siRNA. Human gastric epithelial cells (GES-1) were infected with HP at 24 h after OGG1 inhibition through the use of RNA interference. 24 h and 48 h after HP infection,cytotoxicity was evaluated based on viability using the CCK-8 assay and lactate dehydrogenase (LDH) release. DNA damage was measured using the phosphorylated H2AX (γH2AX) and the comet assays. Apoptosis was determined by TUNEL assay and western blot against poly-ADP-ribose polymerase (PARP). RESULTS: Compared to the control,no significant differences were found in cell viability,LDH release,DNA damage,γH2AX foci formation,TUNEL-positive cells and the expression levels of cleaved PARP in HP infection and OGG1 siRNA groups. However,compared to the control and the HP infection groups,HP infected cells with OGG1 deficiency showed reduction in cell viability and increased LDH release. Moreover,HP infection in OGG1 deficient cells induced DNA damage and γH2AX foci formation,elevated TUNEL-positive cells and expression of cleaved PARP. After statistical analysis,there were significant differences in the above indexes (P < 0.05 or 0.01). CONCLUSION: These findings indicate that OGG1 played a protective role in HP-induced DNA damage in gastric epithelial cells and the information may provide a new strategy for preventing HP infection-related gastric diseases.

OBJECTIVE: The present study was designed to investigate the possible mechanisms of proanthocyanidin-mediated inhibition in lipopolysaccharide (LPS)-induced activation of microglial BV2 cells. METHODS: An inflammation cell model was produced in BV2 cells which were treated with LPS (1.0 μg/mL). In addition,cells were treated with proanthocyanidins (0.1,0.5,2.5,10.0 μg/mL) prior to LPS exposure and MTT assay was used to detect the viability of BV2 cells. The level of inflammatory cytokines TNF-α,IL-1β and IL-6 were examined by ELISA method. TLR4 and p38,p-p38 MAPK protein expressions were examined by the Western blot analysis. RESULTS: Compared with the control group,LPS (1.0 μg/mL) significantly increased the expression of TLR4 and p-p38 protein as well as the release of inflammatory mediator TNF-α,IL-1β and IL-6 (P < 0.05). Proanthocyanidin significantly inhibited LPS-induced production of inflammatory mediators in a dose-dependent manner (P < 0.05). Moreover,PC significantly inhibited the phosphorylation of p38 and TLR4 expression (P < 0.05). CONCLUSION: Proanthocyanidin inhibited inflammatory mediator expression by suppressing TLR4 mediated p38 MAPK signaling pathways in LPS-stimulated BV2 cells.

OBJECTIVE: To understand key molecular events in prognosis of breast cancer,we analyzed the data published by TCGA,and to study the associations between microRNAs and potential target genes at the pathway level. METHODS: To collect and organize the gene expression profile and microRNA sequencing data of breast cancer in TCGA database,we use multiple t-tests to analyze the differentially expressed microRNAs and target genes,and R language package microRNA-mRNA to predict the potential target regulated genes in microRNA. General Applicable Gene-set Enrichment (GAGE) was applied to discover the key genes in essential pathways of breast cancer. Integrated association analysis was used to find the potential targets of the microRNAs. The Cox proportional hazard model was used to evaluate the possible prognostic signatures in breast cancer. RESULTS: The results show that there were abnormal expression of 344 genes and 135 microRNAs. 8 microRNAs with 31 potential target genes might have played key roles in breast cancer through 139 essential pathways. Cox regression risk model results show that increased SFRP1 had a protective effect (HR=0.9,P=0.015) while miR-342-5p had neither protective nor hazard effects (HR=0.99,P=0.144). However interactive analysis suggest that has-mir-342-5p inhibited SFRP1 expression in breast cancer with poor prognosis (HR=1.88,P=0.016). CONCLUSION: Through deep excavation of gene expression and microRNA sequencing data from TCGA database,we identified abnormal expression of certain microRNA and genes in breast cancer,and their involvement in key signaling pathways,suggesting that has-mir-342-5p inhibited the expression of SFRP1 in patients with breast cancer who had poor prognosis.

OBJECTIVE: To explore the damage and recovery of hematopoietic system in mice after total body irradiation (TBI) with different doses of carbon-ion. METHODS: Female BABL/c mice were irradiated with 0.5,1,4,6 Gy carbon-ion beam. DNA damage in bone marrow monocytes cells (BMMCs) were detected by alkaline comet assay and cell cycle distributions were detected by FACS. Peripheral blood cell counts and spleen index were detected at 1^st,3^rd,8^th days after irradiation. RESULTS: Compared to the control group,carbon-ion TBI induced serious BMMNCs DNA damage in a dose-dependent manner (all r ≤ 0.96,P < 0.01). The most serious damage occurred at the 3^rd day post-irradiation. At the 8^th day after irradiation,DNA damage was still serious (P < 0.01). Carbon-ion TBI also caused dose-dependent bone marrow cells S phase and G2/M phase arrests and decrease of peripheral blood nucleated cells and spleen index. At the 8^th day after irradiation,the S phase arrests of bone marrow cells has been alleviated to some extent,but the decrease of peripheral blood nucleated cells and spleen index were still significant. CONCLUSION: Carbon-ion TBI caused serious damage to the hematopoietic system of mice in a dose-dependent manner. At the 8^th day after irradiation,the BMMCs DNA damage was still serious,and hematopoiesis did not recover.

OBJECTIVE: To establish patient-derived non-small cell lung cancer xenegraft models in NOD/SCID or nude mice,and to compare the tumor-forming rates between these mice. METHODS: Non-small cell lung cancer tissues were collected from 23 patients who underwent surgical resection at the Department of Thoracic Surgery of Beijing Chest Hospital from July 2016 to December 2016. Non-small cell lung cancer tissues were subcutaneously implanted into NOD/SCID mice or BALB/c nude mice within 1 hour after surgical resection. Growth processes of the xenotransplanted tumors were observed,the tumor volumes were measured and the growth curves of the xenotransplanted tumors were plotted. The tumor-forming rates,the tumor latent time and the tumor formatting time were calculated. Then,comparison was made on morphology of patient-derived tumor xenegraft tissue and corresponding primary tumor tissue,and the related patients clinical pathological index of two groups,that could form xenegraft tumor group and couldn't form xenergraft tumor group. RESULTS: Xenegrafts were successfully established (55.6%) in NOD/SCID and (20%) nude mice. The tumor-forming rates of NOD/SCID mice models were higher than the nude mice models,and the tumor latent time (37 d,60 d,P=0.002) and the tumor formatting time (79 d,110 d,P=0.002) had significant differences. Tumor factors might associate with engraftment included squamous histology,TNM stage and the lymph node metastasis. Both mice models could keep the morphology of patient-derived tumor xenegraft tissues. CONCLUSION: The xenotransplantation models of patient-derived non-small cell lung cancer in both NOD/SCID mice and nude mice were successfully established. In comparison with the BALB/c nude mice model,the tumor-forming rates of the NOD/SCID mice model was higher,the tumor latent times and the tumor formatting times were shorter,suggesting that the NOD/SICD mice model was more suitable for the establishment of patient-derived NSCLC xenegraft model.

OBJECTIVE: To investigate the correlation of five single nucleotide polymorphisms in the PRKAα1 gene(rs13361707C > T,rs10074991G > A,rs154268T > C,rs3805486T > C and rs6882903C > A) with gastric cancer risk in an East Asian population. METHODS: Publications of correlation between PRKAα1 gene polymorphisms and gastric cancer were searched from PubMed,China National Knowledge Infrastructure (CNKI),Chinese Wan Fang database and Database of Chinese Scientific and Technical Periodicals (VIP). The odds ratios (OR) and 95% confidence intervals (95% CI),and sensitivity and publication bias were analyzed by STATA 12.0 software. RESULTS: The rs13361707C > T polymorphism analysis was mentioned in 9 studies. There was no significant association between rs13361707C > T polymorphism and gastric cancer risk in East Asian populations. The result of subgroup-analysis demonstrates:dominant model,OR=0.687,95% CI(0.614-0.769),P=0.000;recessive model,OR=0.662,95% CI(0.594-0.737),P=0.000; Addictive Model,OR=0.553,95% CI(0.484-0.632),P=0.000;codominant model,OR=0.766,95% CI(0.682-0.859), P=0.000;The subgroup analysis showed that this locus was associated with the decreased risk of gastric cancer in a Korean population under the four models. The rs10074991G > A polymorphism analysis was performed in 3 studies. The result of meta-analysis demonstrates:Dominant model,OR=0.590,95% CI(0.490-0.700),P=0.000;recessive model,OR=0.637,95% CI(0.535-0.759),P=0.000;addictive model,OR=0.478,95% CI(0.385-0.593),P=0.000; codominant model,OR=0.651,95% CI(0.541-0.784),P=0.000. The results show that this locus was associated with the decreased risk of gastric cancer in East Asian populations under the four genetic models. The subgroup analysis show that this locus was associated with the decreased risk of gastric cancer in Korean populations under the four genetic models. In addition to the recessive model,this locus was associated with the decreased risk of gastric cancer in Chinese populations under other three models. Three SNPs of rs154268T > C,rs3805486T > C,rs6882903C > A analysis were performed in 2 published studies. In addition to the recessive model,the rs154268T > C polymorphism was associated with the increased risk of gastric cancer in Korean populations under other three models. rs3805486T > C polymorphism was associated with the decreased risk of gastric cancer in Korean populations under the four genetic models. The rs6882903C > A polymorphism was associated with the increased risk of gastric cancer in Korean populations under the dominance and additive models. Begg's test found no publication bias (P > 0.05),sensitivity analysis showed that the above results were stable. CONCLUSION: The rs10074991G > A,rs154268T > C,rs3805486T > C and rs6882903C > A polymorphisms in PRKAα1 gene were significantly correlated with increased gastric cancer risk.

OBJECTIVE: To construct cells with over-expression of the StAR gene and to study its apoptotic response to di-(2-ethylhexyl) phthalate (DEHP). METHODS: Primers were designed according to cDNA sequence of the StAR gene from GenBank. The gene was amplified using PCR and ligated into the lentiviral vector pLVX-CMV-ZSgreen-PGK-Puro. 293FT cells were transfected with the recombinant vector and then MCF-7 cells were transfected. Cells with over-expression of the StAR gene were identified by gene sequencing,real-time quantitative PCR and western blot. Then the StAR gene over-expression cells and MCF-7 cells were treated with various doses of DEHP for 24 h to detect expression of apoptosis genes including Bax,Caspase-3 and Caspase-8. RESULTS: The sequence contained in the recombinant vector was exactly the same as the StAR gene from GenBank. StAR gene expression level in the transfected MCF-7 cells was 591.9 times higher than that of control MCF-7 cells. StAR protein level in the transfected MCF-7 cells was 190% higher than that of control MCF-7 cells. After DEHP treatment of the transfected and control MCF-7 cells,qPCR results show that mRNA levels of Bax,Caspase-3 and Caspase-8 were significantly higher in the transfected MCF-7 cells. Additionally,mRNA levels of Bax,Caspase-3 and Caspase-8 increased significantly (P < 0.05 or P < 0.01) in the transected compared with the similarly treated non-transfected MCF-7 cells. Western blot results show that the protein expression levels of Bax,Caspase-3 and Caspase-8 also increased significantly in the transfected MCF-7 cells. Protein expression levels of Bax,Caspase-3 and Caspase-8 were also increased significantly (P < 0.05 or P < 0.01) except in the 0.2 mmol/L treatment group. CONCLUSION: The StAR gene over-expression cells were successfully constructed. These cells responded to DEHP treatments by showing significant increase in apoptotic gene expression. These findings indicate that StAR gene promoted DEHP induced-apoptosis.

OBJECTIVE: Local invasion is a leading cause of death in patients with lung cancer. To date,there has been no report on distribution of VEGF,EGFR and serum protein in invasive tissues of lung cancer using in vivo cryotechnique (IVCT) method. METHODS: The human A549 cell line from a non-small cell lung cancer (NSCLC) was used and cells were subcutaneously injected to the dorsal flank of nude mice. In vivo tumor samples were prepared by IVCT method. At the same time,protein distribution of albumin,IgG,IgM,VEGF and EGFR were examined by immunohistochemistry method. RESULTS: The distribution of albumin and IgG in connective tissues around the tumor mass,blood vessels,interstitium and extracellular matrix of NSCLC's invasive parts can be clearly observed by using the IVCT method. IgMs were mainly located in connective tissues around the tumor mass and blood vessels,but their distribution could not be observed in extracellular matrix of cancer cells. VEGF protein was located in cytoplasm of lung cancer cells,and it had fine granular morphology,but there was no distribution of EGFR in cytoplasm or membrane of cancer cells. CONCLUSION: The immunodistribution of different molecular weight of serum proteins in connective tissues around the tumor mass,blood vessels,interstitium and extracellular matrix can be clearly observed. The permeability of NSCLC's invasive part for albumin,IgG1 and IgM in extracellular matrix mainly depended on their respective molecular weights,but not on the protein distribution of VEGF and EGFR.

OBJECTIVE: The in vitro CHO-K1 cells HGPRT gene mutation test and the mouse bone marrow micronucleus test were used to analysis the expression of genetic toxicity of a pesticide,Paichongding. METHODS: In CHO-K1 cells,the positive medium and different concentrations of Paichongding were added into the culture medium (+S9/-S9). After treatment of the cells at low density,mutant selection and colony formation were calculated at the same time with cell survival rates. The 2×10⁵/dish was inoculated with 6-TG,at the same time,the other by the 200/plate were calculated after 7 d,colony number,survival rate and mutation frequency. Mouse bone marrow cell micronucleus test were conducted with high,medium and low Paichongding dose groups,positive control group and negative control group. Femoral bone marrow smears were observed:1 000 polychromatic erythrocytes micronucleus in polychromatic cells and 200 polychromatic erythrocytes (PCE) and the number of mature red blood cells (NCE),red blood cell count percentage calculation PCE. RESULTS: Compared with the control group,the highest dose of Paichongding significantly reduced the relative survival rate of CHO-K1 cells. Paichongding cell gene mutation frequency in each dose group had no significant difference (P > 0.05) while the positive control group (cell gene mutation frequency) had significant difference (P < 0.01). From the Paichongding treated mice,the immature red blood cells accounted for the proportion of the total number of red blood cells was not less than 20% of the negative control group. Compared with the negative control group,there was significant difference between male and female mice in positive control group (P < 0.01),while there was no significant difference in the micronucleus rate of male and female mice in each dose group (P > 0.05). CONCLUSION: Under the experimental conditions,no genetic toxicity was found with Paichongding。

OBJECTIVE: To observe toxicological effects in blood cells in rats after treatment with different doses (0,5,50,500,2 500 mg/kg) DEHP. METHODS: Rats were divided into control and four treatment groups. The treatment groups were exposed to four concentrations of DEHP (5,50,500 and 2 500 mg/kg) for 90 days. At the end of the exposure,whole blood samples were taken to measure the changes of various types of blood cells. RESULTS: Compared with the control group,the leukocyte parameters for the 2 500 mg/kg dose groups were:eosinophils decreased significantly (P < 0.01),the total number of white rats significantly decreased (P < 0.05). Lymphocytes in 500 and 2 500 mg/kg groups were significantly increased (P < 0.05). In the erythrocyte parameters,the total number of red blood cells,hemoglobin (HGB) and hematocrit (HCT) in the 2 500 mg/kg group were significantly decreased (P < 0.01) while the levels of hemoglobin (HGB) in 500 and 2 500 mg (P < 0.05 or P < 0.05). Mean hemoglobin content (MCH) in 500 and 2 500 mg/kg groups decreased significantly (P < 0.05 or P < 0.01). The measured hemoglobin concentration (CHCM) decreased significantly (P < 0.05 or P < 0.01). On the other hand,DEHP had little effect on platelet parameters. CONCLUSION: DEHP exposure affected blood cells of rats under certain dosages.

OBJECTIVE: To establish a stable system for sperm and tumor cell fusion and to provide a biological model for sperm and tumor cell fusion investigation. METHODS: Fusion of sperm and tumor cells (e.g. Hela) was carried out by their co-cultivation in vitro. The 3D-confocal microscopy imaging was used to confirm the fusion of both cells. The transwell insert culture system was used as a control for cell fusion experiment. The fusion model was optimized by using the self-synthesized sperm-tumor cell culture medium (STCM). RESULTS: Fusion between the sperm and the tumor cells was observed in vitro which generally occurred in 1.5 to 2 hours after co-cultivation. The chimeric cells generally displayed a spherical type and floated in the medium as a result of significantly reduced attachment ability. Comparison with RPMI 1640 medium,STCM not only disturbed the growth of tumor cells but greatly improved the fusion rates between the two cell types (P < 0.05) and accelerated the fusion to some extent. CONCLUSION: We have established a fusion model of sperm and tumor cells by using co-cultivation of both cells in vitro.

OBJECTIVE: To develop an in vitro alternative approach to the direct peptide reactivity assay (DPRA) for screening chemical allergens. METHODS: Lysine/cysteine peptides were incubated with test chemicals for 24 h and high performance liquid chromatography was performed. Depletion of the peptide was used as an indication of skin sensitization potential of chemicals. RESULTS: 12 chemicals representing allergens of different potencies caused depletion of greater than 6.38% with different reactivity,6 nonsensitizers gave depletion of less than 6.38%,which was consistent with LLNA sensitization classification. Unknown allergenic chloroform and benzene both yielded less than 6.38% depletion,which were judged as negative. CONCLUSION: The direct peptide reactivity assay appears to be a reliable predictor of sensitization potential for chemicals. In addition,it is simple,rapid and suitable for high throughput screening of chemical sensitization. Therefore,the assay can possibly be used for safety evaluation of chemicals.