OBJECTIVE: To investigate the role of the inhibitor of Wnt production 2 (IWP2) on non-small cell lung cancer (NSCLC) cell migration and invasion and to explore mechanisms for the effects. METHODS: H1299 and 95C cell lines were divided into control (cultured with serum-free RPMI-1640 media) and experiment (treated with 10 μmol/L IWP2) groups. Cell migration ability was examined with wound healing and with Transwell assay without Matrigel. Cell invasion ability was determined using Transwell assay with Matrigel. In addition,Western blot was performed to analyze the effect of IWP2 on β-catenin and epithelial-mesenchymal transition(EMT)-related proteins (ZEB1 and Snail). RESULTS: Compared with the control groups,the wound healing results demonstrated that cells cultured with IWP2 had decreased wound closure both in H1299 and 95C (P < 0.01). Transwell assay without Matrigel showed that fewer cells migrated to the lower chambers after being treated with IWP2 (P < 0.01). The Transwell assay with Matrigel indicated that cell invasion ability was also significantly decreased in the experimental compared with the control groups. Besides,after being treated with IWP2,expression of β-catenin was obviously reduced (41.3% in H1299 and 36.1% in 95C). In addition,IWP2 also decreased the expression of ZEB1 (51.8% in H1299 and 40.9% in 95C) and Snail (43.2% in H1299 and 30.7% in 95C). CONCLUSION: Our study revealed that IWP2 down-regulated EMT via blocking β-catenin which led to the inhibition of NSCLC cell migration and invasion.

OBJECTIVE: To study effects of atrazine (ATR) on neuronal degeneration in adolescent rats. METHODS: 4 weeks old Wistar male rats were randomly subdivided into 4 groups:control and ATR test groups. All rats were treated with ATR:50,100 and 200 mg/kg body weight per day (five times per week) for 45 days by oral gavage. They were sacrificed at 12 h after the last ATR injection. The entire midbrain was collected and stored frozen at -80℃. The presence of tyrosine hydroxylase(TH) was detected by immunohistochemistry SP in dopaminergic neurons. TH expression in mRMA and protein was measured using real-time PCR and Western blot. RESULTS: Immunohistochemical results showed that,compared with the control group,the number of TH positive cells in the 200 mg/kg ATR group was significantly decreased (P < 0.01). In addition,qPCR results show that expression of TH mRNA and Bcl-2 mRNA was significantly decreased in the 100 and 200 mg/kg ATR groups,and expression of p53 mRNA and Caspase-9 mRNA was significantly increased in the 200 mg/kg ATR group,the difference was statistically significant (P < 0.05). Western blot results show that expression of TH and Bcl-2 protein was significantly decreased in the 100 and 200 mg/kg ATR groups compared with the control group. Expression of p53 protein in the ATR group was significantly increased at 100 and 200 mg/kg,and Caspase-9 protein in he 200 mg/kg ATR group was significantly increased,the difference was statistically significant (P < 0.05). CONCLUSION: Atrazine caused damage to the dopaminergic neurons in the substantia nigra of rats and activated the apoptotic pathway which led to the dopaminergic neuronal death.

OBJECTIVE: To investigate the effect of vitamin E succinate (VES) on unfolded protein response (UPR) in human gastric cancer SGC-7901 cells. METHODS: SGC-7901 cells were treated with VES at 5,10 and 20 μg/mL and with tunicamycin (TM) at 3 μg/mL for 24 h or at 20 μg/mL for up to 24 h. RNA-dependent protein kinase (PKR)-like ER kinase (PERK) and activating transcription factor 6 (ATF6) were evaluated using Western blotting. X-box-binding protein 1 (XBP1) and activating transcription factor 4 (ATF4) were evaluated using RT-PCR. RESULTS: VES induced the phosphorylation of PERK in a time-dependent manner and reached the top level at 10 μg/mL. In addition, VES induced the generation of p50ATF6 in SGC-7901 cells in a dose-and time-dependent manner. Like the positive control,VES treatment at 20 μg/mL induced the spliced form of XBP1 from 6 h up to 24 h. Meanwhile,ATF4 mRNA was also up-regulated in cells exposed to VES in a time-dependent manner and reached the top level at 18 h. CONCLUSION: UPR pathways may be activated by VES in human gastric cancer SGC-7901 cells.

OBJECTIVE: To evaluate correlations between serum thyroid hormone levels and exposure to toxic substances from e-waste dismantling regions. METHODS: Randomized-control studies on correlations between serum thyroid hormone levels and exposure to toxic substances from e-waste dismantling areas that were reported on PubMed,Web of Science,Elsevier,CNKI and WanFang Database up to April 2017 were searched and analyzed. The data of the included studies were analyzed using Revman 5.2. RESULTS: 9 randomized-controlled studies were identified including 489 people exposed to toxic chemicals from e-waste dismantling areas and 489 people of control. Serum 1evels of total thyroxine (T4) and free thyroxine (FT4) in the expose groups were significantly lower than those in the control groups (P < 0.05),while there were no significant differences in the serum levels of free triiodothyronine (FT3),total triiodothyronine (T3) and thyrotropin (TSH)(P > 0.05). CONCLUSION: Exposed to toxic chemicals from e-waste dismantling regions may affect the serum level of thyroid hormone.

OBJECTIVE: To construct an inducible lentiviral system which expressed HBx in primary human hepatocytes (PHH) for the study of biological characters of HBx and its influence on PHH. METHODS: We constructed a Tet-on inducible HBx-lentiviral expression system,and optimized it to a relatively high expression level. We applied this system to make LV-HBx and LV-rtTA recombinant lentivirus,to transduce HepG2 hepatoma cells and PHH,and to induce expression of HBx with doxycycline. RESULTS: We successfully constructed the recombinant lentiviral vector of HBx. The ratio of recombinant lentivirus of LV-HBx and LV-rtTA was optimized and a higher transduction efficiency could be approached at the ratio of 1:1 or 2:1 with HBx-positive cell ratio of 67.3% and 66.7%,respectively. This system was successfully applied in transducing HBx gene and inducing HBx expression with doxcycline in HepG2 and PHH. The expression level of HBx in PHH with Dox induction was about 45 fold of the group without Dox induction. HBx significantly increased the expression level of MTA1,VEGFA,and TGFB1 and decreased the expression level of CDH1 and IGFBP3,which is consistent with the previous reports. CONCLUSION: We successfully constructed a Tet-on expression system of LV-HBx for the transduction of HBx into PHH. This system will facilitate studies on influence of HBx and other HBV gene on PHH.

OBJECTIVE: To analyze expression of miR-126 in the tissues and serum of gastric cancer as diagnostic biomarkers. METHODS: Subjects for the study group were 42 cases of gastric cancer patients. The control group were 60 healthy individuals of similar age and gender. Fresh surgical specimens were collected after radical gastrectomy. Blood samples were collected from controls and from the patients before and at one week after the operation. miR-126 expression in tissues and serum were analyzed using quantitative real-time PCR and CEA test in the lab. RESULTS: For gastric cancer tissues,miR-126 expression,which was related to tumor size,clinical stage and differentiation degree,was significantly lower in the cancer tissues than in the distal cancerous contrast tissues,t=7.276,P < 0.05. miR-126 expression in the pre-operative serum,which was related to tumor clinical stage and differentiation degree,was significantly lower than that in the post-operative serum,t=6.316,P < 0.05;and the miR-126 expression in the post-operative serum was significantly lower than that in the healthy control group,t=4.338,P < 0.05. The AUC of the ROC Curve of diagnostic efficiency of miR-126 expression was 0.823 in gastric cancer tissues and 0.776 in serum. The AUC of CEA diagnostic efficiency was 0.537,P > 0.05. Pearson correlation analysis showed positive correlation coefficient of miR-126 expression in tissues and pre-operative serum of gastric cancer at 0.484. CONCLUSION: miR-126 expression was down-regulated in gastric cancer tissues and serum. miR-126 expressions in the serum can possibly be used as an indicator for the severity of gastric cancer. miR-126 expressions in serum had desirable diagnostic values of gastric cancer,better than the serum CEA test. miR-126 expression levels in tissues and serum were positively related,therefore it may become valuable biomarkers for diagnosing early stage of gastric cancer and monitoring of recurrence.

OBJECTIVE: To explore the protein expression efficiency,cellular localization and the amine oxidase activity of exogenous recombinant expressed semicarbazide-sensitive amine oxidase (SSAO) in 293T cells. METHODS: The recombinant plasmid pcDNA3-SSAO was used as template to amplify the entire sequence of SSAO coding area. SSAO and green fluorescent protein (GFP) fusion expression and the co-expression plasmids were respectively constructed and identified by restriction enzyme digestion and sequencing. The recombinant plasmids pcDNA3-SSAO,pEGFP-N3-SSAO,pIRES2-EGFP-SSAO and pcFLAG-SSAO were transiently transfected into 293T cells,then the distribution of recombinant protein in 293T cells was observed under fluorescence microscope. The enzymatic activity of SSAO in 293T cells that were transiently transfected with recombinant plasmids and with corresponding empty plasmids were measured using high-performance liquid chromatography. RESULTS: Sequencing results show that the recombinant eukaryotic expression plasmids pEGFP-N3-SSAO and pIRES2-EGFP-SSAO were constructed correctly. The green fluorescence was observed in 293T cells at 3~36 h after pEGFP-N3-SSAO and pIRES2-EGFP-SSAO plasmids' transfection,with optimal observation time of 36 h. The cells transfected with pIRES2-EGFP-SSAO plasmids showed green fluorescence in cytoplasm. The cells transfected with pEGFP-N3-SSAO and pcFLAG-SSAO showed green fluorescence or red fluorescence on the cellular membranes. SSAO activity of 293T cells transfected with pcFLAG-SSAO,pIRES2-EGFP-SSAO and pcDNA3-SSAO recombinant plasmids were 111.50±7.07,117.22±9.54,215.74±21.44 nmoL/(mg·h),respectively,which were significantly higher than that in the control group (P < 0.01). While,the activity of SSAO in cells transfected with pEGFP-N3-SSAO plasmid was only 2.09±0.29 nmoL/(mg·h). CONCLUSION: Recombinant expressed SSAO proteins were localized on the cellular membranes in 293T cells. Cells transfected with pcDNA3 as the carrier plasmids could generate higher SSAO activity.

OBJECTIVE: To established a method for automated detection of flow cytometry-based biomarker γ-H2AX,and to discussed the prospect of this method in drug screening for early genotoxicity. METHODS: The experiment was divided into two groups:-S9 group (long time treatment for 24 hours) and +S9 group (short time treatment for 4 hours). Then,4 suitable concentrations of Cyclophosphamide and Etoposide were selected using the CCK-8 assay. Treated TK6 cells were harvested according to standard operation procedures. After harvest,cells were double labeled with nucleic acid SYTOX Green and Alexa Fluor 647 conjugated anti-γ-H2AX antibody. BD Accuri C6 Flow cytometry was used to analyze the percentage of phosphorylated histone H2AX (γ-H2AX) cells. RESULTS: In the -S9 long-time treatment group,the ratio of γ-H2AX positive cells increased significantly compared with the solvent control group in different concentrations of Etoposide,and it showed a significant dose-dependent relationship. In the +S9 short-time treatment group,a significant increase in different concentrations of Cyclophosphamide was observed. CONCLUSION: Flow cytometry rapidly and accurately detected changes of γ-H2AX in cultured TK6 cells in vitro. Therefore,it is possible to detect γ-H2AX based on flow cytometry.

OBJECTIVE: To study whether apigenin could induce autophagy in human gastric cancer SGC-7901 cells and whether the induced autophagy would affect apoptosis in these cells. METHODS: SGC-7901 cells were treated with different doses (0,20,40,60,80 μmol/L) of apigenin (API) for 24 h. Cell survival rates were measured using the MTT method. In addition,cells were treated with 60 μmol/L API for different hours(0,12,24,36,48 h). Western blot was used to evaluate the autophagy marker protein microtubule-associated protein 1 light chain 3 (LC3),Beclin-1 and the activities of p-Akt and p-mTOR,the critical targets of Akt/mTOR signaling pathway. Electron microscopy was used to observe autophagy. Furthermore, cells were treated with autophagy inhibitor,chloroquine(CQ) and 3-mehtyladenine (3-MA) and 60 μmol/L API. Western blot analyses were performed to detect changes in LC3,Beclin-1 and PARP. Annexin V-FITC/PI was used to detect the apoptotic rate. RESULTS: SGC-7901 cell viability was inhibited under API treatment in a does-response manner. With increasing doses of API,autophagic vesicles and autophagosome began to appear in electron microscopy. Western blot analysis showed that LC3-Ⅱ and Beclin-1 protein levels increased in cells treated with API (P < 0.05). The phosphorylation levels of Akt and mTOR were reduced. After inhibiting autophagy,the expression of LC3 Ⅱ and Beclin-1 proteins in the API group was significantly higher than that in the control group(P < 0.05). There was no significant difference in the other groups. Cleaved PARP and apoptotic rate increased significantly in API+CQ and API+3-MA then control and API. CONCLUSION: API can induce autophagy in human gastric cancer cells SGC-7901 via the Akt/mTOR signaling pathway. The API-induced autophagy could significantly increase API-induced apoptosis in these cells. Furthermore,API-induced autophagy can protect survival of gastric cancer cells.

OBJECTIVE: To evaluate the preventive and therapeutic effects of Sijunzi decoction on damage from ionizing radiation in hematopoietic and immune systems. METHODS: CCK-8 assay kit was used to detect the toxic effect of AHH-1 cells which were treated with different dosages (0,60,120,600,1 200 μg/mL) of Sijunzi decoction extract for 48 h. In addition,the survival rate of AHH-1 cells which were pretreated with different dosages (0,0.04,0.2,1,25,120 μg/mL) of Sijunzi decoction for 2 h and irradiated with 4.0 Gy ⁶⁰Co γ-rays was investigated. One hundred and sixty BALB/c mice aged 6-8 weeks were divided by random number table method into normal control group,radiation group,and three groups of Sijunzi decoction at dosages of 0.75,2.25 and 6.75 g/kg,respectively. Radiation-damaged mice model were individually-irradiated with 3.5,5.5 Gy γ-rays,respectively. The following activity indexes were investigated:the count and percentage of peripheral lymphocytes,thymus index,survival rate and average survival time in 30 d. RESULTS: Compared to the radiation group,the survival rate of irradiated AHH-1 cells were increased greatly by Sijunzi decoction at the dosage of 120 μg/mL (P < 0.05). At 3 d after irradiation,the count and percentage of peripheral lymphocytes were promoted significantly (P < 0.05) by Sijunzi decoction at the dosage of 2.25 g/kg in mice. The percentage of peripheral lymphocytes was increased significantly (P < 0.05) by Sijunzi decoction at the dosage of 6.75 g/kg,also the count of peripheral lymphocytes was increased. At 3 and 7 d after irradiation,the thymus index was increased significantly (P < 0.05) by Sijunzi decoction at the dosage of 6.75 g/kg. Besides,the survival rate was increased (P < 0.05) and the mean survival time was prolonged significantly (P < 0.05) by Sijunzi decoction at the dosage of 6.75 g/kg. CONCLUSION: Sijunzi decoction could alleviate the ionizing radiation-induced damage,improve mice immune and hematopoietic function,by means of increasing the count and percentage of peripheral lymphocytes and the thymus index. On the other hand,Sijunzi decoction can also improve the 5.5 Gy-irradiated mice's survival rate. Accordingly,Sijunzi decoction might be developed into a new drug for radiation protection.

OBJECTIVE: To detect the expression of β-catenin and its prognostic significance in hepatocellular carcinoma. METHODS: The expression of β-catenin protein was detected by immunohistochemistry in 83 cases of resected HCC tissues,26 cases of adjacent tissues and 5 cases of normal liver tissues. The integrated follow-up data were rerospectively analyzed in patients who had undergone radical hepatectomy. The relationships between β-catenin and clincal variables,HCC pathological differentiation and postoperative survival were statistically analyzed. RESULTS: The positive expression rates of β-catenin in the HCC tissues,adjacent tissues and normal liver tissues were 68.67% (57/83)、30.77%(8/26) and 20%(1/5). The positive rate of β-catenin in HCC was significantly higher than that of adjacent tissues and normal liver tissues (P < 0.05). The positive expression rates of β-catenin was negatively correlated with pathological differentiation (r=-0.34,P=0.027). In the 83 follow-up data,68 cases were effective. In the first 5 years of follow-up data,and among patients with positive expression of β-catenin,the mean survival time was 11.2 months. Among patients with negative expression of β-catenin,the mean survival time was 26.6 months. Kaplan-Meier survival analysis revealed that patients with negative expression of β-catenin had longer survival time and their prognosis was significantly different based on Log-rank test(χ²=15.138,P=0.000). CONCLUSION: Expression of β-catenin was correlated with pathological differentiation in HCC and its high expression predicted poor prognosis.

OBJECTIVE: To investigate the expression of RNA-binding proteins LIN28A,epithelial-mesenchymal transition (EMT) -related proteins E-cadherin and vimentin in invasive breast cancer and to understand their role in development of invasive breast cancer. METHODS: Quantitative real-time PCR (qPCR) and immunohistochemical staining were used to detect expressions of LIN28A,E-cadherin and vimentin mRNAs and proteins in invasive breast cancer tissues and the corresponding adjacent tissues. Relationships between mRNA and protein expressions of LIN28A,E-cadherin and vimentin were analyzed. TUNNEL staining was used to detect apoptosis of invasive breast cancer tissues with high LIN28A expressions. The relationship between LIN28A and EMT-related proteins and survival rate of breast cancer patients were statistically analyzed. RESULTS: qPCR results show that the relative expression level of LIN28A mRNA in breast cancer tissues were higher than adjacent tissues, negatively correlated with E-cadherin mRNA,and positively correlated with vimentin mRNA(P < 0.05). Immunohitochemistry staining results show that in breast cancer tissues,the expression rates of LIN28A,E-cadherin and vimentin protein were 60%,55% and 65%,respectively. The expression of LIN28A protein was negatively correlated with E-cadherin protein,positively correlated with vimentin protein. LIN28A expression was correlated with tumor size,histological grade and lymph node metastasis. TUNNEL assay show that high expression of LIN28A could decrease apoptosis in invasive breast cancer(P < 0.05). The survival rate of patients with positively expressed LIN28A was significantly lower than that of LIN28A negative patients (P < 0.05). CONCLUSION: The RNA-binding protein,LIN28A,may promote the occurrence,development and metastasis of breast cancer via EMT,while high expression of LIN28A may affect prognosis of patients with breast cancer.

OBJECTIVE: To explore the mutagenicity of five different synthetic sport surface chemicals. METHODS: Plastic racetracks from:silicon PU stadium,permeable running tracks,EPDM running tracks,mixed running tracks and prefabricated roll,were extracted using water and dimethyl sulfoxide (DMSO),and were tested in the Ames assays. RESULTS: Using tester strains TA97a and TA100 with S9,the revertant colonies of DMSO-extracted products from permeable running tracks surface were two times higher than that in the controls. Using the same tester strains but with or without S9, the revertant colonies of DMSO-extracted EPDM running tracks surface were also two times higher than that in the controls. On the other hand,water-extracted products did not induce mutagenicity. CONCLUSION: DMSO-extracted products from both permeable and EPDM running tracks surfaces were capable of inducing mutagenicity.

OBJECTIVE: To investigate toxicity of extracts from five kinds of plastic runway surface layer (silicon PU ball surface layer,plastic mat,EPDM plastic track,mixed plastic runway panel,prefabricated plastic track) and to provide a preliminary understanding of its safety to mouse fibroblast L-929. METHODS: Four groups of high density polyethylene negative control,no sample blank control,10% DMSO positive control and 0.5% DMSO extract were used to detect the cytotoxicity of the sample extract on mouse fibroblast L-929 using XTT colorimetry. RESULTS: The relative proliferation rate of the four kinds of samples of silicon PU ball surface layer,plastic mat,EPDM plastic track and mixed plastic runway panel was less than 70%. The relative proliferation rate of the prefabricated plastic runway sample was more than 70%. CONCLUSION: 0.5% DMSO extract showed strong in vitro cytotoxicity of silicon PU ball surface layer,plastic mat,EPDM plastic track,mixed plastic runway panel sample.

OBJECTIVE: Evaluation of di-(2-ethylhexyl)phthalate (DEHP) as raw material for preparing DEHP reference material for medical devices tests. METHODS: Identification and determination of purity of DEHP were carried out for the raw material using the gas chromatography-mass spectrometer(GC-MS) area normalization method. The results of calculation of DEHP reference material was obtained through collaboration with several laboratories. Homogeneity and stability were also carried. RESULTS: The selected raw material had high and stable purity. DEHP reference material's purity was 99.9%±0.2%. The homogeneity determination results were:the content results between and in bottles had no statistical difference. The statistics F was 2.20 and 0.28,which were less than the threshold value F,2.85;P > 0.05. The stability determination results were:the DEHP reference material was unchanged and stable in 12 months. CONCLUSION: The above test results showed that the DEHP reference material was homogeneous,stable,and had high purity and low uncertainty. So it was proper to develop it as DEHP reference material for medical devices tests.