OBJECTIVE: In order to identify the effect and mechanism of miRNAs involved in carcinogenesis， we evaluated the miRNA expression profiles at different stages of HBE cell transformation. METHODS：HBE，HBER and HBERST cells were collected and cultured. The comparison of differential expression profiles among these three cells was performed and analyzed by miRNA microarray. Then，the differentially expressed miRNAs were selected for validation by semi-quantitative real-time PCR. Finally，the functions of these miRNAs were examined using cell growth cure，cell cycle analysis and colony formation assay. RESULTS：856 human miRNAs were tested with microarray analysis in HBE，HBER and HBERST cell lines. Six differentially expressed miRNAs were found in HBERST compared with HBE and HBER cell lines，including 4 down-regulated and 2 up-regulated miRNAs. Among these miRNAs，miR-27a was found to be upregulated by SV40 ST in HBE cells. Suppression of miR-27a expression in HBERST cells inhibited the rate of cell growth (P＜0.01) and led to cell cycle arrested in the G0-G1 phase (P＜0.01). In addition，suppression of miR-27a in HBERST cells attenuated the capacity of cell colony formation (P＜0.01). CONCLUSION：Promotion of cell growth by miR-27a overexpression might be responsible for the viral oncoprotein small T antigen-induced malignant transformation.

OBJECTIVE: To study the apoptotic effect and regulation of 1，4-benzoquinone(PBQ) on human embryonic lung fibroblast (HELF)， cells. METHODS：MTT assay was used to detect the relative growth rate (RGR)，cell cycle was determined by single-stained assay with PI and apoptosis was determined by double-stained assay with Annexin-Ⅴand PI using flow cytometry. Bax，Bcl-2，p53 mRNA levels were determined with real-time PCR. RESULTS：The RGRs of HELF cells exposed to different PBQ concentrations after 24 h decreased (P < 0.05)，cell cycle distribution was changed at 40 and 60 μmol/L after 24 h (P <0.05). The RGRs of HELF cells exposed to 40 μmol/L PBQ after different times decreased (P <0.05)，the rates of G0/G1 phase increased with time (P < 0.05). Apoptosis was induced with PBQ higher than 20μmol/L，and increased with PBQ concentration (P < 0.05). The relative expression levels of Bax，Bcl-2，p53 increased with concentration after exposure for 24 h (P < 0.05). The relative expression levels of Bax and p53 exposed at 40μmol/L increased with time (P < 0.05)，while bcl-2 decreased with time (P < 0.05)，with Bax/Bcl-2 ratio increased with time (P < 0.05). CONCLUSION：PBQ was able to change cell cycle distribution of HELF cells，and induce apoptosis，with dose-response and time-response effects. The up-regulation of mRNA expression level of p53 and the elevated Bax/Bcl-2 ratio may be the mechanisms for apoptosis of HELF cells after PBQ exposure.

OBJECTIVE: To investigate the effect of p38 MAPK signaling pathway on the apoptosis induced by olaquindox in human hepatoma G2 (HepG2) cells. METHODS：The HepG2 cells were treated with different concentrations (0，200，400，800 μg/ml) of olaquindox for 24 h or with 800 μg/ml olaquindox for different time points (0，0.5，1，2，4，6，12，24 h). Then the expression of p38 and phosphorylation of p38 (p-p38) were determined by Western blot. The HepG2 cells which were pretreated with different concentrations (0，10，20 μmol/L) of SB203580 for 1 h were subsequently treated with 800 μg/ml olaquindox for 24 h. The changes of apoptosis were analyzed by Annexin V-FITC and propidium iodide (PI) staining. RESULTS：The expression of p-p38 increased gradually with the dose and time of olaquindox treatment. Moreover，compared with control group，the expression of p-p38 was observably apparent with the treatment of 800 μg/ml olaquindox for 24 h (P＜0.01). Compared with olaquindox control group (23.1%±3.59%)，the apoptosis of olaquindox-treated HepG2 cells was enhanced by different concentrations (10，20 μmol/L) of SB203580 with the apoptotic rate of 35.4%±2.83% and 40.2%±3.98% respectively (P＜0.05). CONCLUSION：p38 MAPK signaling pathway was activated by olaquindox，which played a role in inhibiting apoptosis induced by olaquindox-treated HepG2 cells.

OBJECTIVE: To observe the effects of paclitaxel on human lung cancer cell lines with different p53 genotype in vitro. METHODS：A549 (wild-type p53)，H322 (mutant-type p53) and H1299 (absence-type p53) were exposed to paclitaxel at different concentrations (0.1，1.0，10，100，1 000 nmol/L) or different treatment times (4，12，24，48，96 h)，then MTT assay，flow cytometry and western blotting were used to analyze the effects of paclitaxel on cell growth rate，cell cycle progression，apoptosis and the expressions of acetyl-tubulin and p53 protein. At the same time，negative control group (no paclitaxel) and blank control group (only culture medium) were set up. RESULTS：The growth-inhibition effects of paclitaxel on the three human lung cancer cell lines increased along with the response time extension (P＜0.05). Paclitaxel of different concentrations also had different effects on each lung cancer cell line (P＜0.05). The IC50 (50% inhibitory concentration) of paclitaxel for A549 was lowest in the three cell lines，but the difference of chemosensitivity of those cells to paclitaxel was not significant (P＞0.05). For the time-dependent experiment，the induction of apoptosis was time-dependent (P＜0.05) and the percentage of G2/M phase was highest at 12 h when paclitaxel was 10 nmol/L. When treated with 1 000 nmol/L paclitaxel，A549 showed the time-dependent G2/M arrest (P＜0.05)，while H322 and H1299 had the greatest G2/M arrest at 24 h. Prolonging paclitaxel treatments (48 h or more) resulted in appearance of polyploidy cells in H1299. The two concentrations could induce time-dependent apoptosis，but the higher one caused apoptosis later than the lower one. During concentration-dependent experiments， the proportion of apoptosis was highest at 10 nmol/L paclitaxel，while the percentage of G2/M phase increased in parallel with the paclitaxel concentration，when the latter changed from 0.1 nmol/L to 100 nmol/L (P＜0.05). Apoptosis had no relationship with G2/M arrest (P＞0.05). Paclitacxel up-regulated acetyl-tubulin and wild-type p53 protein expressions in time-and concentration-dependent manner，with no influence on mutant-p53 protein. CONCLUSION：Paclitaxel could inhibit the growth of the three human lung cancer cell lines with different p53 status in vitro in time-dependent manner. Different paclitaxel concentrations also had different effects on each lung cancer cell line. Paclitaxel could up-regulate wild-p53 protein expression and induce both p53-dependent and p53-independent apoptosis pathways. p53 genotype of the cells altered the effect of high concentration paclitaxel on cycle arrest，but did not influence paclitaxel chemosensitivity.

OBJECTIVE: To observe the effects of the ethanol and water extracts of Forsythia suspensa in which root，stem and leaf were tested， on the proliferation and apoptosis of human esophageal carcinoma cells in vitro， and to investigate its probable mechanisms. METHODS： The ethanol and water extracts of the root ，leaf and fruit of Forsythia suspensa were prepared. The growth-inhibitory effect of extracts of Forsythia suspensa on esophageal cancer cells were examined by MTT assay. Morphological changes of cancer cells were studied by light microscope and Wright-Giemsa staining. The apoptotic rate was analyzed with Annexin/PI double staining. Western blotting was employed to assess the changes of PARP expression. RESULTS ： All extracts of Forsythia suspensa could inhibit the proliferation of esophageal cancer cells，and the inhibitory effect of FSEER was the most obvious among them. After treatment with FSEER，TE-13 cells showed typical apoptosis morphological changes and the apoptotic rate was increased as compared to control group (P<0.05). Meanwhile， the cleavage of PARP was visible， and all of these changs were in a time-and dose-dependent manner. CONCLUSION： FSEER could significantly inhibit human esophageal carcinoma cell growth in vitro by inducing apoptosis.

OBJECTIVE: Oxidative DNA-damaging effects of mono-2-ethylexyl phthalate (MEHP) on MCF-7 cells were investigated. METHODS：MCF-7 cells were treated with MEHP at various concentrations (6.25，12.5，25，50 and 100 μmol/L) or dimethyl sulphoxide alone (DMSO，solvent control，final concentration <1‰). Cell proliferation was measured by the MTT assay at 12 and 24 h after the treatment. At the time points (1.5，3，6，12 and 24 h)，the content of malondialdehyde (MDA) and activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were measured using the corresponding kits. The level of (8-hydroxy-2'-deoxyguanosine，8-OHdG) in MEHP-treated cells was estimated using high-pressure liquid chromatography with electrochemical detection HPLC-ECD method. RESULTS：At 12 h after treatment， cell proliferation rates significantly increased in MEHP-treated groups (6.25 and 12.5μmol/L，P<0.05 for both). However，cell proliferation rates significantly decreased at 24 h after treatment，in the higher MEHP-treated groups (25-100μmol/L，P<0.05 for all). There were no significant changes in the levels of MDA，GSH-Px and 8-OHdG (P>0.05 for all).After MCF-7 cells were treated with MEHP for short periods (1.5，3 and 6 h)，the levels of MDA and 8-OHdG in all treatment groups were increased (P<0.05)，but SOD and GSH-Px activities were reduced (P<0.05 for all). CONCLUSION：Short periods(≤6 h) of treatment with MEHP at a certain concentration induced oxidative stress in MCF-7 cells，and accelerated DNA oxidative damage. The underlying mechanisms of MEHP mediated oxidative DNA damage MCF-7 cells need to be investigated.

OBJECTIVE: To study the role of NOX (NADPH oxidase) in X-ray- induced damage of human androgen-independent prostate cancer PC-3 cells damage，search for potential targets for radiation sensitization. METHODS：The viability of human androgen-independent prostate cancer PC-3 cells induced by 0，1，2，4 and 12 Gy of X rays after 24，48 and 96 h was detected by the MTT assay. The level of ROS after X-ray irradiation for 15，30，60 and 120 min ，and the expression of NOX1-5 protein in PC-3 cells induced by 0，1 and 4 Gy of X rays was analyzed by using DCFH-DA as a probe and by Western blot，respectively. RESULTS：Compared with non-irradiated，the viability of human prostate cancer PC-3 cells induced by 1，2，4 and 12 Gy of X rays was significantly decreased (P<0.05) . The level of ROS reached a maximum at 60 min after 1 and 4 Gy of X-ray irradiation. NOX inhibitor DPI and antioxidant NAC pretreatment could reduce the generation of ROS. Western blotting showed the expressions of NOX1，NOX2 and NOX5 were increased after irradiation. CONCLUSION：X-ray-induced the homologs NOX1，NOX2 and NOX5 of the catalytic subunit gp91phox of NADPH oxidase over-expression，resulting in excessive intracellular ROS which is a new mechanism of X-ray-induced damage of prostate cancer cells.

OBJECTIVE: The aim of this study was to investigate the association of Toll-like receptor 9 protein expression with cervical cancer pathogenesis. METHODS：Levels of TLR9 protein in chronic cervicitis，cervical intraepithelial neoplasia (CIN) and cervico-squamous cell carcinoma (CSCC) tissues from 97 Uighur patients were examined by immunohistochemistry. RESULTS：Immunohistochemical staining showed that TLR9 expression was mainly observed as cytoplasmic staining and was undetectable (88.2%) or weak (11.8%) in chronic cervicitis tissues，gradually increased in accordance with the histopathological grade in the following order：chronic cervicitis (2/17，11.8%)，CIN I (4/19，28.6%)，CIN II(3/10，30.0%)，CIN III(12/24，50.0%)，CSCC(17/32，53.1%). There were statistical differences among five groups (P＜0.05). The difference was statistically significant for CIN III and CSCC compared with cervicitis (P＜0.05)，but not for CIN I and CIN II (P＞0.05). CONCLUSION：This study showed that the TLR9 protein expression was，to some extent，associated with the development of cervical precancerous lesion and cancer.

OBJECTIVE: To investigate the effects of cadmium chloride on differentiation of mouse neural stem cells (mNSCs) in vitro. METHODS：Primary culture of mNSCs was used as research model. mNSCs were exposed to cadmium chloride (0，0.1，0.3，1.0，3.0 and 10.0 μmol/L). MTT assay was used to access cytotoxicity at 24 and 48 h，the differentiation of mNSCs after 48 h exposure of cadmium chloride was determined by immunocytochemistry staining. RESULTS：With the increase of cadmium chloride dose，cytotoxicity on mNSCs was accentuated in a dose-dependent manner (24 h，r＝－0.91；48 h，r＝－0.96；P＜0.05). The cell viability at 10.0 μmol/L after 24 hours and at 1.0，3.0 and 10.0 μmol/L after 48 h were lower than the control (P＜0.05 or P＜0.01). Cadmium chloride influenced differentiation of mNSCs. 10.0 μmol/L significantly decreased the rate of Sox2-positive cells (P＜0.05) after 48 h exposure to cadmium chloride. The GFAP-positive also decreased in a dose-dependent manner (r＝－0.99，P＜0.05). CONCLUSION：Cadmium chloride could inhibit the differentiation of mNSCs to astrocytes.

OBJECTIVE: To investigate the genotoxicity induced by two types of disinfection by-products (DBPs) trihalomethanes (chloroform，bromodichloromethane，chlorodibromomethane and bromoform) and haloacetonitriles (trichloroacetonitrile and dibromoacetonitrile). METHODS：Human hepatocellular carcinoma cell line HepG2 cells were treated with the DBPs mentioned above at four concentrations to evaluate their effects on the formation of micronuclei and nuclear division index (NDI) by means of cytokinesis-block micronucleus test (CBMNT). Dimethyl sulfoxide and benzo(a)pyrene were used as solvent control and positive control，respectively. RESULTS：Chloroform， bromodichloromethane and chlorodibromomethane were found to induce statistically significant increases in the frequency of micronuclei at the concentrations of 10 000 and 3 000 μmol/L(P＜0.05 )，while bromoform did not show similar effect on HepG2 cells in comparison with the solvent control. Trichloroacetonitrile and dibromoacetonitrile also induced statistically significant increases in the frequency of micronuclei and decreases in NDI at the lowest observed adverse effect levels of 2 000 and 30 μmol/L(P＜0.05 )，respectively. CONCLUSION：CBMNT could be used to detect the genotoxicity of trihalomethanes and haloacetonitriles，which were found to cause chromosome breakage in HepG2 cells.

OBJECTIVE: To evaluate the teratogenicity and developmental toxicity of recombinant B lymphocyte stimulating factor antagonist–antibody fusion protein (RCT-18) in Sprague-Dawley (SD) rats. METHODS：Mated female rats were randomly segregated into four groups，including three RCT-18 groups (11，37 and 129 mg/kg) and one negative control group (sodium chloride injection)，with more than 25 rats in each group. RCT-18 was administered via subcutaneous injection to timed pregnant rats on gestation days (GD) 6-15 every two days. Clinical signs，body weights，and feed consumption were monitored during the whole gestation. Caesarean section and autopsy were performed on GD20. Uterine content were evaluated for number of implantations，resorptions，live and dead fetuses. The number of corpora lutea in each ovary was also recorded．Live fetuses were examined for gender，body weights，body lengths，tail lengths，and gross external，visceral and skeletal changes. RESULTS：There were no significant differences in maternal and embryo-fetal parameters. CONCLUSION：No maternal toxicity and embryo-fetal toxicities were found when RCT-18 was administered to SD rats during GD 6–15.

OBJECTIVE: To establish post-implantation rabbit whole embryo culture for studying teratogenic agents. METHODS：Gestation day 9(GD9) rabbit embryos were cultured mainly according to the method of Carney and Naya，embryos dissected，and cultured one per bottle，and morphology was compared with results in the literature after culturing 48 h in vitro. 5 mmol/L methoxyacetic acid was used as positive control. The scoring system was a new refined morphological scoring system by Carney. RESULTS：23 GD9 embryos cultured in vitro all survived，the average growth of head length was 2.15 times，the visceral yolk sac closure，flexion，somites， spontaneous malformation，average of morphology scores etc were close to the literature. The positive control of 5 mmol /L MAA induced developmental toxicity consisted of reducing closure rate of yolk sac，swollen head regions，lack of facial development，disordered somites，delayed closure of caudual neural tube etc，the scoring was lower than the solvent control (P<0.05). CONCLUSION：The post-implantation rabbit whole embryo culture method and conditions established in our laboratory was reliable，which could be an efficient tool to study teratogenic agents.

OBJECTIVE: To study mRNA expression of oncogenes (c-fos，c-myc，K-ras，p53) in peripheral blood of patients allergic to trichloroethylene (TCE). METHODS：Peripheral blood samples were collected from healthy workers (control group) and allergic patients (case group). Real-time quantitative PCR was applied to detect mRNA expression of oncogenes c-fos，c-myc，K-ras，p53. RESULTS：The level of c-fos mRNA expression increased by 352% in TCE patients when compared with control (P<0.01)，c-myc increased by 41%，K-ras by 136% and p53 increased by 64%. mRNA expression levels of these oncogenes showed significant differences between case and control groups (P<0.01 or P<0.05). CONCLUSION：Trichloroethylene could induce oncogene expression in patients with allergic dermatitis， indicating that TCE might be potentially carcinogenic.

OBJECTIVE: To investigate the association between lymphomas and Epstein-Barr virus(EBV) infection. METHODS: We collected 171 lymphomas including diffuse large B-cell lymphoma (DLBC) 106 cases; extranodal NK/T cell nasal type lymphoma 22 cases; Hodgkin's lymphoma (HL) 19 cases; angioimmunoblastic T- cell lymphoma (AITL) 13 cases and mucosal-associated lymphoid tissue (MALT) 11 cases. The expressions of EBV protein LMP-1 and EBER1 mRNA in 171 lymphomas were analyzed by immunohistochemistry (IHC) and in situ hybridization (ISH). RESULTS: The positive results of EBV-LMP-1 protein expression and EBER1 mRNA expression were 11.1%(19/171) and 25.7%(44/171) respectively，including 30.8%(4/13) and 61.5%(8/13) in AITL；47.4%(9/19) and 57.9%(11/19) in HL；22.7%(5/22) and 81.8%(18/22) in extranodal NK/T cell nasal type lymphoma；0.94%(1/106) and 5.7%(6/106) in DLBC. The expression rates of EBV in AITL，HL and nasal-type extranodal NK/T cell lymphoma were significantly higher than in MALT and DLBC(P<0.05). ISH was more sensitive than IHC in identification of EBV(P<0.01). CONCLUSION: There was a close association between EBV infection and lymphomas， with variation among different types of lymphomas.

OBJECTIVE: To explore the prognostic significance of the number of negative lymph node on survival rates in patients with thoracic esophageal cancer. METHODS：Clinicopathological and survival data were collected retrospectively in 239 patients with thoracic esophageal cancer in the Shantou Central Hospital between 2000 and 2006. Kaplan-Meier curves and the Cox regression analysis were used to evaluate the association between survival and each clinicopathological factor. RESULTS：The 5-year survival rate was 44.1% for the entire cohort. Univariate analysis indicated that both the positive and negative lymph node numbers were significant prognostic factors. Patients with 4 or more negative nodes had a better 5-year survival rate than those with fewer than 4 negative nodes (49.1% vs. 32.5%， respectively，χ2 = 6.042，P = 0.014). Based on multivariate regression analysis，positive and negative lymph node numbers were independent factors. CONCLUSION：The number of negative lymph nodes has significant prognostic impact on the survival rate of patients with thoracic esophageal cancer and could be integrated into the TNM staging consideration for esophageal cancer.

OBJECTIVE: To evaluate the mutagenicity and anti-mutagenicity of aloe-emodin and aloe extract by in vitro micronucleus test in L5178Y cells. METHODS：For 4 concentration of both aloe-emodin and aloe extract，mutagenicity and anti-mutagenicity groups were set up，as well as solvent control，positive control and anti-mutagenicity control. The L5178Y cells were treated respectively for 12 h，and in vitro micronucleus test was analyzed by routine method. RERULTS：Aloe-emodin showed mutagenic effect at 6.67 μg/ml. Micronucleus rate at this dose showed a significant difference compared with the control group (P＜0.05)，but aloe extract showed non-mutagenic effect in this study. Within a certain dose range，both aloe-emodin(0.22 μg/ml) and aloe extract(20 μg/ml) could exert antagonistic effect on the increase of micronucleus rate induced by MMS，showing a significant difference compared with the control group (P＜0.01). CONCLUSION：Under our experimental conditions，aloe-emodin showed weak mutagenic effect， while aloe extract was not found to have mutagenic effect. Both of them displayed anti-mutagenic activity.

OBJECTIVE: To evaluate the use of polymerase chain reaction-denaturing high performance liquid chromatography technology (PCR-DHPLC) in Down syndrome screening. METHODS：Primers of D21S1409， D21S1422，D21S1435 and GATA163G03 loci on chromosome 21 were designed. The samples of the children with Down's syndrome or the normal ones were amplified using PCR method. The PCR amplification products were identified by denaturing high performance liquid chromatography technology. At the same time the same samples were analyzed with karyotyping，to evaluate the accuracy of PCR-DHPLC. RESULTS：The column temperature was 50 ℃，the Down syndrome samples showed specific DHPLC peak of Down syndrome. CONCLUSION：The PCR-DHPLC method edmonstrated high sensitivity and specificity. It could be used in high-throughput rapid screening for Down's syndrome.